US 20140370007A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0370007 A1 McCabe et al. (43) Pub. Date: Dec. 18, 2014

(54) METHOD OF DIRECTED DIFFERENTATION Publication Classification PRODUCING CORNEAL ENDOTHELLAL CELLS, COMPOSITIONS THEREOF, AND (51) Int. Cl. USES THEREOF CI2N5/079 (2006.01) A619/00 (2006.01) (71) Applicant: Advanced Cell Technology, Inc., CI2N 5/0797 (2006.01) Marlborough, MA (US) A6135/30 (2006.01) A6II 45/06 (2006.01) (72) Inventors: Kathryn L. McCabe, Westborough, MA (52) U.S. Cl. (US); Shi-Jiang Lu, Shrewsbury, MA CPC ...... CI2N5/0621 (2013.01); A61K 35/30 (US); Robert P. Lanza, Clinton, MA (2013.01); A61K 45/06 (2013.01): CI2N (US) 5/0623 (2013.01); A61 K9/0048 (2013.01); CI2N 2506/08 (2013.01); C12N 2501/135 (2013.01); C12N 2501/415 (2013.01); C12N (73) Assignee: Advanced Cell Technology, Inc., 2506/02 (2013.01); C12N 2501/40 (2013.01); Marlborough, MA (US) CI2N 2501/999 (2013.01); C12N 2500/60 (2013.01); C12N 2500/90 (2013.01); C12N (21) Appl. No.: 14/362,775 2533/50 (2013.01); C12N 2506/45 (2013.01) USPC ...... 424/133.1; 435/377; 435/368; 435/325: (22) PCT Fled: Dec. 6, 2012 424/93.7; 424/278.1; 424/173.1 (86) PCT NO.: PCT/US12A68305 (57) ABSTRACT This disclosure generally relates to cell-based therapies for S371 (c)(1), treatment of visual disorders, including disorders of the cor (2), (4) Date: Jun. 4, 2014 nea. Methods are exemplified for directed differentiation of corneal cells from stem cells. Compositions of corneal endot helial cells and uses thereof are also provided. Exemplary Related U.S. Application Data compositions exhibit improved cell density and/or more (60) Provisional application No. 61/567,479, filed on Dec. “youthful” gene expression relative to cells obtained from 6, 2011. donated tissue. Patent Application Publication Dec. 18, 2014 Sheet 1 of 45 US 2014/0370.007 A1

FIG. 1

o VO O O O O Example 1 O O Noggin O. O. SB41s42 u (i.AG) Poss exagonal cols in ESFederreo KK creuards

Day O. Day 2 M day 9 O O. O O O O Example2 O O) PDGFBB O O ck2

Dissociate hexagonal cells Non-enzymatic or mechanical Patches of Progenitors Replate Day 3 Oay 10

Patent Application Publication Dec. 18, 2014 Sheet 2 of 45 US 2014/0370.007 A1

FIG.2

Patent Application Publication Dec. 18, 2014 Sheet 3 of 45 US 2014/0370.007 A1

FIG. 3

E

N Patent Application Publication Dec. 18, 2014 Sheet 4 of 45 US 2014/0370.007 A1

FIG. 4

Na/K ATPase

A B i

i N-F - -w WNKN W Patent Application Publication Dec. 18, 2014 Sheet 5 of 45 US 2014/0370.007 A1

FIG. 5

A. B

2 s 2

Patent Application Publication Dec. 18, 2014 Sheet 6 of 45 US 2014/0370.007 A1

FIG. 6

HUVEC A A

A

N N Patent Application Publication Dec. 18, 2014 Sheet 7 of 45 US 2014/0370.007 A1

FIG. 7

C 2000 1800 1600 140 1200 cy Collagen C OOO WI 800 SO 400

hESC Corneal O Endo ESC Corneal B Edo

27.5 25.0 22.5 20,0 E. 7.5 15.0 SLC6A3 12.5 10.0 7.5 SO 2.5 0.0 hESC Corneat Endo Patent Application Publication Dec. 18, 2014 Sheet 8 of 45 US 2014/0370.007 A1

FG. 8

DS3 DSA Days of exposure to Dual Smad Patent Application Publication Dec. 18, 2014 Sheet 9 of 45 US 2014/0370.007 A1

FIG. 9

ColVIII (Endo) cONGFR (NC) choSox10(NC) to Nanog (hESC)

hESC Day2 Day3 Day4 Day5 Day6 Day7 Day9 Patent Application Publication Dec. 18, 2014 Sheet 10 of 45 US 2014/0370.007 A1

FIG. 10

PTX2 FOXC1

Figure 10. Corneal endothelial cells express the transcription factors PITX2 and FOXC1, markers of ocular neural crest during development. RQS relative quantification by QPCR. Patent Application Publication Dec. 18, 2014 Sheet 11 of 45 US 2014/0370.007 A1

F.G. 11

Patent Application Publication Dec. 18, 2014 Sheet 12 of 45 US 2014/0370.007 A1

FIG. 12

A E. D D

N N N A1

N N N A1

N N N A-1 Patent Application Publication Dec. 18, 2014 Sheet 13 of 45 US 2014/0370.007 A1

FIG. 13

A B C

A1 A-1

1. A1

A W . . . . 3'' -ax8 '' : s - . c -w'

-1 Patent Application Publication Dec. 18, 2014 Sheet 14 of 45 US 2014/0370.007 A1

e

uosseudopazutor aarea Patent Application Publication Dec. 18, 2014 Sheet 15 of 45 US 2014/0370.007 A1

-S4 s N H t C All

O) 3 3 Ss 3Y S Svs. w N y uosse depezius Lutor Aeg Losseudopazelio emea Patent Application Publication Dec. 18, 2014 Sheet 16 of 45 US 2014/0370.007 A1

e. Lossottpozeunion era

Solitic sfsis

g r c lossodpareucose lossed apezetuso ages C U Patent Application Publication Dec. 18, 2014 Sheet 17 of 45 US 2014/0370.007 A1

F.G. 17

Gene Expressa

is ESC as Poit is Rock as Rock so iD Rock

ca

e A. t r r

Ap larget hSC a P 1 Rack to 5 Rack Rack s Patent Application Publication Dec. 18, 2014 Sheet 18 of 45 US 2014/0370007 A1

FIG. 18

ZO-1 DAP MERGE A B C

1. 1. 1. Patent Application Publication Dec. 18, 2014 Sheet 19 of 45 US 2014/0370.007 A1

FIG. 19

COL8A1

Patent Application Publication Dec. 18, 2014 Sheet 20 of 45 US 2014/0370.007 A1

FIG. 20 (1)

genesymbol genedescription Accession number Fold Change PRKCZ. protein kinase C, Zeta NM002744 1645 GRHL3 grainyhead-like 3 (Drosophila) NM 198173 12.71 CNKSR conncctor enhancer of kinase suppressor of Ras NM_006314 O SFN stratifin NM 006142 10.50 SCARNA small Cajal body-specific RNA 0.02 TNAGL tubulointerstitial nephritis antigen-like l NM 022164 9.97 PTPRF protein tyrosine phosphatase, receptor type, F NM 002840 9.85 NADL lmaD-like (Drosophila) NM 176877 9.79 ARD3B AT rich interactive domain 3B (BRIGHT-like) NM_006.465 9.66 TMEM56 transmembrane protein 56 NM 152487 9.54 LPPRA lipid phosphate phosphatase-related protein type 4 NM_014839 9.42 Clorf3 chromosome 1 open reading frame 88 9.4 PHGDH phosphoglycerate dehydrogenase NM_006623 9.39 Tr. 7904959 9.35 BNP BCL2/adenovirus El B 19kD interacting protein like NM 001159642 9.31 CGN cingulin NM 020770 9.27 RALGPS2 Ral GEF with PH domain and SH3 binding motif 2 NM 152663 9.22 E74-like factor 3 (ets domain transcription factor, epithelial ELF3 specific) NM_001114309 9.17 FAM4.6B family with sequence similarity 46, member B 8.78 NKANE Na+/K+ transporting ATPase interacting 1 8.76 RSPO1 R-spondinhomolog (Xenopus laevis) NM 00038633 8.52 Corf210 chronosome l open reading frame 210 8.10 KANK.4 KN motif and ankyrin repeat domains 4 8.07 SYDE2 . synapse defective 1, Rho GTPase, homolog 2 (C. elegans) NM 032184 8.03 ABCA4 ATP-binding cassette, sub-family A (ABC), member 4 NM 000350 3.03 F3 coagulation factor lll (thromboplastin, tissue factor) NM 001993 8.02 FRRS1 ferric-chelate reductase 1 NM_001013660 7.98 TBX15 T-box 5 NM 152380 7.94 CRABP2 cellular retinoic acid binding protein 2 NM_001878 7.89 F11 receptor thiosulfate sulfurtransferase (rhodanese)-like domain F11RTSTD containing NM 016946 7.86 PWRL4 poliovirus receptor-related 4 NM 03.096 7,83 APOA2 apolipoprotein A-II NM_001643 7.82 TNNT2 troponin Ttype 2 (cardiac) NM_000364 7.80 LAD ladinin 7.65 LMOD leiomodin l (smooth muscle) NM 012134 7.59 IRF6 interferon regulatory factor 6 NM 006147 7.58 Tr. 7924069 7.49 LBR lamin B receptor 7.47 Clorf198 chromosome 1 open reading frame 198 7.44 FAM89A family with sequence similarity 89, member A 7.38 GNG4 guanine nucleotide binding protein (G protein), gamma 4 NM_00109872) 7.34 NET neuroepithelial cell transforming NM_001047160 7.7 BMP and activin membrane-bound inhibitor homolog (Xenopus BAMBI laevis) NM 012342 7.15 Patent Application Publication Dec. 18, 2014 Sheet 21 of 45 US 2014/0370.007 A1

FIG. 20 (2)

genesynbol genedescription Accession number Fold Change annexin A8-like 2 annexin A8 annexin A8-like

annexin A8 annexin A8-like lannexin A8-like 2 7.07 tet oncogene l NM 03.0625 7.06

ENTPD ectonucleoside triphosphate diphosphohydrolasel NM_001776 ATRNL) attractin-like NM 207303 1TH5 inter-alpha (globulin) inhibitor H5 NM 030569 integrin, alpha 8 NM_003638 membrane protein, palmitoylated 7 (MAGUK p55 subfamily member 7) 6.96 ANXA8LlANXA8L2 annexin A8-like 1 annexin A8 annexin A8-like 2 ACTA2 actin, alpha 2, smooth muscle, aorta NM_00141945 694 ANKRD1 ankyrin repeat domain l (cardiac muscle) NM 04391 6.9 cytochrome P450, family 2, subfamily C, polypeptide 8 cytochrome P450, family 2, subfamily C, polypeptide 19 NM 000770 6.87 PDLM PDZ and LIM domain l NM_020992 KIAA1598 NM00272 6.8S chromosome 1 open reading frame 35 hypothetical locus C11orf35LOC692247 LOC692247 insulin-like growth factor 2 (somatomedin A) NS-IGF2 1GF2INS-IGF2 readthrough transcript NM 000612 6.69 SNORA3 small nucleolar RNA, H/ACA box 3 666 SNORA23 small nucleolar RNA, H/ACA box 23 6.6 PDE3B phosphodiesterase 3B, c0MP-inhibited NM_000922 6.58 NAV2 neuron navigator 2 NM 182964 6.53

tetraspanin 18 6.50

cAMP responsive element binding protein 3-like 1 NM 052854 6.49 PTPRU protein tyrosine phosphatase, receptor type, J NM 002843 6.48 STX3 syntaxin3 NM004in 6.46 C11orf chromosome l l open reading frame 9 NM_001 127392 6.45 CST6 cystatin E/M NM 001323 6.45 purinergic receptor P2Y, G-protein coupled, 2 NM 176072 6.42 pyrimidinergic receptor P2Y, G-protein coupled, 6 NM 176796 PWIL4 piwi-like 4 (Drosophila) NM 152431 6.38 ST4 suppression of tumorigenicity 14 (colon carcinoma) NM021978 NM_001012302 SOX6 SRY (sex determining region Y)-box 6 NM 017508 6.34 PLEKHAT pleckstrin homology domain containing, family A member 7 NM 175058 6.30 SLC5A12 solute carrier family 5 (sodium/glucose cotransporter), member 12 NM 178498 6.26 RCOR2 REST corepressor 2 NM 173587 6.25 SHANK2 SH3 and multiple ankyrin repeat domains 2 NMO 12309 6.23 exophilin 5 NM_015065 6.20 dopamine receptor D2 NM 000795 6.4 FXYD6 FXYD domain containing ion transport regulator 6 NM 001164836 6.13 USP2 ubiquitin specific peptidase 2 NM_004205 6.09 Patent Application Publication Dec. 18, 2014 Sheet 22 of 45 US 2014/0370007 A1

FIG. 20 (3)

genesymbol Fold Change CACNAC calcium channel, voltage-dependent, L type, alpha 1 C subunit CCND2 cyclin D2 SCARNA10 small Cajal body-specific RNA 10 602 PIK3C2G phosphoinositide-3-kinase, class 2, gamma polypeptide NM_004570 5.94 METTLA methyltransferase like 7A NM014033 s.93 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) NM_001982 HMGA2 high mobility group AT-hook 2 NM 003483 5.93 RAB3IP RAB3A interacting protein (rabin3) NM 175623 s 9 MIR492|KRT19P2 microRNA 492 keratin 19 pseudogene 2 NM 002276 5.87

SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 NM_006931 S.86 C-type lectin domain family l, member B NM 016509 5.82 DUSP6 dual specificity phosphatase l6 NM 030640 SRY (sex determining region Y)-box 5 NM 52989 FAM60A family with sequence similarity 60, member A 8 PRICKLE prickle homolog 1 (Drosophila) NM 153026

ADAMTS20 ADAM metallopeptidase with thrombospondin type 1 motif, 20 NM025003 5.7380 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N acetylgalactosaminyltransferase 6 (GalNAc-T6) NM_007210 5.71 keratin 8 NM_002273 TAC3 tachykinin 3 NR 033654 LUM lumican NM 002345 NUAK1 NUAK family, SNF)-like kinase, 1 NM_014840 RNU4-1 RNA, U4 small nuclearl 5.65 SGCG sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) NM_000231 C3orf5 chromosome 13 open reading frame 15 NM 014059 MIR622|KRT18 microRNA 622 keratin 18 NM 000224 MIR17HG|MIR19AM1RMIR17 host gene (non-protein coding) microRNA 17 microRNA 19Bl|MIR20AMIR92Al 19a microRNA 19b-l microRNA 20a microRNA 92a-1 |M1R18A microRNA 18a 5.59 GPC6 glypican 6 5.56 fms-related tyrosine kinase l (vascular endothelial growth FLT1 factor/vascular permeability factor receptor) NM_002019 5,55 solute carrier family 7 (cationic amino acid transporter, y+ system), SLC7Al member 1 NM_003045 5.54 protocadherin 9 NM 203487 5.54 POTE ankyrin domain family, member M POTE ankyrin domain family, member GPOTE ankyrin domain family, member El POTEMPOTEGPOTEEPOTE ankyrin domain family, member F POTE ankyrin domain POTEF|POTEH family, member H SLC39A2 solute carrier family 39 (zinc transporter), member 2 NM_014579 5.52 dapper, antagonist of beta-catenin, homolog 1 (Xenopus laevis) NM 016651 5.52 AKAPS A kinase (PRKA) anchor protein 5 NM 004857 5.50 DO3 deiodinase, iodothyronine, type III NM_001362 5.43 Patent Application Publication Dec. 18, 2014 Sheet 23 of 45 US 2014/0370007 A1

FIG. 20 (4)

genesymbol genedescription Fold Change POTE ankyrin domain family, member GPOTE ankyrin domain POTEGPOTEM)POTEH family, member M POTE ankyrin domain family, member H 5.39

RNASE1 ribonuclease, RNase A family, l (pancreatic) 5.39 nidogen 2 (osteonidogen) NM 198686 TC2N tandem C2 domains, nuclear S.36 CLMN calmin (calponin-like, transmembrane) SNORD 16-25 small nucleolar RNA, C/D box 116-25 SNORD16-26 small nucleolar RNA, C/D box 1 16-26

SNORD 15 |SNORD115 13|SNORD 15- small nucleolar RNA, C/D box 115-1 small nucleolar RNA, C/D 16SNORD 15- box 115-13 small nucleolar RNA, C/D box 115-16 small l2SNORD115- nucleolar RNA, C/D box 115-12 small nucleolar RNA, C/D box 26SNORD15- 115-26 small nucleolar RNA, C/D box 115-40 small nucleolar 40SNORD15- RNA, C/D box 115-41 | small nucleolar RNA, C/D box 115-5 41SNORD15- small nucleolar RNA, C/D box l 15-9 small nuclear 5|SNORD115-9|SNRPN ribonucleoprotein polypeptide N AF4.00495 SNORD15 12SNORD 15 5|SNORD 15 9|SNORD 15 11SNORD15 29SNORD 15 36SNORD15- small nucleolar RNA, C/D box 15-12 small nucleolar RNA, C/D 43SNORD15- box 115-5 small nucleolar RNA, CID box 115-9 small nucleolar 20 SNORD115- RNA, C/D box 115-11 small nucleolar RNA, C/D box 115-29 | 17|SNORD115- small nucleolar RNA, C/D box 115-36 small nucleolar RNA, C/D 18|SNORD 15- box 115-43 small nucleolar RNA, C/D box 115-20 small 19 SNORD 15- nucleolar RNA, C/D box 115-17 small nucleolar RNA, C/D box 13SNORD115- 115-18 small nucleolar RNA, C/D box 115-19 small nucleolar 25 SNORD115- RNA, C/D box llS-l small nucleolar RNA, C/D box 115-13 40SNORD115- small nucleolar RNA, C/D box 115-25 small nucleolar RNA, C/D 42 SNORD 15- box 15-40 small nucleolar RNA, C/D box 115-4 small OSNORD 5- nucleolar RNA, C/D box 115-42 small nucleolar RNA, C/D box 14|SNORD15- 115-10 small nucleolar RNA, C/D box l l 5-14 small nucleolar 15|SNORD 15-24 RNA, C/D box 115-15 small nucleolar RNA, C/D box 115-24

Patent Application Publication Dec. 18, 2014 Sheet 25 of 45 US 2014/0370.007 A1

FIG. 20 (6)

SNORD 5 2 SNORD15 5|SNORD 115 9|SNORD 115 SNORDS SNORD 5 SNORD 15 SNORD 5 SNORD 15 small nucleolar RNA, C/D box 115-12 small nucleolar RNA, CID SNORD 15 box 115-5 small nucleolar RNA, C/D box 5-9 small nucleolar SNORD 5 RNA, C/D box 115-11 small nucleolar RNA, CID box 115-29 SNORD 15 small nucleolar RNA, C/D box 115-36 small nucleolar RNA, CID SNORD5 box 115-43 small nucleolar RNA, C/D box 15-22 small SNORDS nucleolar RNA, C/D box 115-20 small nucleolar RNA, C/D box SNORD15 115-39small nucleolar RNA, C/D box 115-17 small nucleolar SNORD 15 RNA, C/D box 115-18 small nucleolar RNA, CID box 115-19 SNORD115 small nucleolar RNA, C/D box li5-3 small nucleolar RNA, CID SNORD115 box 115-1 small nucleolar RNA, C/D box 115-13 small SNORD15 nucleolar RNA, C/D box IS-16 small nucleolar RNA, C/D box SNORD 15 115-25 small nucleolar RNA, C/D box 115-40 small nucleolar SNORD15 RNA, C/D box 115-44 small nucleolar RNA, C/D box 115-34 small nucleolar RNA, C/D box 115-4 small nucleolar RNA, C/D O SNORD15 box 115-42 small nucleolar RNA, C/D box 15-6 small 14 SNORD15 nucleolar RNA, C/D box 115-10 small nucleolar RNA, C/D box SNORD15 15-14 small nucleolar RNA, C/D box 115-15 small nucleolar SNORD5-24 RNA, C/D box 115-21 small nucleolar RNA, C/D box 115-24 SNORD5. 1SNORD115 13 SNORD 15 16 SNORD15 small nucleolar RNA, C/D box 115-1 small nucleolar RNA C/D 12 SNORD15 box 115-13 small nucleolar RNA, C/D box 15-16 small 40 SNORD 5 nucleolar RNA, C/D box 115-12 small nucleolar RNA, C/D box I lis-40 small nucleolar RNA, C/D box 1 15-5 small nucleolar RNA, C/D box 115-9 small nucleolar RNA, C/D box 115-10 SNORD15 small nucleolar RNA, C/D box 115-14 small nucleolar RNA, CID SNORD 15 box 115-21 small nucleolar RNA, C/D box 115-4 small 21 SNORD 15 nucleolar RNA, C/D box 15-42 small nuclear ribonucleoprotein SNRPN polypeptide N AF400495 SNORD 5 |SNORD) 15 13|SNORD 15 6 SNORDS 26 SNORD15 small nucleolar RNA, C/D box 115-1 small nucleolar RNA, C/D box 115-13 small nucleolar RNA, C/D box 115-16 small nucleolar RNA, C/D box 15-11 small nucleolar RNA, C/D box 115-26 small nucleolar RNA, CID box 115-29 small nucleolar 40 SNORD15 RNA, C/D box ll5-36 small nucleolar RNA, C/D box 115-39 41SNORDE 5 small nucleolar RNA, C/D box l 15-40 small nucleolar RNA, C/D 43SNORD 15 box 115-4 small nucleolar RNA, C/D box 115-43 small 34|SNORDt 15 nucleolar RNA, C/D box 15-34 small nucleolar RNA, C/D box 6SNRPN li5-6 small nuclear ribonucleoprotein polypeptide N AF4.00495

Patent Application Publication Dec. 18, 2014 Sheet 27 of 45 US 2014/0370.007 A1

FIG. 20 (8)

genesymbol genedescription Accession number Fold Change SNORD 15 SNORD115 SNORD15 SNORD15 SNORD15 SNORD15 5|SNORD115 9|SNORD115- small nucleolar RNA, C/D box 115-11 small nucleolar RNA, C/D SNORD15- box l 15-12 small nucleolar RNA, C/D box l 5-29 small SNORD115- nucleolar RNA, C/D box 115-36 small nucleolar RNA, C/D box SNORD15- 115-43 small nucleolar RNA, C/D box 115-5 small nucleolar SNORED115- RNA, C/D box 115-9 small nucleolar RNA, CID box 115-26 SNORD15- small nucleolar RNA, C/D box 115-22 small nucleolar RNA, CID SNORD115- box 115-20 small nucleolar RNA, C/D box 115-39 small SNORD15- nucleolar RNA, C/D box 115-3 small nucleolar RNA, C/D box SNORD15- 115-41 small nucleolar RNA, C/D box 115-17 small nucleolar SNORD115- RNA, C/D box 115-18 small nucleolar RNA, C/D box 115-19 SNORD115- small nucleolar RNA, C/D box 115-1 small nucleolar RNA, C/D SNORD 15- box 15-13 small nucleolar RNA, C/D box liS-16 small SNORD 15- nucleolar RNA, C/D box 115-40 small nucleolar RNA, CID box SNORD15- 115-44 small nucleolar RNA, C/D box 115-10 small nucleolar SNORD115- RNA, C/D box 115-14 small nucleolar RNA, CID box 115-34 SNORD115- small nucleolar RNA, C/D box 115-4 small nucleolar RNA, C/D SNORD15- box 15-42 small nucleolar RNA, C/D box llS-6 small 6|SNORD) 15-24 nucleolar RNA, C/D box 15-24 SNORD 15 SNORD15 SNORD15 SNORD115- small nucleolar RNA, C/D box 115-11 small nucleolar RNA, C/D SNORED15- box l 15-26 small nucleolar RNA, C/D box 115-29 small SNORD15- nucleolar RNA, C/D box 115-36 small nucleolar RNA, C/D box SNORED115- l 15–39 small nucleolar RNA, C/D box l l 5-43 small nucleolar SNORD15- RNA, C/D box 115-41 small nucleolar. RNA, C/D box 115-22 SNORD15- small nucleolar RNA, C/D box 115-16 small nucleolar RNA, C/D SNORD115- box 115-14 small nucleolar RNA, C/D box 115-38 small SNORD15- nucleolar RNA, C/D box 115-44 small nucleolar RNA, C/D box SNORD15-44 15-3 SNORD 15 42 SNORD115 6|SNORD115 3OSNORD115- w 34ISNORD15- small nucleolar RNA, C/D box 115-42 small nucleolar RNA, C/D SISNORD115- box 15-6 small nucleolar RNA, CID box 115-4 small nucleolar 21SNORD115- RNA, C/D box 115-30 small nucleolar RNA, CID box 115-34 12 SNORD115- small nucleolar RNA, C/D box 115-15 small nucleolar RNA, C/D 13SNORD115- box 15-21 small nucleolar RNA, C/D box 115-12 small 40SNORD115- nucleolar RNA, C/D box 115-13 small nucleolar RNA, C/D box 5|SNORD) 15- l li5-40 small nucleolar RNA, C/D box l 15-5 small nucleolar 9|SNORD115- RNA, C/D box 115-9 small nucleolar RNA, C/D box 115-10 1OSNORD115- small nucleolar RNA, C/D box 115-14 small nucleolar RNA, C/D 14|SNORD115-24 box 15-24 Patent Application Publication Dec. 18, 2014 Sheet 28 of 45 US 2014/0370.007 A1

FIG. 20 (9)

genesymbol genedescription Accession number Fold Change SNORD 15 - SNORD15 12|SNORD15 29SNORD 15 36SNORD 5 43SNORD 5 SISNORD115 9|SNORD115 small nucleolar RNA, C/D box 115-11 small nucleolar RNA, CID 26SNORD15 box 115-12 small nucleolar RNA, C/D box 115-29small SNORD 15 nucleolar RNA, C/D box 115-36 small nucleolar RNA, C/D box SNORD15 115-43 small nucleolar RNA, C/D box 115-5 small nucleolar SNORD15 RNA, C/D box 115-9 small nucleolar RNA, C/D box 115-26 SNORD15. small nucleolar RNA, C/D box 115-22 small nucleolar RNA, C/D SNORD115 box 115-20 small nucleolar RNA, C/D box 115-39small SNORD5. nucleolar RNA, C/D box 115-3 small nucleolar RNA, C/D box SNORD15. 15-41 small nucleolar RNA, C/D box 115-17 small nucleolar SNORD115 RNA, C/D box 115-18 small nucleolar RNA, C/D box 115-19 SNORD115 small nucleolar RNA, C/D box 115-1 small nucleolar RNA, C/D SNORD115 box 115-13 small nucleolar RNA, CID box 115-16 small SNORD15. nucleolar RNA, C/D box 115-40 small nucleolar RNA, CID box SNORD115 115-44 small nucleolar RNA, C/D box 115-34 small nucleolar 6|SNORD 15 RNA, C/D box 115-6 small nucleolar RNA, CD box l 15-10 OSNORD 5-24 small nucleolar RNA, C/D box 115-24 C15orfA. chromosome 15 open reading frame 41 PAK6 p21 protein (Cdc42/Rac)-activated kinase 6 NM 020168 SPINT serine peptidase inhibitor, Kunitz type 1 NM 181642 creatine kinase, mitochondrial la creatine kinase, mitochondrial CKMT1ACKMT1B 1B NM_001015001 creatine kinase, mitochondrial lA creatine kinase, mitochondrial CKMTIACKMT1B B NM 00101500 SLC27A2 solute carrier family 27 (fatty acid transporter), member 2 NM 003645 talin 2 NM 0.15059 tropomyosin l (alpha) NM 000366 nuclear receptor subfamily 2, group F, member 2 NM021005

RAS guanyl releasing protein 1 (calcium and DAG-regulated) NM_005739 glycine amidinotransferase (L-arginine:glycine amidinotransferase)NM 001482 cytochrome P450, family 19, subfamily A, polypeptide 1 NM 031226 4,93 myosin VC 4.93 RNA binding protein with multiple splicing 2 4.92 GRAM domain containing 2 4.92 activating transcription factor 7 interacting protein 2 NM O24997 49 acyl-CoA synthetase medium-chain family member 3 ER1 exoribonuclease family member 2 NM_005622 4.90 4.38 LPCAT2 lysophosphatidylcholine acyltransferase 2 NM 017839 - 4.86 GPR56 G protein-coupled receptor 56 NM 201524 4.86 MMP5 matrix metallopeptidase 5 (membrane-inserted) NM 002428 4.83 MARVELD3 MARVEL domain containing 3 4.82 periplakin NM_002705 4.8 Patent Application Publication Dec. 18, 2014 Sheet 29 of 45 US 2014/0370.007 A1

FIG.20 (10)

genesymbol genedescription Accession number Fold Change GPRC5B G protein-coupled receptor, family C, group 5, member B 4.79 Tr. 8000480 4.78 CDH8 cadherin 8, type 2 NM_001796 4.77 ESRP2 epithelial splicing regulatory protein 2 NM 024939 4.75 solute carrier family 7 (cationic amino acid transporter, y+ system), SLC7A5 member 5 NM_003486 4.75 TMEM88 transmembrane protein 88 4.71 RABFIP4 RABll family interacting protein 4 (class ll) NM 032932 470 MYL4 myosin, light chain 4, alkali, atrial, embryonic NM_001002841 470 TBX4 T-box 4. NM_018488 4.69

TANC2 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 LLGL2 lethal giant larvae homolog2(Drosophila) NM_001031803 TGB4 integrin, beta 4 NM 000213 nucleoredoxin NM_022463 Go.LOC10O3O876 " - solute carrier family 6 (neurotransmitter transporter, serotonin), SLC6A4 member 4 NM_001045 SLFN3 schlafen family member 13 KRT23 keratin 23 (histone deacetylase inducible) KRT19 keratin 9 NM_002276 WNT3 wingless-type MMTV integration site family, member 3 NM 030753 homeobox B2 NM 002145 homeobox 33 NM 002146 ring finger protein 43 solute carrier family lé, member 6 (monocarboxylic acid SLC6A6 transporter 7) NM_001 17466 C17orf28 chromosome 7 open reading frame 28

ARHGAP28 Rho GTPase activating protein 28 NM_001010000 DSG2 desmoglein 2 NM_001943 transthyretin NM 000371 ASXL3 additional sex combs like 3 (Drosophila) NM 030632 EPB4L3 erythrocyte membrane protein band 4.1-like 3 NM 012307 NM_005.559 PSTPP2 proline-serine-threonine phosphatase interacting protein 2 MYO5B myosin VB NM_001080467 ATPase, aminophospholipid transporter, class 1, type 8B, member ATP8B l NM 00:5603 APK2 alpha-kinase 2 NM 052947 CCBEl collagen and calcium binding EGF domains ) NM_133459 CYB5A cytochrome b5 type A (microsomal) NM 148923 ARD3A AT rich interactive domain 3A (BRIGHT-like) NM_005224 KAA1543 NM 00108.0429 zinc finger protein 93 NM 031218 ZNF90ZNF93 zinc fingerprotein 90 zinc finger protein 93 NM 007138 ZNF74 zinc finger protein 714 NM 1825.15 Patent Application Publication Dec. 18, 2014 Sheet 30 of 45 US 2014/0370.007 A1

FIG. 20 (11)

RPSAP58 ribosomal protein SA pseudogene 58 in

chorionic gonadotropin, beta polypeptide 5 chorionic CGB5ICGB8ICGB7ICGBgonadotropin, beta polypeptide 8 chorionic gonadotropin, beta polypeptide 7 chorionic gonadotropin, beta polypeptide 1 NM 000737 FLJ45949 FBN3 fibrillin 3 RG-3 ral guanine nucleotide dissociation stimulator-like 3 NM 001161616 NOTCH3 notch 3 NM 0004:35 4.35 EPHX3 epoxide hydrolase 3 NM_024794 4.35 inositol-3-phosphate synthase 1 NM 016368 RHPN2 rhophilin, Rho GTPase binding protein 2 NM_033103 4.33 DMKN dermokine 4.3 chorionic gonadotropin, beta polypeptide chorionic gonadotropin, beta polypeptide 5 chorionic gonadotropin, beta polypeptide 8 CGBCGB5ICGB8ICGB7chorionic gonadotropin, beta polypeptide 7 chorionic CGB2 gonadotropin, beta polypeptide 2 NM 000737 chorionic gonadotropin, beta polypeptide chorionic CGBlCGB5ICGB8CGBgonadotropin, beta polypeptide 5 chorionic gonadotropin, beta 2 polypeptide 8 chorionic gonadotropin, beta polypeptide 2 BCO6059 chorionic gonadotropin, beta polypeptide 5 chorionic gonadotropin, beta polypeptide 8 chorionic gonadotropin, beta CGB5CGB8ICGB7 polypeptide 7 NM 000737 Tchorionic gonadotropin, beta polypeptide 7 chorionic gonadotropin, beta polypeptide 5 chorionic gonadotropin, beta CGB7ICGB5ICGB2CGBpolypeptide 2 chorionic gonadotropin, beta polypeptide 8 8NTF4 neurotrophin 4 NM 000737 ZNF702P zinc finger protein 702, pseudogene NR 003578 v-myc myelocytomatosis viral related oncogene, neuroblastoma MYCN derived (avian) NM_005378 PKDCC protein kinase domain containing, cytoplasmic homolog (mouse) NM 138370 chromosome 2 open reading frame 15 mitochondrial ribosomal C2orflis MRPL30 protein L30 AK26402 IL1R1 interleukin l receptor, type I NM_000877 4.25 EPB4ELS erythrocyte membrane protein band 4.1 like 5 NM_020909 4.24 C2orfl4LOC440905|LO chromosome 2 open reading frame 14 hypothetical LOC440905 C002820 Uncharacterized protein C2Orfl4-like 2 ACVR2A activin A receptor, type IIA NM_001616 4.24 LYPD6 LY6/PLAUR domain containing 6 GCA grancalcin, EF-hand calcium binding protein NM 012198 4.22 CDCA7 cell division cycle associated 7 NM 031942 4.2 FSP2 fibrous sheath interacting protein 2 4.2 fibrous sheath interacting protein 2 4.21 COL3A collagen, typell, alpha NM 000090 4.2 SLC39AO solute carrier family 39 (zinc transporter), member 10 NM OO127257 4.21 CPS carbamoyl-phosphate synthase l, mitochondrial NM_001875 4.20 Patent Application Publication Dec. 18, 2014 Sheet 31 of 45 US 2014/0370.007 A1

FIG. 20 (12)

chemokine (C-X-C motif) receptor 7 NM_020311 4.19 FAM49A family with sequence similarity 49, member A SLC8A solute carrier family 8 (sodium/calcium exchanger), member 1 NM 021097 FSHR follicle stimulating hormone receptor NM_000145 BCLA B-celcLLymphoma 1A (zinc finger protein) NM_02289

C2orf14LOC440905|LO chromosome 2 open reading frame 14 hypothetical LOC440905 C10028270 Uncharacterized protein C2orf14-like 2 POTE ankyrin domain family, member E POTE ankyrin domain family, member F POTE ankyrin domain family, member H POTEEPOTEF|POTEH POTE ankyrin domain family, member MPOTE ankyrin domain POTEMPOTEGIPOTEB family, member GPOTE ankyrin domain family, member B fidgetin COB-like UDP-N-acetyl-alpha-D-galactosamine:polypeptide N acetylgalactosaminyltransferase 3 (GalNAc-T3) chimerin (chimaerin) 1 solute carrier family 19, member 3 NM 025243 potassium inwardly-rectifying channel, subfamily J, member 13 NM_002242 ADP-ribosylation factor-like 4C NM_005737

WAP four-disulfide core domain 2 NM_006103 teashirt zinc finger homeobox2 NM 173485 teashirt zinc finger homeobox2 NM 173485 transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) NM 003222 FLRT3 fibronectin leucine rich transmembrane protein 3 NM 198391 4.0 transglutaminase 2 (C polypeptide, protein-glutamine-gamma glutamyltransferase) NM_004613 4.00 bone morphogenetic protein 7 NM_001719 3.99 hormonally up-regulated Ncu-associated kinase NM 014586 3.99 T-cell lymphoma invasion and metastasis NM_003253 3.99 cystathionine-beta-synthase NM_001 I78008 3.98 feat eye syndrome chromosome region, candidate 2 NM 031413 3.97 adrenergic, beta, receptor kinase 2 NM_005160 Tr. 8073680 POTE ankyrin domain family, member M POTE ankyrin domain family, member H. POTE ankyrin domain family, member G POTEMPOTEHPOTEGPOTE ankyrin domain family, member E POTE ankyrin domain |POTEEPOTEF family, member F 3.94

C1q and tumor necrosis factor related protein 6 LIM and cysteine-rich domains NM_014.583 tripartite motif-containing 7l NM_0010391 | 1 ACVR2B activin A receptor, type IIB NM 001106 Patent Application Publication Dec. 18, 2014 Sheet 32 of 45 US 2014/0370.007 A1

FIG. 20 (13)

NBEAL2 neurobeachin-like 2 3.90 DOCK3 dedicator of cytokinesis 3 3.88 ST3GAL6 ST3 beta-galactoside alpha-2,3-sialyltransferase 6 NM 006100 3.87 UPK1B uroplakin B NM006952 3.86 SLC5A2 solute carrier family 15 (HF/peptide transporter), member 2 NM021082 3.86

ARHGEF26 NLGN neuroligin l NM 014.932 3.84

thyroid hormone receptor, beta (erythroblastic leukemia viral (v- THRB erb-a) oncogene homolog2, avian) NM_0012876 3.83 ADAMTS9 ADAM metallopeptidase with thrombospondin type 1 motif, 9 NM 182920 WGLL3. vestigial like 3 (Drosophila) FLIP1L filamin A interacting protein 1-like NM 182909 immunoglobulin superfamily, member 10 mediator complex IGSF10MED12L subunit 12-like NM 178822 SST somatostatin NM_001048 D4S234E DNA segment on chromosome 4 (unique) 234 expressed sequence NM_014392 CPZGPR78 carboxypeptidase Z G protein-coupled receptor 78 NM_001014448 SLT2 slit homolog2 (Drosophila) NM_004787 C4orf19|RELL chromosome 4 open reading frame 19 RELT-likel

SHROOM3 shroom family member 3 NM 020859 Fraser syndrome 1 NM 025074 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 NM_001263 FAM90A family with sequence similarity 190, member A DAPP1 dual adaptor of phosphotyrosine and 3-phosphoinositides NM_014.395 MAB2L2 mab-21-like 2 (C. elegans) NM 006439 FHDC FH2 domain containing 1 NM 033393 GUCY1B3 guanylate cyclase l, soluble, beta 3 NM 000857 CPE carboxypeptidase E NM001873 NBLA0030 NbaoSO1 LDB2 LIM domain binding 2 NM_001130834 ARAP2 ArfQAP with RhoGAP domain, ankyrin repeat and PH domain 2 NM_015230 RBM47 RNA binding motif protein 47 RASGEFB RasGEF domain family, member 1B NM 52545 MOP ABCG2 ATP-binding cassette, sub-family G (WHITE), member 2 NM_004.827 PRSS2 protease, serine, 12 (neurotrypsin, motopsin) NM 003619 TMEM154 transmembrane protein ls.4 FGA fibrinogen alpha chain NM 000508 AADAT aminoadipate aminotransferase NM_016228 HAND2 heart and neural crest derivatives expressed 2 NM_021973 Patent Application Publication Dec. 18, 2014 Sheet 33 Of 45 US 2014/0370.007 A1

FIG. 20 (14)

genedescription Accession number Fold Change 3.64 3.63 3.62

NM_005983 3.62 NM 52403 3.62 NM_005921 3.6 NM_001038603 3,61 NM_006633 3.6

NM_001882 3.59 3.58 NM 000679 3.57 NM_001161661 3.56 Tr. 81 10668 ELOVL family member 7, elongation of long chain fatty acids ELOVLT (yeast) NM_024930 ENC1 ectodermal-neural cortex 1 (with BTB-like domain) NM_003633 FAMI69A family with sequence similarity I69, member A

GCNT4 glucosaminyl (N-acetyl) transferase 4, core 2 NM 016591 MEF2C myocyte enhancer factor 2C NM_002397 EPB4L4A erythrocyte membrane protein band 4.1 like 4A NM 022140 PPP2R2B protein phosphatase 2, regulatory subunit B, beta NM 004576 3.52

SLT3 slit homolog 3 (Drosophila) NM_003062 3.51

synaptonemal complex protein 2-like transmembrane protein 14B 3.49

3.47 NM 003524 3.47 3.46

NM 152753 3.46 3.45 PRSS35 protease, serine, 35 NM_001170423 3.45 PKIB protein kinase (cAMP-dependent. catalytic) inhibitor beta NM 181794 RSPO3 R-spondin 3 homolog (Xenopus laevis) NM 032784 NM_003206 NM 012454 SPARC related modular calcium binding 2 3.43 NM_004155 3.42 tubulin, beta 2B NM 178012 E.3.443.40 HIST1H3F histone cluster 1, H3f NM 021018 340 ZNF204P zinc finger protein 204, pseudogene 3.39 Tr, 8124.604 3.39 C6orf132 chromosome 6 open reading frame 132 3.38

KH domain containing, RNA binding, signal transduction KHDRBS2 associated 2 NM 152688 3.37

SRSF2 serinefarginine-rich splicing factor 12 NM 080743 3.37 BTB and CNC homology l, basic leucine zippertranscription BACH2 factor 2 3.36 NM 021813 FYN NM_002037 3.36 Patent Application Publication Dec. 18, 2014 Sheet 34 of 45 US 2014/0370007 A1

FIG. 20 (15)

genesymbol genedescription Accession number Fold Change microtubule-associated protein 7 NM_003980 ezrin. NM_003379 small nucleolar RNA, H/ACA box 20 sidekick homolog l, cell adhesion molecule (chicken) NM_l 52744 GLCC glucocorticoid induced transcript l tetraspanin 13 sorting nexin 10 NM_013322 proline rich 15 NM 175887 AQP1 aquaporin l (Colton blood group) NM 198098 WBSCR 7 Williams-Beuren syndrome chromosome region 17 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 NM_002069 filamin C, gamma mesoderm specific transcript homolog (mouse) NM_002402 TRIM24 tripartite motif-containing 24 NM 015905 Rho guanine nucleotide exchange factor (GEF) 5 Rho guanine ARIHGEF5|LOC728377 nucleotide exchange factor (GEF) 5 pseudogene NM_005435 lCAl islet cell autoantigen 1, 69kDa NM_004968 SKAP2 src kinase associated phosphoprotein 2 NM_003930 HOXA1 homeobox Al NM_005522 TRIL TLR4 interactor with leucine-rich repeats NM 014.817 ELMO engulfment and cell motility 1 NM_014800 TNS3 tensin 3 NM_022748 COBL cordon-bleu homolog (mouse) DLX5 distal-less homeobox 5 NM_005221 BAAP2L1 BAIl-associated protein 2-like l NM_018842 WNT2 wingless-type MMTV integration site family member 2 NM_00339 TSPAN2 tetraspanin 12 NM 012338 Rho guanine nucleotide exchange factor (GEF) 5 Rho guanine nucleotide exchange factor (GEF) 35 Rho guanine nucleotide ARHGEF5ARHGEF35 exchange factor (GEF) 5 pseudogene olfactory receptor, family 2, LOC728377|OR2A7 subfamily A, member 7 NM_005435 olfactory receptor, family 2, subfamily A, member 7 olfactory OR2A7OR2A4LOC728 receptor, family 2, subfamily A, member 4 Rho guanine 377 nucleotide exchange factor (GEF) 5 pseudogene NM00005328 XKR4 XK, Kell blood group complex subunit-related family, member 4 ADHFEl alcoholdehydrogenase, iron containing, l NM 144650 CRISPLOl cysteine-rich secretory protein LCCL domain containing l MATN2 matrilin 2 NM 002380 c8orf.42 chromosome 8 open reading frame 42 NM 175075 SGK223 homolog of rat pragma of Rnd2 NM_001080826 PPP1R3B protein phosphatase 1, regulatory (inhibitor) subunit 3B NM_024607 MTMR7 myotubularin related protein 7 NM 004686 SFRP1 secreted frizzled-related protein 1 NM_003012 TOX thymocyte selection-associated high mobility group box PDE7A phosphodiesterase 7A NM_002603 JPH junctophilin 1 NM_020647 Patent Application Publication Dec. 18, 2014 Sheet 35 of 45 US 2014/0370.007 A1

FIG. 20 (16)

genesymbol genedescription Accession number Fold Change ZNF704 zinc finger protein 704 3.8 PAGl phosphoprotein associated with glycosphingolipid microdomains i NM_018440 3.8 runt-related transcription factor ; translocated to, (cyclin D RUNXT related) NM 00:1986.25 3.18 RSPO2 R-spondin2 homolog (Xenopus laevis) NM 178565 3.17 KCNV1 potassium channel, subfamily V, member 1 NM_014379 3.16 KANK. KN motif and ankyrin repeat domains NM 153186 3.6 TEK NM 000459 3.6 KRT8 keratin 18 NM 000224 3.6 TP2 tight junction protein 2 (zona occludens 2) NM_004817 3.16 PCSK5 proprotein convertase subtilisin?kexin type 5 NM_001 190482 3.6 PSAT phosphoserine aminotransferase 1 NM 058179 3.5 transmembrane protein with EGF-like and two follistatin-like TMEFF domains 1 NM_003692 3.4 RALGPS Ral GEF with PH domain and SH3 binding motif) NM 014636 3.13 GARNL3 GTPase activating Rap/RangAP domain-like 3 NM 032293 3.3 AIFL allograft inflammatory factor 1-like 3,12 FAM69B family with sequence similarity 69, member B 3.12 MOBKL2B MOB1, Mps One Binder kinase activator-like 2B (yeast) 3. KIAA61 NM 020702 3. RASEF RAS and EF-hand domain containing NM 52573 3. ROR2 receptor tyrosine kinase-like orphan receptor 2 NM_004560 3.1 CORO2A coronin, actin binding protein, 2A NM_003389 3.0 NOTCH1 notch NM_017617 3.10 colony stimulating factor 2 receptor, alpha, low-affinity CSF2RA (granulocyte-macrophage) 3.09 TBLIX transducin (beta)-like X-linked NM_005647 3.09 SHROOM2 shroom family member 2 NM_001649 3.09 SYTL5 synaptotagmin-like 5 NM 138780 3.09 CLCNS chloride channel 5 NM_001 127899 3.08 XAGE2XAGE2B X antigen family, member 2X antigen family, member 2B 3.07 LPAR4 lysophosphatidic acid receptor 4 NM_005296 3.07 PCDH1XPCDH 1Y protocadherin ) X-linked protocadherin Y-linked NM 032967 3.06 Tr. 868614 3.06 PLP proteolipid protein 1 NM 000533 3.05 NRK Nik related kinase NM198465 3.05 MST4 NM 016542 3.05 CSAG2CSAG3 CSAG family, member 2 CSAG family, member 3 NM_001080848 3.04. MD midline I (Opitz/BBB syndrome) NM 00038 3.04. TMEM27 transmembrane protein 27 NM_020665 3.04. AKAP4 A kinase (PRKA) anchor protein 4 NM_003886 3.04 XAGE2XAGE2B X antigen family, member 2X antigen family, member 2B 3.04 ITM2A integral membrane protein 2A 3.04 BEX brain expressed, X-linked 1 NM 08476 3.03 HS6ST2 heparan sulfate 6-O-sulfotransferase 2 NM_001077188 3.0 MR503 microRNA 503 3.01 Patent Application Publication Dec. 18, 2014 Sheet 36 of 45 US 2014/0370.007 A1

FIG. 20 (17) genesymbol genedescription Accession number Fold change GABRA3 gamma-aminobutyric acid (GABA) A receptor, alpha 3 NM 000808 3.01 CSAG family, member 2 CSAG family, member 3 NM_001080848 - colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage) PCDHYIPCDHX protocadherin 11 Y-linked protocadherin X-linked NM 03297 NM_013230

ZNF471 zinc finger protein 471 NM 020813 -3.0 PRA domain family, member 2WD repeat domain 45 NM007213 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 NM_004566 -3.02 hect domain and RLD 6 NM 017912 SAMD's sterile alpha motif domain containing is -304 HRH histamine receptor Hl NM_001098.213 sparcfosteonectin, cwcv and kazal-like domains proteoglycan SPOCK (testican) 1 NM 004598 COP22 coatomer protein complex, subunit zeta 2 NM 016429 nuclear receptor subfamily 4, group A, member 1 NM 002135 LOC645166|LOC654342 lymphocyte-specific protein pseudogene CLP4 CAP-GLY domain containing linker protein family, member 4 LRRK2 leucine-rich repeat kinase 2 NM 98.578 glucoside xylosyltransferase 2 NM_001080393 -3.07 DHRs dehydrogenase/reductase (SDR family) member 3 NM_004753 -3.07 chromosome open reading frame 54 ABCC4 ATP-binding cassette, sub-family C (CFTR/MRP), member 4 NM 005845 proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7) NM_004159 proteasome (prosome, macropain) subunit, beta type, 8 (large PSMB8 multifunctional peptidase 7) NM_004159 proteasome (prosome, macropain) subunit, beta type, 8 (large PSMB8 multifunctional peptidase 7) NM00459 MDGA MAM domain containing glycosylphosphatidylinositol anchor NM 153487 ZNF562 zinc finger protein 562 NM 001130.031 NDP Norrie disease (pseudoglioma) NM000266 SPO translocator protein (18kDa) NM 000714 Tr, 8163253 TPRIP inositol 1,4,5-triphosphate receptor interacting protein p21 protein (Cdc42/Rac)-activated kinase 3 NM_002578

NM_07848) glutaminase NM_014.905

zinc finger, NFX1-type containing 1

transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) NM 018833 MORC family CW-type zinc finger 4

neuropilin NM_003873 Patent Application Publication Dec. 18, 2014 Sheet 37 of 45 US 2014/0370007 A1

FIG. 20 (18)

signal transducer and activator of transcription 1,91kDa NM007315 PCDHB13 protocadherin beta 13 NM_018933 PC pyruvate carboxylase NM_001040716 FKBP5|LOC285847 FK506 binding protein 5 hypothetical LOC285847 NM 001145775 MPHOSPH6 M-phase phosphoprotein 6 NM_005792 -3,6 NPAL2 NlPA-like domain containing 2 -3.7 SLC9A solute carrier family 9 (sodium/hydrogen exchanger), member 7 NM_032591 ANOS anoctamin 5 NM 213599 SLCO3A solute carrier organic anion transporter family, member 3A NM 013272 C16orf25 chromosome 16 open reading frame 45 DNAJB4 DnaJ (Hsp40) homolog, subfamily B, member 4 NM_007034 SNX25 sorting nexin 25 NM_031953 BDNF brain-derived neurotrophic factor NM 170732 TBC1D8B TBC1 domain family, member 8B (with GRAM domain) NM 017752 CGALTC C1GALT1-specific chaperonel WDR52 WD repeat domain 52 Tr. 8043500 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NM 000176 PDE8A phosphodiesterase 8A NM 002605 CMPK2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial NM 20735 ANKRD42 ankyrin repeat domain 42 TBX2 T-box2 NM_005994 small AfCAP2 NM 022733 -3.24.

AE binding protein l NM 001 129 protein kinase C, alpha NM_002737 -3.25 WDR3S

WD repeat domain 35 ADAM15 ADAM metallopeptidase domain 15 NM 207196

SNORD56B small nucleolar RNA, C/D box 56B arylsulfatase B

NM 000046

dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 NM_001004023

PDZ and LM domain 5 NM_006457

zinc finger protein 585B

NM 152279 contactin associated protein l NM 003632

neuron navigator 1 NM 020443

podocan-like 1 -3.32 NFB nuclear factor I/B NM_001 190737 -3.32 G6PD glucose-6-phosphate dehydrogenase NM 000402 -3.33 syncoilin, intermediate filament protein NM_030786

intercellular adhesion molecule 1

NM 00020 -3.34 paired-like homeodomain 2

NM_153426 NM 021229 Patent Application Publication Dec. 18, 2014 Sheet 38 of 45 US 2014/0370.007 A1

FIG. 20 (19)

TAP binding protein-like vesicle-associated membrane protein TAPBPLIVAMP (synaptobrevin l) NM_018009 -3.38 guanine nucleotide binding protein (G protein), gamma 12 FLJ38109 hypothetical Loc86627 .340

FAM46C -3.44

NRG1 LOC100507358 neuregulin hypothetical protein LOC100507358 AF76921 -3.44 RNF112 ring finger protein l l 2 -3.45

T. 807963 346 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein NM 002167 EFR3 homolog B (S. cerevisiae) GLBTL galactosidase, betal-like NM 024506

PLCB4 phospholipase C, beta 4 SP 110 pregnancy-associated plasma protein A, pappalysin l PAPPA PAPPA|PAPPAS antisense RNA (non-protein coding) NM_00258 -3.52 EML6 echinoderm microtubule associated protein like 6 352 ADAM2 ADAM metallopeptidase domain 12 NM_003474 -353

potassium voltage-gated channel, shaker-related subfamily, beta KCNAB2 member 2 NM_003636 MCB MHC class I polypeptide-related sequence B NM_005931 ABCA8 ATP-binding cassette, sub-family A (ABC1), member 8 NM 007168 solute carrier family 12 (sodium/potassium/chloride transporters), SLC2A2 member 2 NM_001046 ULBP2 UL16 binding protein 2 NM 025217 DNAJC2 DnaJ (Hsp40) homolog, subfamily C, member 12

transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) NM 000544 caspase 4, apoptosis-related cysteine peptidase NM 033306

SPON spondin l, extracellular matrix protein NM_006108 GLS GL1S family zinc finger 1 NM 471.93 apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like APOBEC3G 3 G NM 021822 -359 PSD3 pleckstrin and Sec7 domain containing 3 NM 05310 Patent Application Publication Dec. 18, 2014 Sheet 39 of 45 US 2014/0370.007 A1

FIG. 20 (20)

genesymbol genedescription Accession number Fold Change interferon-induced protein with tetratricopeptide repeats 5 NM_012420 ARHGAP26 Rho GTPase activating protein 26 NM 015071 chromosome 3 open reading frame 55 BH3-like motif containing, cell death inducer NM 001001786 GAP43 growth associated protein 43 NM_001130.064 Tr. 7977951 USP8 ubiquitin specific peptidase 18 NM_017414 TAP2 transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) NM 000544 APBA amyloid beta (A4) precursorprotein-binding, family A, member i NM_001163 SUSD2 Sushi domain containing 2 NM_019601 ANKRD44 ankyrin repeat domain 44 ACVRI activin A receptor, type I NM_001105 -3.64 ME1 malic enzyme 1, NADP(+)-dependent, cytosolic NM 002395 -3.64 PARP3 poly(ADP-ribose) polymerase family, member 3 NM00003931 DYNC1 dynein, cytoplasmic l, intermediate chain l NM004411 TCDD-inducible poly(ADP-ribose) polymerase NM_015.508 Friend leukemia virus integration 1 Ewing sarcoma breakpoint FL11EWSR1 region 1 NM002017 regulator of G-protein signaling 4 NM 001102445 chromosome 4 open reading frame 26 ubiquitin-like modifier activating enzyme 7 NM_003335 NTM neurotrim in NM_016522 AMGO2 adhesion molecule with g-like domain 2 NM_001143668 TBC1 domain family, member 19 NM 018317 mannose receptor, C type 2 NM_006039 cell cycle progression NM 004748 cytoplasmic polyadenylation element binding protein 4 DCC deleted in colorectal carcinoma NM_005215 -3.76 cathepsin F NM_003793 FAM10B family with sequence similarity 101, member B pleckstrin and Sec7 domain containing 3 NM 015310 non-metastatic cells 5, protein expressed in (nucleoside diphosphate kinase) NM 003551 CPEB2 cytoplasmic polyadenylation element binding protein 2 NM 182485 APOL2 apolipoprotein L, 2 NM 030882 TRIM14 tripartite motif-containing 14 NM 014788 RBFOX1 RNA binding protein, fox-l homolog (C. elegans) l NM 018723 wingless-type MMTV integration site family, member 5A NM003392 ZNF248 zinc finger protein 248 NM_021045 DUSP4 dual specificity phosphatase 4 NM_001394 fibroblast growth factor 2 (basic) nudix (nucleoside diphosphate linked moiety X)-type motif6 NM002006 Kruppel-like factor 9 NM 001206 sprouty homolog4 (Drosophila) NM 030964 NSG insulin induced gene l NM_005542 Patent Application Publication Dec. 18, 2014 Sheet 40 of 45 US 2014/0370.007 A1

FIG. 20 (21)

genesymbol genedescription Fold Change ZEB1 zinc finger E-box binding homeobox 1 NM 030751 acyl-CoA dehydrogenase family, member 11 nephronophthisis 3 ACAD NPHP3 (adolescent) NM 0321.69 MCB MHC class I polypeptide-related sequence B NM 005931 adaptor-related protein complex 1, sigma 3 subunit NM_001039569 leupaxin NM_0048ll chemokine (C-X-C motif) ligand 11 NM_005409 SMAD9 SMAD family member 9 NM_001 127217 -3.94 solute carrier family 9 (sodium/hydrogen exchanger), member 3 SLC9A3R2 regulator 2 NM 001130012 platelet-derived growth factor receptor, alpha polypeptide NM_006206 aldehyde oxidase 1 NM_001.59 Tr. 8045347 contactin 6 NM_014461 LMO7 LIM domain 7 NM_005358 ADAMTSL1 ADAMTS-like TMEM22 transmembrane protein 22 -4.00

transmembrane protein 100 NM_001099640 BMP2 bone morphogenetic protein 2 NM 001200 Tr. 796909 cholinergic receptor, nicotinic, alpha 5 NM 000745 PID1 phosphotyrosine interaction domain containing l sushi, nidogen and EGF-like domains 1 NM_001080437 tripartite motif-containing 22 NM_006074 potassium inwardly-rectifying channel, subfamily J, member 15 NM_002243 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 NM_014314

FBN fibrillin NM_000138 - 4.13 CNRP cannabinoid receptor interacting protein -4.13 nuclear receptor subfamily l, group D, member 1 thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) NR1D1 THRA oncogene homolog, avian) w NM 021724 TGFBR3 transforming growth factor, beta receptor III NM_003243

HIST1HA histone cluster I, Hla NM_005325 C9orf150 chromosome 9 open reading frame 150 NFASC neurofascin NM 001005388 multiple EGF-like-domains 9 NM 001080497

MIR 155MIRl55HG microRNA 155 MIR 155 host gene (non-protein coding) .

ATP-binding cassette, sub-family A (ABC1), member 6 NM 080284 zinc finger, SWIM-type containing 5 NOG NM_005450 FLRT2 fibronectin leucine rich transmembrane protein 2 NM_013231 Patent Application Publication Dec. 18, 2014 Sheet 41 of 45 US 2014/0370.007 A1

FIG. 20 (22)

genesymbol genedescription Accession number Fold Change sulfiredoxin NM 080725 Fas apoptotic inhibitory molecule 2 NM 012306 dual specificity phosphatase 1 NM_004417 nicroRNA 29a RORA RAR-related orphan receptor A NMI34260 keratinocyte growth factor-like protein 2 keratinocyte growth KGFLP2KGFLP1 (FGF7factor-like protein fibroblast growth factor 7 hypothetical FLU20444 protein FLJ20444 NR 003670 K(lysine) acetyltransferase 2B NM_003884 Tr. 80993.93 -4.3 acyl-CoA oxidase 2, branched chain NM 003500 4.32 stromal interaction molecule 2 NM_001169.18 4.33 NM_020404 mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl glucosaminyltransferase NM 002410 Tr, 8002301 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 NM 182943 -4.37 HIST2H2BFIHIST2H2B histone clustcr2, H2bf histone cluster 2, H2ba histone cluster 2, AHIST2H2BE H2e NM_001024599 PTPRG protein tyrosine phosphatase, receptor type, G NM 002841 GK glycerol kinase NM_001 1281.27 Tr, 82.884 SAT spermidine?spermine N1-acetyltransferase 1 NR 027783 COMT catechol-O-methyltransferase NM 000754 -44 EGF, latrophilin and seven transmembrane domain containing 1 NM022159 melanoma cell adhesion molecule NM_006500 solute carrier family 20 (phosphate transporter), member 1 NM_005415

LTEBP3 latent transforming growth factor beta binding protein 3 NM_001130 144 4.48 SNORD6- small nucleolar RNA, C/D box 116-21 small nucleolar RNA, C 21|SNORD116G. box 6 cluster -4.48 Tr. 865692 -4.49 TGFB transforming growth factor, beta 1 NM 000660 -4.49 SLCA8 solute carrier family 7 (amino acid transporter, L-type), member 8 NM_012244 -4.5 vitamin D (1,25-dihydroxyvitamin D3) receptor NM_001017535 -451 GKGK3P glycerol kinase glycerol kinase 3 pseudogene NM_001 128127 -4.52 solute carrier family 6 (neurotransmitter transporter, taurine), SLC6A6 member 6 NM 003043 4.52 cytoplasmic polyadenylation element biriding protein NM 030594 4.54 poly(rC) binding protein 3 NM 020528 -4.54 PRNP prion protein NM000311 GFBP4 insulin-like growth factor binding protein 4 NM_001552 ZNF454 zinc finger protein 454 NM_001178089 LGALSE lectin, galactoside-binding, soluble, NM 002305 CSGALNACT chondroitin sulfate N-acetylgalactosaminyltransferase 2 NM 018590 HBEGF heparin-binding EGF-like growth factor NM_001945 Patent Application Publication Dec. 18, 2014 Sheet 42 of 45 US 2014/0370.007 A1

FIG. 20 (23)

genesymbol genedescription Accession number Fold Change GEM GTP binding protein overexpressed in skeletal muscle NM 00:5261 ZNF676 zinc finger protein 676 NM_001001411 -4.65 LOC100 128252|LOC100 2884 hypothetical LOC100128252 hypothetical LOC100283114 –4.68 C3orf

DDX60L DEAD (Asp-Glu-Ala-Asp) box polypeptide 60-like NM 000930 4.77 NM 178833 4.79

CORO2B

BHMT2 TMEM13 transmembrane protein 173 NM198282 - 4.82 NM 006528 NM 002178

MAPA microtubule-associated protein 1 A 4.90 PROCR protein C receptor, endothelial NM0064.04 -4.90 ETV5 ets variant 5 NM 004.454 4.95 ABCAS ATP-binding cassette, sub-family A (ABC1), member 5 NM 018672

ATOH8 atonal homolog 8 (Drosophila) NM 032827 CTSK cathepsin K NM_000396 LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein C10orf 16AGAP1)an GTPaseAR" domain Arf GTPase activating protein ll lossNM 006829 -S.05so

CDYL2 chromodomain protein, Y-like 2 NM 152342 PARP2 poly (ADP-ribose) polymerase family, member 12

SULF1 sulfatase 1 NM 00I 128205 CDH3 cadherin 13, H-cadherin (heart) Tr. 8079742 —- -5.27

empty spiracles homeobox2 NM 004.098 -S.34 BICC bicaudal Chomolog 1 (Drosophila) NM 001080512 -S.36 DDX60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 -5.37 TMTC transmembrane and tetratricopeptide repeat containing 1 -5.37 PARP9 poly (ADP-ribose) polymerase family, member 9 NM 031458 -S.40 SERPINB8 serpin peptidase inhibitor, clade B (ovalbumin), member 8 NM 002640 -S.42 SRPX2 sushi-repeat-containing protein, X-linked 2 NM O14467 -S.43 CD44 NM 000610 5.44 NNMT nicotinamide N-methyltransferase Patent Application Publication Dec. 18, 2014 Sheet 43 of 45 US 2014/0370.007 A1

FIG. 20 (24)

genesymbol gene description Accession number Fold Change

NCAM1 neural cell adhesion molecule 1 NM 181351 -S47 GLS3 GLIS family zinc finger 3 NM 52629 -5.52 CPB2 carboxypeptidase B2 (plasma) GPR a protein-coupled receptor NM005279 -5.60 QPCT glutaminyl-peptide cyclotransferase NM01243 -5.64 AHI. Abelson helperintegration site 1 -5.65 SAMD5 sterile alpha motif domain containing 5 PAPSS2 3'-phosphoadenosine 5'-phosphosulfate synthase 2 F35 interferon-induced protein 35 NM005533 -S.68 DKK dickkopfhomolog 1 (Xenopus laevis) NM 012242 -5.70 RANBP3L RAN binding protein 3-like NM_001161429 low density lipoprotein receptor-related protein 8, apolipoproteine receptor

protein tyrosine phosphatase, receptor type, M NM_00105244 epoxide hydrolase 4 MALL mal, T-cell differentiation protein-like NM_005434 -5.85 ARHGAP20 Rho GTPase activating protein 20 NM_020809 -5.90 major facilitator superfamily domain containing 2A NM 001136493 cytochrome P450, family l, subfamily B, polypeptide 1 NM 00004 myxovirus (influenza virus) resistance 2 (mouse) NM002463 fibroblast growth factor 10 nuclear factor I/C (CCAAT-binding transcription factor) NM 205843 phosphoglucomutase 2-like 1 NM 173582 synaptotagmin XI

testis expressed 15 NM 031271 USP18|USP41 ubiquitin specific peptidase 18 ubiquitin specific peptidase 41 NM_017414 CD302|LY75 CD302 molecule lymphocyte antigen 75 ENSTOOOOO409803 CD68 PDCDG2 programmed cell death 1 ligand 2 NM 025239 MCA MHC class polypeptide-related sequence A NM_001177519 -6.42 FHL four and a half LIM domains l NM 001159702 major histocompatibility complex, class l-related NM_001531 LMF |lipase maturation factor NM_022773 -6.0 beta-2-microglobulin NM 004.048 dual specificity phosphatase 6 NM 00 1946

interferon-induced protein with tetratricopeptide repeats 2 NM_001547 -6.77 TM4SF8 transmembrane 4 L six family member 18 NR4A2 nuclear receptor subfamily 4, group A, member 2 NM_006186 -7.14 C5orf62 chromosome 5 open reading frame 62 -7.6 XYLT1 xylosyltransferase I NM 022166 Tr. 8048976 MHC class polypeptide-related sequence A NM 000247 NAD(P)H dehydrogenase, quinone l NM_000903 Patent Application Publication Dec. 18, 2014 Sheet 44 of 45 US 2014/0370007 A1

FIG. 20 (25)

genesymbol AB3BP AB family, member 3 (NESH) binding protein 18, 14 synuclein, alpha (non A4 component of amyloid precursor)

PLCD4 phospholipase C, delta 4 NM 032726 complement component l, r subcomponent NM_001733 Patent Application Publication Dec. 18, 2014 Sheet 45 of 45 US 2014/0370.007 A1

O C) - U Jas O So is U?) S. - at VH 3 NN Sis 8 O O Li ooz Og OO S. d o

S) so UI Eais s S. st was O s CN u- Ol - CD C5 s U O S ... O O ooi O9. O S O und US 2014/0370.007 A1 Dec. 18, 2014

METHOD OF DIRECTED DIFFERENTATION and cell density thereafter decreases rapidly as the eyes grow PRODUCING CORNEAL ENDOTHELLAL (reflecting the same number of cells covering a larger area). CELLS, COMPOSITIONS THEREOF, AND Thereafter, corneal cell density gradually declines with age, USES THEREOF apparently reflecting the gradual loss of cells which are not replaced. As cell density decreases, each cell spreads out and RELATED APPLICATION DISCLOSURE covers a larger area to maintain the cell layer's barrier and pump functions. However, once the cell density drops too low 0001. This application claims the benefit of U.S. Provi (lower than about 500 to 1000 cells/mm) its function is sional Application Ser. No. 61/567,479, filed Dec. 6, 2011, compromised, resulting in corneal clouding, Stromal edema, which is hereby incorporated by reference in its entirety. loss of visual acuity and eventual blindness. Specifically, the cell density of tightly packed corneal endothelium in vivo has FIELD been reported to be as high as 5624 cells/mm in infants two 0002 This disclosure generally relates to methods for months of age, falling to 4252 cells/mm within the first year directed differentiation of corneal cells from stem cells. Com from birth, and Subsequently decreasing rapidly during early positions of corneal endothelial cells, including pharmaceu childhood (associated with the increase incorneal size as eyes tical compositions, and uses thereof, are also disclosed. Also grow). By 5 years of age, corneal endothelium density falls to exemplified are cell-based therapies for treatment of visual approximately 3591 plus or minus 399 cells/mm, and falls disorders, including disorders of the cornea. farther to approximately 2697 plus or minus 246 cells/mm. by 10 years of age, and further declines by approximately BACKGROUND 0.6% per year throughout adulthood. See Peh et al., Trans 0003. The cornea performs functions critical for normal plantation. 2011 Apr. 27; 91 (8):811-9. vision and maintenance of eye health, including providing 0006 Primary diseases that affect the corneal endothelium about two-thirds of the optical power of the eye and protecting include Fuchs dystrophy, iridocorneal endothelial syn the eye from injury or infection. Corneal disease and injury is drome, posterior polymorphous dystrophy, and congenital a leading cause of blindness worldwide. Many corneal dis hereditary endothelial dystrophy. Secondary diseases for ease and injuries can be treated by transplantation of donor which the most effective treatment is replacement of the corneas. The cornea is the most transplanted organ in the body corneal endothelium include several corneal dystrophies, and has a high Success rate over 15 years. For example, contact lens usage, cataract Surgery, and late endothelial fail approximately 40,000 corneal transplantations are performed ure in cornea transplantation. The preferred treatment when per year in the U.S. However, demand for corneas for trans only the corneal endothelium is compromised is Descemet's plantation greatly exceeds the current Supply worldwide, and stripping with endothelial keratoplasty (DSEK), which the limited quality and quantity of available donor tissue includes the removal of Descemet's membrane and the cor hinders treatment. One factor contributing to the inadequate neal endothelium, and Subsequent transplantation of donor supply of donated corneas is that up to 30% of donated cor tissue. Alternatively, in penetrating keratoplasty (PKP) the neas are rejected for transplantation due to poor quality of the entire cornea is removed and replaced. corneal endothelium. Quality of the corneal endothelium 0007 Generally, corneal transplantation includes obtain generally decreases with donor age because, as the cornea ing a donor cornea (e.g., from a post-mortemanatomical gift), ages or is injured, the endothelial cells die and are not determining whether the donor cornea is of Sufficient quality replaced. Therefore, as the population ages, the Supply of and otherwise Suitable for use, and Surgical replacement of donor tissue having Suitably healthy corneal endothelium the damaged or diseased cornea. Procedures have been devel decreases. Moreover, the number and quality of donated cor oped to replace the entire cornea (penetrating keratoplasty) or neas is expected to decline as the popularity of LASIK sur leave the patient’s Descemet's membrane and endothelium gery increases (these corneas are rejected for transplanta and replace the remaining layers with donated tissue (lamel tion). larkeratoplasty); the latter procedure may decrease the risk of 0004 Diseases of the cornea may involve one or more of transplant rejection but may also give inferior visual acuity the cornea's five layers: the corneal epithelium, Bowman's post-transplant. Additionally, lamellar keratoplasty may not layer, the corneal stroma, Descemet's membrane, and the be suitable for treatment of some conditions for which corneal endothelium. The corneal epithelium, corneal replacement of the patient’s corneal endothelium and/or stroma, and corneal endothelium are cellular layers, while Descemet's membrane may be the indicated treatment. See, Bowman's layer and Descemet's membrane are primarily generally, U.S. Pat. No. 5,755,785, U.S. Pat. No. 5,649,944, composed of collagen fibrils. The corneal endothelium is a U.S. Pat. No. 7,147,648, U.S. Pat. No. 7,300,653, U.S. Pat. single layer of cells on the inner Surface of the cornea. It faces No. 5,584.881, U.S. Pat. No. 5,686,414, U.S. Pat. No. 7,300, the chamber formed between the cornea and the iris and keeps 654, U.S. patent application Ser. No. 10/525,391, each of the cornea transparent by regulating fluid levels. Without which is incorporated by reference in its entirety. Additional functional corneal endothelium, the cornea becomes cloudy methods of corneal endothelial Surgical replacement are and vision is lost. Properly functioning corneal endothelial under development, including Descemet's Membrane Endot cells maintain the proper fluid levels in the cornea, e.g., the helial Keratoplasty (DMEK), in which the donor tissue con balance between “leakage' offluid into the stroma and active sists only of Descemet's membrane and corneal endothelium. pumping that continuously operates to move fluid from the Another potentially promising therapeutic avenue is corneal stroma to the anterior chamber of the eye. endothelial reconstruction, in which corneal endothelial cells 0005 Corneal endothelial cells have been reported to have are cultured in vitro prior to transplantation. For example, little or no capacity to proliferate in vivo, such that they are donated human corneal cells were cultured on a polymer, not replaced when injured or otherwise lost. In humans, the released onto a bioadhesive gelatin disc, and then Success corneal endothelial cell layer is most densely packed at birth fully integrated into denuded rabbit corneas, with the gelatin US 2014/0370.007 A1 Dec. 18, 2014 disc dissolving after transplantation (Hsiue et al., Transplan nucleic acid encoding PDGFAB, Phorbol 12-myristate tation. 2006 Feb. 15: 81(3):473-6, which is incorporated by 13-acetate (PMA), VEGF, and any combination thereof. reference herein in its entirety). However, methods utilizing 0014. Theat least one factor that induces differentiation of culture cells presuppose a source of said cells, and thus are said neural crest cells into CEC may comprise at least one affected by the shortage of suitable donated tissues as DKK2 agonist and at least one PDGFB agonist. described above. Additionally, due to differences among 0015 The at least one DKK2 agonist may comprise donated cells, it may prove difficult to produce corneal endot DKK2 polypeptide. helial cell cultures of consistent quality and efficacy. Regula 0016. The at least one PDGFB agonist may comprise tory hurdles may also make such methods logistically diffi PDGFB polypeptide. cult to perform on a large scale, due to the possibility that 0017. The at least one DKK2 agonist may comprise extensive testing for safety and/or efficacy may be required DKK2 polypeptide and said at least one PDGFBagonist may for the cells obtained from each donor. These and additional comprise PDGFB polypeptide. therapeutic methods are further described in Thomas John, 0018. The concentration of said DKK2 polypeptide may Corneal Endothelial Transplant: DSAEK, DMEK & DLEK be between 1 ng/ml and 15 g/ml, between 10 ng/ml and 15 (JP Medical Ltd, 2010), which is incorporated by reference ug/ml, between 1 ng/ml and 1 ug/ml, between 1 ng/ml and herein in its entirety. 100 ng/ml, between 2 ng/ml and 20 ng/ml, between 5 ng/ml 0008. Additional disclosures generally related to methods and 20 ng/ml, or about 10 ng/ml. of obtaining and using corneal cells, including therapeutic (0019. The concentration of said PDGFB polypeptide may methods, culture methods, preservation methods, composi be between 0.1 ng/ml and 250 ng/ml, between 0.5 ng/ml and tions containing or that that may be used in conjunction 150 ng/ml, between 1 ng/ml and 50 ng/ml, between 2 ng/ml therewith, and the like are included in U.S. 2007/0275365, and 20 ng/ml, or about 10 ng/ml. US 2010/0209402, US 2010/0233240, US 2011/000.9488, 0020. The method may comprise culturing said CEC on a US 2009/0232772, U.S. Pat. No. 5,166,048, US 2007/ matrix. 0092550, US 2005/0214259, US 2007/0148137, U.S. Pat. 0021. The matrix may be selected from the group consist No. 4,959,319, U.S. Pat. No. 5,310,728, U.S. Pat. No. 5,589, ing of laminin, fibronectin, vitronectin, proteoglycan, entac 451, US 2010/0215717, U.S. Pat. No. 5,703,047, US 2009/ tin, collagen, collagen I, collagen IV, collagen VIII, heparan 0222086, US 2009/0263465, US 2006/0228693, US 2006/ sulfate, Matrigel (a soluble preparation from Engelbreth 0240552, US 2009/0270982, U.S. Pat. No. 5,269,812, U.S. Holm-Swarm (EHS) mouse sarcoma cells), a human base Pat. No. 7,371,513, US 2010/0069915, and US 2011/ ment membrane extract, and any combination thereof. 0166650, each of which is incorporated by reference herein in 0022. The matrix may be of human or non-human animal its entirety. origin. 0023 The matrix may be of bovine, mouse or rat origin. SUMMARY 0024. The matrix may comprise Matrigel. 0025. The subsequent to commencement of step (a), said 0009. It may be one object of the present disclosure to cells may be passaged. provide a method of producing corneal endothelial cells 0026. The passaging may be effected between 1 hour and (CEC), which may comprise (a) contacting neural crest stem 5 days, between 2 hours and 4 days, between 3 hours and 3 cells (NCSCs) with at least one factor that induces differen days, between 4 hours and 2 days, or about 1 day Subsequent tiation of said neural crest cells into CEC. to commencement of step (a). 0010. Theat least one factor that induces differentiation of 0027. The passaging may be effected by a method which said neural crest cells into CEC may comprise at least one may comprise contacting the cells with a cell dissociation DKK2 agonist and/or at least one PDGFBagonist. buffer, or by a method which may comprise mechanical dis 0011. The at least one DKK2 agonist may include a factor sociation of the cells or a subset thereof, or by a method which selected from the group consisting of LRP5/6 antagonists; may comprise optical isolation the cells or a Subset thereof. Kremen antagonists; Dkk 1: Dkk 3: Dkk 4: Soggy; secreted 0028. The cell dissociation buffer may be non-enzymatic. frizzled related proteins (Frzb); Wnt inhibitor factor (WIF); a 0029. The cell dissociation buffer may comprise ethylene Wnt modulator, Casein Kinase 1-7B catenin antagonists; diaminetetraacetic acid (EDTA). LEF/TCF transcription factor members modulators; IWR: 0030 The duration of said contacting NCSCs with a factor pyrvinium; ICG-001; PKF 115-584; IWP; Ant1.4Br/Ant that induces differentiation of said neural crest cells into CEC 1.4Cl; Niclosamide: apicularen; bafilomycin; XAV939: may be at least 2 days, between 2 and 25 days, or may be NSC668036: 2,4-diamino-quinazoline; Quercetin; and any between 2 and 10 days. combination thereof. 0031. In another exemplary embodiment, the CEC may be 0012. The at least one DKK2 agonist may include a factor purified using affinity-based depletion of other cell types, selected from the group consisting of Wnt proteins, nucleic which may be effected concurrently with passaging or at an acids encoding Wnt proteins, LiCl, AXin antagonists; APC earlier or later time. Depletion methods may utilize a binding antagonists; norrin; R-spondin2; (hetero)arylpyrimidines; molecule having affinity for a marker of a non-CEC cell type, IQ1; BIO(6-bromoindirubin-3'-oxime); 2-amino-4-3,4- e.g., an antibody or fragment thereof, which may be directly (methylenedioxy)benzyl-amino-6-(3-methoxyphenyl)pyri or indirectly coupled to a bead or other substrate. Such as a midine; a Wnt modulator; WAY-316606: QS11; SB-216763: magnetic microbead. For example, a Substrate may be SB-2 16763; DCA; and any combination thereof. coupled to one or more antibodies that can deplete stem cells 0013 The at least one PDGFB agonist may include a or fibroblasts. Exemplary markers that may be utilized factor selected from the group consisting of: a PDGFRB include CD271, SSEA-1, TRA-1-60, SSEA-4, and/or agonist, a PKC pathway agonist, PDGFAA polypeptide, a CD326. Potentially suitable commercially available reagents nucleic acid encoding PDGFAA, PDGFAB polypeptide, a include Anti-SSEA-1 MicroBeads (Miltenyi Biotech, US 2014/0370.007 A1 Dec. 18, 2014

cati130-094-530), Anti-TRA-1-60 MicroBead Kit (Miltenyi 0047. The neural crest stem cells may be produced from Biotech, cath 130-095-816), Anti-SSEA-4 MicroBeads ES cells by a method further which may comprise contacting (Miltenyi Biotech, cath 130-097-855), and Anti-fibroblast ES cells with at least one Wntagonist selected from the group CD326 (Miltenyi Biotech, cath 130-050-601). consisting of Wnt proteins, nucleic acids encoding Wnt pro 0032 Step (a) further may comprise culturing said NCSCs teins, LiCl, AXin antagonists; APC antagonists; norrin; on a matrix, which may be selected from the group consisting R-spondin2: (hetero)arylpyrimidines; IQ1; BIO(6-bromoin of laminin, fibronectin, vitronectin, proteoglycan, entactin, dirubin-3'-oxime); 2-amino-4-3,4-(methylenedioxy)ben collagen, collagen I, collagen IV, collagen VIII, heparan Sul Zyl-amino-6-(3-methoxyphenyl)pyrimidine: WAY-316606: fate, Matrigel (a soluble preparation from Engelbreth-Holm QS11; SB-216763; SB-216763; DCA; and any combination Swarm (EHS) mouse sarcoma cells), a human basement thereof. membrane extract, and any combination thereof, may be of 0048. The neural crest stem cells may be produced from human or non-human animal origin, may be of bovine, mouse ES cells by a method which may comprise contacting ES cells or rat origin, or may comprise Matrigel. with a first inhibitor of SMAD protein signaling and a second 0033. The NCSCs may be obtained from an animal tissue, inhibitor of SMAD protein signaling. which may be selected from the group consisting of the gut, 0049. The first inhibitor of SMAD protein signaling may dorsal root ganglia, bone marrow, skin, heart, cornea, caratoid be selected from the group consisting of Noggin polypeptide, body, neural tube, teeth, and Sciatic nerve. The animal tissue dorsomorphin, LDN-193189, and any combination thereof. may be human tissue. 0050. The first inhibitor of SMAD protein may comprise 0034. The NCSCs may be produced by transdifferentia Noggin polypeptide. tion of a Somatic cell. 0051. The Noggin polypeptide may be present in a con centration between 10 ng/ml and 5,000 ng/ml, between 100 0035. The NCSCs may be obtained from cultures of neural ng/ml and 700 ng/ml, between 400 ng/ml and 600 ng/ml, or rOSettes. about 500 ng/ml. 0036. The neural crest stem cells may be produced from 0.052 The first inhibitor of SMAD protein signaling may ES cells by a method which may comprise culturing ES cells be selected from the group consisting of antagonists of with MS5 Stromal feeder cells. BMP2; antagonists of BMP4; antagonists of BMP7; and 0037. The NCSCs may be produced from ES cells. antagonists of TGFB; 0038. The neural crest stem cells may be produced from 0053. The second inhibitor of SMAD protein signaling ES cells by a method which may comprise contacting ES cells may comprise an inhibitor of an anaplastic lymphoma kinase with one or more inhibitors of SMA/Mothers Against Deca signaling pathway. pentaplegic (SMAD) protein signaling. 0054 The second inhibitor of SMAD protein signaling 0039. The one or more inhibitors of SMAD protein sig inhibits a signaling pathway selected from the group consist naling may prevent the binding of a TGF-3 family ligands to ing of Lefty, Activin, and TGFbeta. its corresponding receptor. 0055. The second inhibitor of SMAD protein signaling 0040. The one or more inhibitors of SMAD protein sig inhibits both activin and nodal signaling. naling may prevent the activation of a TGF-B receptor. 0056. The second inhibitor of SMAD protein signaling 0041. The one or more inhibitors of SMAD protein sig inhibits the Lefty/Activin/TGFbeta pathways by blocking naling inhibits one or more SMAD intracellular proteins/ phosphorylation of the ALK4, ALK5 and ALK7 receptors. transcription factors. 0057 The second inhibitor of SMAD protein signaling 0042. The one or more inhibitors of SMAD protein sig may be an ALK4 receptor inhibitor. naling may comprise Leukemia Inhibitory Factor (LIF), 0058. The second inhibitor of SMAD protein signaling GSK3 inhibitor (CHIR 99021), Compound E (Y secretase may be selected from the group consisting of 4-4-(1,3- inhibitor XXI), SB431542, or any combination thereof. benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-ylbenza 0043. The one or more inhibitors of SMAD protein sig mide (SB431542) and derivatives thereof. naling may comprise Chordin, Follistatin, dominant negative 0059. The second inhibitor of SMAD protein signaling receptors or blocking antibodies that sequester BMP2, may comprise SB431542. BMP4, and/or BMP7, dorsomorphin (or Compound C), SIS3 0060. The SB431542 may be present in a concentration (6,7-Dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo between 10 nM and 100 uM, between 0.1 uM and 50 uM, 2,3-bipyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroiso between 0.1 and 20 uM, between 1 and 20 uM, or about 10 quinoline, Specific Inhibitor of Smad3 (SIS3), an inhibitor uM. SMAD, SMAD6, SMAD7, SMAD10, an antagonist of a 0061 The second inhibitor of SMAD protein signaling receptor SMAD, an antagonist of SMAD1, an antagonist of blocks phosphorylation of ACTRIB, TGFBR1, and ACTRIC SMAD2, an antagonist of SMAD3, an antagonist of SMAD5, receptors. an antagonist of SMAD8/9, or any combination thereof. 0062. The second inhibitor of SMAD protein signaling 0044) The duration of said contacting ES cells with one or inhibits TGFB/Activin/Nodal signaling. more inhibitors of SMAD protein signaling may be at least 2 0063. The second inhibitor of SMAD protein signaling days, between 1 and 10 days, or may be between 2 and 6 days. blocks endogenous Activin and BMP signals. 0045. The neural crest stem cells may be produced from 0064. The first inhibitor of SMAD protein signaling and/ ES cells by a method further which may comprise contacting or said second inhibitor of SMAD protein signaling may be ES cells with at least one Wntagonist. each selected from the group consisting of Chordin, Follista 0046. The neural crest stem cells may be produced from tin, dominant negative receptors or blocking antibodies that ES cells by a method further which may comprise contacting sequester BMP2, BMP4, and/or BMP7, dorsomorphin (or ES cells with at least one Wntagonist which may comprise Compound C), SIS3 (6,7-Dimethoxy-2-((2E)-3-(1-methyl (2'Z.3E)-6-bromoindirubin-3'-oxime (BIO) and/or Wnt3a. 2-phenyl-1H-pyrrolo2,3-bipyridin-3-yl-prop-2-enoyl))-1.2. US 2014/0370.007 A1 Dec. 18, 2014

3,4-tetrahydroisoquinoline, Specific Inhibitor of Smad3 SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, (SIS3), an inhibitor SMAD, SMAD6, SMAD7, SMAD10, an PITX2, and FOXC1, and may not express the markers v WF antagonist of a receptor SMAD, an antagonist of SMAD1, an and CD31. antagonist of SMAD2, an antagonist of SMAD3, an antago I0084. The CEC may form a monolayer of uniformly sized nist of SMAD5, and an antagonist of SMAD8/9. cells with a predominantly hexagonal shape, such as at least 0065. The first inhibitor of SMAD protein signaling may 50%, at least 60%, at least 70%, at least 80%, or at least 90% comprise Noggin and said second inhibitor of SMAD protein of said CEC may exhibit said hexagonal shape. signaling may comprise SB431542. I0085. The CEC may allow unidirectional leakage of sol 0066. The duration of said contacting ES cells with said utes and nutrients. first inhibitor of SMAD protein signaling and said second I0086. The CEC may actively pump water in the opposite inhibitor of SMAD protein signaling may be at least 2 days, direction of said unidirectional leakage. between 1 and 10 days, or may be between 2 and 6 days. I0087. The CEC may exhibit a high level of metabolic 0067. The ES cells may be human ES cells. activity that may be comparable to animal-derived CEC. 0068. The ES cells may be iPS cells. I0088. The CEC may be human CEC. 0069. The ES cells may not exhibit changes or mutations I0089. The CEC may exhibit a culture density of at least in genes associated with a disease of corneal endothelial cells. 1000 cells/mm, at least 2000 cells/mm, at least 3000 cells/ 0070 The ES cells may exhibit a normal karyotype. mm, at least 4000 cells/mm. at least 5000 cells/mm, at least 6000 cells/mm, at least 7000 cells/mm, at least 8000 cells/ (0071 Prior to differentiation into NCSCs, said ES cells mm, at least 9000 cells/mm, between 2000 and 9000 cells/ may be maintained in culture in the absence of feeder cells. mm between 2000 and 8000 cells/mm between 2000 and 0072 Prior to differentiation into NCSCs said ES cells 7000 cells/mm, between 2000 and 6000 cells/mm, between may be cultured on a matrix, which may be selected from the 2000 and 5,000 cells/mm, between 2000 and 4000 cells/ group consisting of laminin, fibronectin, vitronectin, pro mm, between 2000 and 3500 cells/mm, or about 2500 cells/ teoglycan, entactin, collagen, collagen I, collagen IV, heparan mm sulfate, Matrigel (a soluble preparation from Engelbreth (0090. The CEC may exhibit a decreased level of accumu Holm-Swarm (EHS) mouse sarcoma cells), a human base lated oxidative stress and/or DNA damage compared to CEC ment membrane extract, and any combination thereof, or may isolated from a living host. be of human or non-human animal origin, or may be of 0091. The level of oxidative stress and/or DNA damage bovine, mouse or rat origin, or may comprise Matrigel. may be detected by measuring the quantity of one or more of: 0073. The NCSCs may express one or more markers nuclear DNA damage foci: level of expression of p21Cip1, selected from the group consisting of: Sox10, AP2, HNK1, level of expression of p16INK4a: level of expression of cyto PAX7, p75 (NGFR), and any combination thereof. globin protein, level of expression of GPX-1 protein, and 0074 The CEC may express one or more markers selected level of 8-hydroxy-2 -deoxyguanosine (8-OHdG). from the group consisting of Na+/K+ ATPase, ZO-1, KLF13, 0092. The CEC may comprise at least 50%, at least 60%, AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2/ at least 70%, at least 80%, or at least 90% of the cells in the SCL4A2, SCL16A1, CA12, CA4, FoxC1, and any combina resulting culture. tion thereof. 0093. In another aspect, the present disclosure provides a 0075. The CEC may express the markers Collagen VIII, composition comprising corneal endothelial cells (CEC) pro Na+K+-ATPase pump, and ZO-1, and may not express the duced according to any method as described herein. markers v WF and CD31. 0094. In another aspect, the present disclosure provides a 0076. The CEC may express one or more corneal endot composition comprising a sheet of CEC having a culture helial pump markers. density of at least 1000 cells/mm, at least 2000 cells/mm. 0077. The CEC may express one or more periocular neural between 2000 and 6000 cells/mm, between 2000 and 5,000 crest markers. cells/mm, between 2000 and 4000 cells/mm, between 2000 0078. The CEC may express one or more cell adhesion and and 3500 cells/mm, or about 2500 cells/mm. matrix proteins. 0095. In another aspect, the present disclosure provides a 007.9 The CEC may express at least one corneal endothe composition comprising CEC that exhibit a decreased level of lial pump marker, at least one periocular neural crest marker, accumulated oxidative stress and/or DNA damage compared and at least one cell adhesion and matrix protein. to CEC isolated from a living host. 0080. The one or more corneal endothelial pump markers 0096. In another aspect, the present disclosure provides a may be selected from the group consisting of: AQP1, CA2, composition comprising corneal endothelial cells (CEC) pro CA4, CA12, SCL14A2, SLC16A1 SLC16A3, SLC16A7, duced from neural crest stem cells (NCSCs). CFTR, NHE1, ADCY10, voltage-dependent anion channels (0097. The NCSCs may be produced from embryonic stem VDAC2 and VDAC3, chloride channel proteins CLCN2 and (ES) cells. CLC. (0098. The NCSCs may be produced from ES cells by a 0081. The periocular neural crest markers may be selected method which may comprise contacting said ES cells with from the group consisting of PITX2, and FOXC1. one or more inhibitors of SMAD signaling. 0082. The cell adhesion and matrix proteins may be 0099. In another aspect, the present disclosure provides a selected from the group consisting of Occludin, Connexin composition comprising corneal endothelial cells (CEC) pro 43, 9.3E antigen, Collagen III, Collagen IV, N cadherin, VE duced from embryonic stem (ES) cells. cadherin, E cadherin, beta catenin, p120, p.190 Laminin alpha 0100. The ES cells may be human ES cells. 4. Nidogen-2, and Netrin 4. 0101. The ES cells may be iPS cells. 0083. The CEC may express the markers Collagen VIII, 0102 The ES cells may not exhibit changes or mutations Na+K+-ATPase pump, AQP1, CA2, CA4, CA12, SCL14A2, in genes associated with a disease of corneal endothelial cells. US 2014/0370.007 A1 Dec. 18, 2014

0103) The ES cells may exhibit a normal karyotype. 6000 cells/mm, at least 7000 cells/mm. at least 8000 cells/ 0104 Prior to differentiation into NCSCs, said ES cells mm, at least 9000 cells/mm, between 2000 and 9000 cells/ may be maintained in culture in the absence of feeder cells. mm between 2000 and 8000 cells/mm between 2000 and 0105. Prior to differentiation into NCSCs said ES cells 7000 cells/mm, between 2000 and 6000 cells/mm, between may be cultured on a matrix. Such as a matrix may be selected 2000 and 5,000 cells/mm, between 2000 and 4000 cells/ from the group consisting of laminin, fibronectin, vitronec mm, between 2000 and 3500 cells/mm, or about 2500 cells/ tin, proteoglycan, entactin, collagen, collagen I, collagen IV. mm heparan Sulfate, Matrigel (a soluble preparation from Engel I0123. The CEC may exhibit a decreased level of accumu breth-Holm-Swarm (EHS) mouse sarcoma cells), a human lated oxidative stress and/or DNA damage compared to CEC basement membrane extract, and any combination thereof, isolated from a living host, which may for example be which may be of human or non-human animal origin, such as detected by measuring the quantity of one or more of nuclear bovine, mouse or rat origin, or Matrigel. DNA damage foci: level of expression of p21Cip1, level of 0106 The NCSCs may express one or more markers expression of p16INK4a: level of expression of cytoglobin selected from the group consisting of: Sox10, AP2, HNK1, protein, level of expression of GPX-1 protein, and level of PAX7, p75 (NGFR), and any combination thereof. 8-hydroxy-2-deoxyguanosine (8-OHdG). 0107 The CEC may express one or more markers selected 0.124. The CEC may comprise at least 50%, at least 60%, from the group consisting of Na+/K+ ATPase, ZO-1, KLF13, at least 70%, at least 80%, or at least 90% of the cells in the AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2/ culture in which they are contained. SCL4A2, SCL16A1, CA12, CA4, FoxC1, and any combina (0.125. The CEC may be contained in a sheet of CEC. tion thereof. 0.126 The sheet of cells may comprise or consists essen 0108. The CEC may express the markers Collagen VIII, tially of an approximately circular disc of cells having a Na+K+-ATPase pump, and ZO-1, and may not express the diameter of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mm. markers v WF and CD31. I0127. The CEC may exhibit a culture density of at least 0109 The CEC may express one or more corneal endot 1000 cells/mm, at least 2000 cells/mm, at least 3000 cells/ helial pump markers. mm, at least 4000 cells/mm. at least 5000 cells/mm. at least 0110. The CEC may express one or more periocular neural 6000 cells/mm, at least 7000 cells/mm. at least 8000 cells/ crest markers. mm, at least 9000 cells/mm, between 2000 and 9000 cells/ 0111. The CEC may express one or more cell adhesion and mm between 2000 and 8000 cells/mm between 2000 and matrix proteins. 7000 cells/mm, between 2000 and 6000 cells/mm, between 0112 The CEC may express at least one corneal endothe 2000 and 5,000 cells/mm, between 2000 and 4000 cells/ lial pump marker, at least one periocular neural crest marker, mm, between 2000 and 3500 cells/mm, or about 2500 cells/ and at least one cell adhesion and matrix protein. mm. 0113. The one or more corneal endothelial pump markers I0128. The total number of cells (for example in a sheet or may be selected from the group consisting of: AQP1, CA2, dissociated cells) may be between about 800 and about 800, CA4, CA12, SCL14A2, SLC16A, SLC16A3, SLC16A7, 000 cells, e.g., at least about 10,000, at least about 20,000, at CFTR, NHE1, ADCY10, voltage-dependent anion channels least about 50,000, at least about 100,000, at least about VDAC2 and VDAC3, chloride channel proteins CLCN2 and 200,000, at least about 300,000, at least about 400,000, at CLC. least about 500,000, at least about 600,000 or at at least about 0114. The periocular neural crest markers may be selected 700,000 cells, such between about 100,000 and about 800, from the group consisting of PITX2, and FOXC1. 000 cells, between about 150,000 cells and about 675,000 0115 The cell adhesion and matrix proteins may be selected from the group consisting of Occludin, Connexin cells, between about 250,000 cells and about 550,000 cells, as 43, 9.3E antigen, Collagen III, Collagen IV, N cadherin, VE well as other numerical ranges within these values. cadherin, E cadherin, beta catenin, p120, p.190 Laminin alpha I0129. The CEC may be situated on a carrier. 4. Nidogen-2, and Netrin 4. 0.130. The CEC may be cultured on a substrate and 0116. The CEC may express the markers Collagen VIII, released onto a carrier. Na+K+-ATPase pump, AQP1, CA2, CA4, CA12, SCL14A2, I0131 The substrate may comprise a thermoresponsive SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, polymer orathermoresponsive poly(N-isopropylacrylamide) PITX2, and FOXC1, and may not express the markers v WF (PNIPA Am)-grafted surface. and CD31. 0.132. The carrier may comprise gelatin, fibrin-based 0117 The CEC may form a monolayer of uniformly sized matrixes, endothelium-denuded corneal buttons, denuded cells with a predominantly hexagonal shape, Such as at least Descemet's membrane, devitalized stromal cornea, fresh cor 50%, at least 60%, at least 70%, at least 80%, or at least 90% neal stromal discs, and/or an amniotic membrane. of said CEC may exhibit said hexagonal shape. I0133. The CEC may be in suspension. 0118. The CEC may allow unidirectional leakage of sol I0134. The composition may further comprise an inhibitor utes and nutrients. of Rho-associated kinase (ROCK). 0119 The CEC may actively pump water in the opposite 0.135 The inhibitor of Rho-associated kinase may com direction of said unidirectional leakage. prise Y-27632. 0120) The CEC may exhibit a high level of metabolic 0.136 The CEC may exhibit a decreased level of accumu activity that may be comparable to animal-derived CEC. lated oxidative stress and/or DNA damage compared to CEC 0121. The CEC may be human CEC. isolated from a living host, which may for example be 0122) The CEC may exhibit a culture density of at least detected by measuring the quantity of one or more of nuclear 1000 cells/mm, at least 2000 cells/mm, at least 3000 cells/ DNA damage foci: level of expression of p21Cip1, level of mm, at least 4000 cells/mm. at least 5000 cells/mm, at least expression of p16INK4a: level of expression of cytoglobin US 2014/0370.007 A1 Dec. 18, 2014

protein, level of expression of GPX-1 protein, and level of 0152 The disease of corneal endothelial cells may be 8-hydroxy-2-deoxyguanosine (8-OHdG). selected from the group consisting of Fuchs dystrophy, iri 0.137 The composition may comprise at least 2000, at docorneal endothelial syndrome, posterior polymorphous least 3000, at least 8000, at least 1000, at least 18000, at least dystrophy, and congenital hereditary endothelial dystrophy, 25000, at least 31000, at least 44000, at least 49000, at least and secondary diseases for which an effective treatment may 69000, at least 71000, at least 96000, at least 99000, at least be replacement of the corneal endothelium including corneal 126000, at least 135000, at least 159000, at least 176000, at dystrophies, contact lens usage, cataract Surgery, and late least 196000, at least 223000, at least 275000, at least 237000, endothelial failure in cornea transplantation. at least 283000, at least 332000, or at least 396000 CEC. 0153. The method of treatment may comprise administer 0.138. The composition may comprise at least 50%, at least ing an immunosuppressive agent or immune tolerizing agent 60%, at least 70%, at least 80%, or at least 90% CEC. to said patient. 0.139. The composition may further comprise a pharma 0154 The immunosuppressive agent or immune toleriz ceutically acceptable excipient. ing agent may be administered in an amount Sufficient to 0140. The composition may further comprise an immuno reduce the risk of rejection of said CEC. Suppressive or immune tolerizing agent. 0155 The immunosuppressive agent or immune toleriz ing agent may be administered prior to, concurrently with, 0141. The immunosuppressive or immune tolerizing and/or subsequent to administration of said CEC to said agent may comprise one or more of mesenchymal stem cells, patient. anti-lymphocyte globulin (ALG) polyclonal antibody, anti 0156 The composition comprising CEC further may thymocyte globulin (ATG) polyclonal antibody, azathioprine, comprise an immunosuppressive agent or immune tolerizing BASILIXIMABR) (anti-IL-2RC. receptor antibody), agent. cyclosporin (cyclosporin A), DACLIZUMABOR (anti-IL 0157. The immunosuppressive or immune tolerizing 2RC. receptor antibody), everolimus, mycophenolic acid, agent may comprise one or more of mesenchymal stem cells, RITUXIMAB(R) (anti-CD20 antibody), sirolimus, tacroli anti-lymphocyte globulin (ALG) polyclonal antibody, anti mus, mycophemolate mofetil, and corticosteroids. thymocyte globulin (ATG) polyclonal antibody, azathioprine, 0142. The composition may be free of detectable bacterial BASILIXIMABR) (anti-IL-2RC. receptor antibody), contaminants, mycoplasmal contaminants, and viruses. cyclosporin (cyclosporin A), DACLIZUMABOR (anti-IL 0143. The composition may comprise human CEC. 2RC. receptor antibody), everolimus, mycophenolic acid, 0144. The composition may be suitable for transplantation RITUXIMAB(R) (anti-CD20 antibody), sirolimus, tacroli into the eye of a patient in need thereof, such as a human mus, mycophemolate mofetil, and corticosteroids. patient. 0145 The composition may be used in the manufacture of BRIEF DESCRIPTION OF THE DRAWINGS a medicament. 0158 FIG. 1A schematically illustrates two methods that 0146 The medicament may be for the treatment of a dis were used to generate corneal endothelial cells from embry ease of corneal endothelial cells. onic stem cells. FIG. 1B is a phase micrograph showing a 0147 The disease of corneal endothelial cells may com circular field of hES-derived corneal endothelium (outlined prise Fuchs dystrophy, iridocorneal endothelial syndrome, by a dashed circle), which were identifiable by their hexago posterior polymorphous dystrophy, or congenital hereditary nal morphology (Hex). The circular field of corneal endothe endothelial dystrophy, and/or secondary diseases for which lium was surrounded by non-hexagonal cells (P) which were an effective treatment may be replacement of the corneal thought to include progenitor cells that had not adopted a endothelium including corneal dystrophies, contact lens hexagonal corneal morphology. The corneal endothelial cells usage, cataract Surgery, and late endothelial failure in cornea in FIG. 1B were generated from hESCs using the method transplantation. described in Example 1 and are shown at day 9. FIG. 1C is a 0148. The disease of corneal endothelial cells may com micrograph showing a field of hESC-derived corneal endot prise pleomorphism, a significant disruption in the regular helium (Hex) that was generated using the method described hexagonal pattern of the endothelium that can cause a in Example 2 (i.e., with the passaging step). Relative to the decrease in endothelial mosaic stability. Pleomorphism may method described in Example 1, the Method described in occur secondary to another disease of the cornea, such as Example 2 produced a greater proportion of corneal endot physiological stress from ocular disease, contact lens wear or helial cells, with small patches of non-hexagonal cells (P) normal aging changes. present among the corneal endothelial cells. 014.9 The medicament may be adapted for administration 0159 FIG. 2A-D shows populations of recently differen by a method which may comprise Descemet's stripping with tiated corneal endothelial cells. FIG. 2A shows cell popula endothelial keratoplasty (DSEK), Penetrating Keratoplasty tions produced using the method described in Example 1, in (PKP), lamellar keratoplasty, Descemet's Membrane Endot which at day 9, corneal endothelial cells formed circular helial Keratoplasty (DMEK), DSAEK, and DLEK. colonies (arrow), with isolated corneal endothelial cells also 0150. In a further aspect, the disclosure provides a method present outside of the circular colonies (4x magnification). of treatment of a disease of corneal endothelial cells or injured FIG. 2B shows representative cells derived using the method corneal endothelial cells, which may comprise administering described in Example 1 at higher magnification (20x) to a composition comprising CEC to a patient in need thereof. illustrate the hexagonal or polygonal shape indicative of 0151. The CEC may be administered by a method which endothelial cells (arrow). FIG. 2C shows cell populations may comprise Descemet's stripping with endothelial kerato produced as described in Example 2, in which at day 10, plasty (DSEK), Penetrating Keratoplasty (PKP), lamellar culture corneal endothelial cells were predominant in the keratoplasty, Descemet's Membrane Endothelial Kerato culture (4x magnification). FIG. 2D shows representative plasty (DMEK), DSAEK, DMEK and DLEK. cells derived as described in Example 2 at higher magnifica US 2014/0370.007 A1 Dec. 18, 2014 tion (20x) to illustrate the hexagonal or polygonal shape this figure were produced as described in Example 1 and are indicative of endothelial cells (arrow). shown at day 9. These results demonstrate that the CEC do not 0160 FIG.3A-F shows positive expression and tightjunc detectably express the vascular endothelial markers CD31 tion localization of the marker ZO-1 by corneal endothelial and VWF, further confirming their identity as corneal endot cells differentiated from the hESC line H1GFP that provides helial cells. confirmation of their corneal endothelial identity. Corneal 0164 FIG. 7A-C shows upregulation of mRNAs charac endothelial cells (CEC) were stained for the tight junction teristic of corneal endothelial cells differentiated by hESC marker ZO-1 (red: FIG.3A, 10x: FIG. 3D, 40x) and using the derived corneal endothelial cells. Gene expression was nuclear stain DAPI (blue; FIG.3B, 10x: FIG.3E, 40x). ZO-1 detected by qPCR, and normalized to the level of expression exhibited a polygonal or hexagonal localization consistent detected from hESCs and the endogenous control PGK1. with the expected Staining of tight junctions. Merged views Expression of Collagen VIII, a major component of Descem (FIG. 3C, 10x; FIG. 3F, 40x) demonstrate the expected spa et's membrane that is secreted by corneal endothelial cells, tial relationship between ZO-1 and DAPI staining, i.e., gen was upregulated by approximately 20-fold (FIG.7A); expres erally a single nucleus contained within each polygonal or sion of the corneal endothelial cell markers CFTR, NBC1, hexagonal ZO-1 stained cell. The corneal endothelial cells in and SLC16A3 were upregulated by approximately 3-, 6-, and this figure were produced using the method described in 23-fold, respectively (FIG. 7B, left, center, and right bar in Example 1 and are shown at day 9. each group, respectively); and expression of AQP1, a major 0161 FIG. 4A-F shows expression and localization of the component of a corneal endothelial pump, was upregulated marker Na+K+-ATPase by cells differentiated from the hESC by about 1500-fold. RQ indicates quantity detected relative to line H1 GFP that provides confirmation of their corneal endot the amount detected from hESCs (average of multiple mea helial identity. Undifferentiated H1GFP hESCs exhibited a Surements, with error bars indicating the minimum and maxi relatively unorganized distribution of Na+K+-ATPase (FIG. mum of the RQ). CEC in this experiment were produced as in 4A, Na+K+-ATPase staining shown in red; FIG. 4B, DAPI Example 1. staining shown in blue; FIG. 4C, merged view of panels A and 0.165 FIG. 8 shows upregulation of the neural crest genes B). By contrast, CEC (differentiated from the hESC line NGFR and Sox 10 over the course of six days after commenc H1GFP) exhibited a localized distribution of Na+K+-ATPase ing exposure of hESC to the dual SMAD inhibitors Noggin (FIG. 4D, Na+K+-ATPase staining shown in red; FIG. 4E, and SB431542 (4-4-(1,3-benzodioxol-5-yl)-5-(2-pyridi DAPI staining shown in blue; FIG. 4F, merged view of panels nyl)-1H-imidazol-2-ylbenzamide). Expression of each gene D and E). The Na+K+-ATPase staining in the CEC exhibited a was markedly increased at day 2 (“DS2) and remained polygonal or hexagonal localization consistent with the elevated through day 6 (“DS6'). RQ indicates quantity expected Staining of tight junctions, generally with a single detected relative to the amount detected from hESCs and the nucleus within each cell. The corneal endothelial cells in this endogenous control PGK1 (average of multiple measure figure were produced as described in Example 1 and are ments, with error bars indicating the minimum and maximum shown at day 9. of the RQ): values are indicated with square symbols for 0162 FIG.5A-D shows expression and localization of the NGFR and triangular symbols for Sox 10. Gene expression marker KLF13 by cells differentiated from the hESC line was measured by qPCR analysis of samples collected on the H1GFP that provides confirmation of their corneal endothe indicated days (DS2 through DS6 referring to days 2 through lial identity, and additionally illustrates that the cells adopted 6, respectively, after commencing exposure of hESC to the a more regular and uniform hexagonal shape after a greater dual SMAD inhibitors). time in culture. Corneal endothelial cells were differentiated (0166 FIG. 9 shows induction of neural crest and corneal from hESCs using as described in Example 1 and are shown gene expression over the course of production of corneal after 25 days in culture (CEC Long). Cells were stained for endothelial cells from embryonic stem cells as described in the transcription factor KLF13 (FIG. 5A, red) and with the Example 1. Collagen VIII (ColVIII), a corneal endothelium nuclear stain DAPI (FIG. 5C, blue). GFP fluorescence was gene, was expressed at high levels after two days of dual also visualized (FIG. 5B, green) and was visible at both the SMAD inhibitor exposure. The neural crest genes NGFR and cell nucleus and outlining the boundaries of the cells. Relative SOX10 were expressed early and maintained during the cul to the day 9 cultures, the CEC Long cells had adopted a more ture period. Nanog (a gene indicative of pluripotency) was regular and uniform hexagonal shape. The merged view (FIG. greatly reduced early in the culture process. Gene expression 5D) shows the expression of KLF13 as expected. was measured by qPCR on the indicated days of the differ 0163 FIG. 6A-D shows the absence of vascular endothe entiation protocol or for untreated hESC, as labeled. RQ lial marker expression from corneal endothelial cells derived indicates quantity detected relative to the amount detected from H9 hESCs (CEC). Human vascular endothelial cells from hESCs, i.e., the level of expression in hESC is set to 1. (HUVEC) were used as a positive control. Cells were stained RQ values shown are the average of multiple measurements, for the vascular endothelial cell marker von Wildebrand fac with error bars indicating the minimum and maximum of the tor (VWF, red), corneal endothelial cell marker ZO-1 (green), RQ): values are indicated with square symbols for ColVIII, and nuclear stain DAPI (blue). Merged views show that diamond symbols for NGFR, triangular symbols for SOX10. HUVEC (FIG. 6A) were positive for vWF and negative for and circular symbols for Nanog. ZO-1, while CEC (FIG. 6B) were negative for vWF and 0.167 FIG. 10 shows expression of the transcription fac positive for ZO-1. Cells were stained for the vascular endot tors PITX2 and FOXC1 (markers of ocular neural crest) at helial cell marker CD31 (red), corneal endothelial cell marker day 9 of induction of corneal endothelial cell differentiation ZO-1 (green), and nuclear stain DAPI (blue). Merged views using the method as described in Example 1. RQ indicates show that HUVEC (FIG. 6C) were positive for CD31 and quantity detected relative to the amount detected from hESCs negative for ZO-1, while CEC (FIG. 6D) were negative for and the endogenous control PGK1, i.e., the level of expres CD31 and positive for ZO-1. The corneal endothelial cells in sion in hESC is set to 1. RQ values shown are the average of US 2014/0370.007 A1 Dec. 18, 2014

multiple measurements, with error bars indicating the mini reflect the production of extracellular matrix. B. AQP1 mum and maximum of the RO). mRNA expression increased with time in culture as the cells 0168 FIG. 11. Polygonal shape continues to refine with matured and paralleled the time-course of adoption of hex time. Phase contrast pictures at low (4x, panels A-D) and high agonal morphology as illustrated in FIG. 11 above. power (20x, panels E-H) of hESC derived corneal endothelial 0173 FIG. 16. Many corneal endothelial pumps are cells after increasing time in culture for cells produced as upregulated over 1-4 weeks of culture. All samples were described in Example 2. A., E. Polygonal hESC derived cor normalized to stem cells (hESC). “PD1T indicates cells neal endothelial cells are clearly visible after 1 week of dif produced as in Example 2, i.e. with the replating step during ferentiation. B., F. Cell shape continues to become more differentiation. A. Expression of upregulated pumps over regular at 2 weeks of differentiation. C., G. The cells were time. B. Corneal endothelial pump markers that are present, more uniform in size and shape at 3 weeks differentiation. D., but not at increased levels compared to hESC. C. Expression H. Even greater uniformity of cell shape is present at 4 weeks of COL8A2 is lower than the related gene, COL8A1. of differentiation. 0.174 FIG. 17. Rock inhibitor improves morphology of (0169 FIG. 12. Tightjunctions are presentinhESC derived hESC CEC after harvesting and replating and changes levels corneal endothelial cells. ZO-1 (red) outlines the tight junc of corneal endothelial genes by QPCR. A-D. Phase contrast tions between corneal endothelial cells. Nuclei are stained pictures of hESC CEC cells that have been trypsinized and with DAPI (blue). Arrows indicate hexagonal cell. A. E. I. As then replated in the presence of Rock inhibitor. A. The mor expected, ZO-1 was present at generally uniform intensity at phology of the hESC CEC is variable with the presence of 1 the junctions between cells, and cells contained a single uM Rock inhibitor. B. The hESC CEC are somewhat more stained nucleus. ZO-1 positive cells were polygonal after 1 uniform with 5 uM Rock inhibitor. C. The hESC CEC are week of differentiation. B. F. J. Polygonal ZO-1 cells contin uniform and polygonal/hexagonal with 10 uM Rock inhibi ued to be present with some variation in cell size at 2 weeks of tor. D. The hESC CEC are uniform and polygonal/hexagonal differentiation. C, G, K. After 3 weeks of differentiation, the with 20M Rock inhibitor. E-F. All samples were normalized CEC became more regular in size and increasing numbers of to stem cells (hESC). E. COL8A1 expression is relatively hexagonal cells appeared in the culture, indicating a Switch unchanged with the presence of Rock inhibitor compared to from polygonal to hexagonal morphology. D. H. L. After 4 PD1T that has not been harvested. SLC16A3 is upregulated weeks of differentiation, refinement of tight junctions for 1-10 uM Rock inhibitor, but unchanged for 20 uM Rock between cells continued and increasing numbers of hexago inhibitor, compared to PD1T that has not been harvested. nal cells were observed. ("PD1T indicates cells produced as in Example 2, i.e. with (0170 FIG. 13. hESC derived corneal endothelial cells the replating step during differentiation.) express the water pump Aquaporin1 (AQP1), indicative of (0175 FIG. 18. hESC CEC grown on UPCELL treated CEC identity. AQP1 (red) was localized to the cell borders at tissue culture dish can be removed as a sheet. hESC CEC were all time-points examined. Nuclei were stained with DAPI grown on UPCELL treated culture dishes, removed, and (blue). Arrows indicate hexagonal cell. A. E. I. 1 week of stained for ZO-1. Arrows indicate hexagonal cell. A. ZO-1 differentiation. B. F. J. 2 weeks of differentiation. C, G, K. 3 expression (red) indicates that tight junctions are maintained weeks of differentiation. D. H. L. 4 weeks of differentiation. after removal of cell sheet. B. Nuclei are stained with DAPI Consistent with mRNA expression levels shown below (see (blue). C. Merge of ZO-1 and DAPI. FIG. 15), AQP1 protein was present between weeks 1 and 4. (0176 FIG. 19. COL8A1 and COL8A2 are secreted to (0171 FIG. 14A-C. Expression of AQP1 (left column in form an extracellular-like matrix by hESC derived CEC. The each group) and COL8A1 (right column in each group) underlying subcellular matrix below the hESC CEC was col mRNA by hESC-derived CEC (generated using the method lected and analyzed by Western Blot. A. COL8A1 is present of Example 2) differed depending on the method used for as detected in an approximate 50 kDa band in the subcellular harvesting the cells. Expression levels of these genes were matrix. A band greater than 250 kDa was detected, and is detected using qPCR and normalized to stem cells (hESC) likely insoluble. B. COL8A2 is present as detected in an and compared to hESC derived corneal endothelial cells gen approximate 50 kDa band in the subcellular matrix. erated using the method of Example 2 prior to harvest 0177 FIG. 20. Global comparison of gene expression (PD1T). All methods of harvesting increased levels of between hESC CEC and adult-derived CEC. The genes COL8A1 and decreased amounts of AQP1 compared to cells shown exhibited at least a 3-fold difference in expression prior to harvesting. Man manual dissociation (cell Scraping); between hESC CEC and Adult CEC at a significance thresh “0.05% T=0.05% Trypsin; “0.25% 5 min’=0.25% Trypsin old of p<0.05. Gene symbol, gene description, accession for 5 minutes: “0.25% T'=0.25% Trypsin for 10 minutes: number, and fold difference are listed for each gene. The “0.25%T EDTA'=0.25% Tryspin diluted 1:1 with Cell Dis microarray additionally included positions which were not sociation buffer. Acc-accutase, ColB=collagenase B over annotated and the results for these positions is included in the night; ColB EDTA=Collagenase B treatment 1 hr followed able with the designation “Tr” (e.g., “Tr. 7904959) indicat by cell dissociation buffer. ing the expression result for the specified Transcripts Cluster 0172 FIG. 15. COL8A1 and AQP1 mRNA levels in hESC Id on the array. A negative value indicates that the expression CEC cultured for 1-4 weeks. All samples were normalized to is lower in the adult CEC compared to the hESC CEC. stem cells (hESC). “PD indicates cells produced as in (0178 FIG. 21. Improved purity of hESC-derived CEC Example 1, i.e., without passaging during differentiation. using magnetic bead subtraction. CD271+ positive cells were “PD1T indicates cells produced as in Example 2, i.e. with removed from corneal endothelial cultures. Phase contrast the replating step during differentiation. A. COL8A1 was (20x) view of cells that have been cultured for a total of 2 highly expressed at all time-points tested. The temporary weeks of differentiation. A. Control cells produced as in increase in COL8A1 expression detected at week 2 coincides Example 2 but not passaged at Day 10. B. Control for CD271 with the transfer to a new tissue culture well and is thought to subtraction. Cells were trypsinized on Day 10 and immedi US 2014/0370.007 A1 Dec. 18, 2014

ately replated. C. The majority of the CD271 + cells were a human basement membrane extract, or any combination removed by magnetic bead separation. The processing of the thereof. In another embodiment, the matrix is from human or cells has altered the morphology of the corneal endothelial non-human animal (e.g., bovine, mouse or rat origin). In cells. D. Flow Cytometry analysis of CD271+ cells on exemplary embodiments, cells (e.g., hES cells, NCSCs, and/ unsorted by magnetic beads. Approximately 23% of all cells or CEC) may be cultured on feeder cells (including but not expressed CD271 at relatively low levels. E. CD271+ cells limited to MEFs, NHDFs, other fibroblasts, or other non were depleted by magnetic bead separation. FLA4 is the APC fibroblast cell types), which may be mitotically inactivated channel where CD271 was detected. CD271 is also known as (e.g., by gamma irradiation, Mitomycin C treatment, or other neural crest gene NGFR. methods known in the art). Suitable matrixes and/or feeder cells generally permit the maintenance of hES cells. hES cell DETAILED DESCRIPTION differentiation into NCSCs, or differentiation of NCSCs into 0179 The present application provides methods for CEC. obtaining corneal endothelial cells through directed differen 0183. Additional exemplary embodiments may include tiation of pluripotent or multipotent stem cells, including sensitive and specific detection of other cell types (e.g., ES human embryonic stem cells (hESC), somatic cells (includ cells and/or neural crest stem cells) in a CEC cell population, ing transdifferentiated cells and stem cells such as neural crest using methods disclosed in co-owned international applica stem cells), and induced human pluripotent stem cells tion ser. no. PCT/US 11/45232 and U.S. Provisional Applica (hiPSC). It is expected that these cells can provide an alter tions Ser. Nos. 61/414,770 and 61/367,038, each of which is native to the burdensome collection of donated corneas for incorporated by reference herein in its entirety. For example, therapeutic use. a cell population may be stained for two markers indicative of 0180. As further detailed below, monolayers of corneal ES cells (e.g., stained for alkaline phosphatase (AP) expres endothelium were produced by directed differentiation of sion and stained expression of an ES cell-specific marker Such human embryonic stem cells (hESC). hESC were induced to as Oct-4, Nanog, etc.), and cells may be examined to detect differentiate into neural crest progenitors, and Subsequently, any cells that express those ES cell markers. Likewise, the the neural crest progenitors were exposed to growth factors population may be stained for the presence of two or more that induced the formation of corneal endothelial cells. Cor markers indicative of neural crest stem cells and examined to neal endothelial cell identity was confirmed based on charac detect any cells that express those neural crest stem cell teristic morphological features and gene expression, includ markers. As described in the aforementioned applications, ing presence of markers of corneal endothelial cells, and highly sensitive detection can be attained using these methods absence of certain markers of vascular endothelial cells through examination of a Sufficiently large numbers of cells, (whose marker expression has some overlap with corneal e.g., between at least one million cells and at least 10 million endothelial cells). cells. Preferably, at least one stain is detectable under visible 0181. In exemplary embodiments, populations of corneal light, permitting the cells to be viewed under visible light to endothelial cells may be tested for presence of other cell detect at least one of the markers, which may greatly increase types, e.g., any remaining pluripotent cells and/or neural crest throughput; the second marker may be visible under UV light stem cells. Exemplary methods that may be used to detect and any cell positive for said first marker may be visualized other cell types in a corneal endothelial cell population under UV light to detect expression of said second marker, include methods that can detect expression of markers of wherein a cell detected to express both markers is scored as a pluripotent cells, such as Northern blotting, Western blotting, cell of the type to be detected. immunostaining, PCR, and other methods known in the art. 0.184 Alternatives and Additional Factors for Production See, generally, Ausubel, Current Protocols in Molecular Biol of Corneal Endothelial Cells from Embryonic Stem Cells ogy (Current Protocols, 1988); Ausubel et al., Short Protocols 0185. As further described in the Examples below, Appli in Molecular Biology (Current Protocols: 5th Edition, 2002); cants have demonstrated methods for production of corneal Sambrook et al., Molecular Cloning: A Laboratory Manual endothelial cells (CEC) by differentiation of hES cells (Cold Spring Harbor Laboratory Press, 3rd edition, 2001); through contact with certain factors. In addition to the specific Sambrook et al., The Condensed Protocols from Molecular factors used in the examples, it is expected that other factors, Cloning: A Laboratory Manual (Cold Spring Harbor Labora e.g., equivalents, agonists, etc. may be used in place of or in tory Press, 2006), each of which is incorporated by reference addition to any of the identified factors. Suitable combina herein in its entirety. tions of factors can be readily and concentrations thereof can 0182. In exemplary embodiments cells (e.g., hES cells, be readily determined by those of ordinary skill in the art. For NCSCs, and/or CEC) may be cultured on a matrix. The matrix example, hES cell populations can be readily contacted with may comprise one or more of transforming growth factor candidate compositions and monitored for adoption of neural beta (TGF-beta), epidermal growth factor (EGF), insulin-like crest stem cell fate, such as expression of genetic markers growth factor 1, fibroblast growth factor 2 (FGF2, sometimes thereof including those identified herein and in WO/2010/ also referred to as bFGF), platelet-derived growth factor 096496, which is incorporated by reference herein in its (PDGF), laminin (e.g., laminin-511, which may be recombi entirety. Additionally, neural crest stem cell populations (in nant), fibronectin, vitronectin (e.g., recombinant Zebrafish cluding populations of ES cells undergoing the process of modified vitronectin), proteoglycan, entactin, collagen, col neural crest induction) can be contacted with candidate com lagen I, collagen IV, collagen VIII, heparan Sulfate, Matrigel positions and monitored for adoption of corneal endothelial (a soluble preparation from Engelbreth-Holm-Swarm (EHS) cell fate. Such as the characteristic morphology (see, e.g., mouse sarcoma cells, the major components of which include FIG. 2), as well as expression of one or more markers char laminin, collagen IV, entactin and heparan Sulfate proteogly acteristic of corneal endothelial cells, including those identi can and which also includes growth factors, collagenases, fied herein and others known in the art. Cells may be further plasminogen activators and may include other components), monitored for decreased or absent expression of one or more US 2014/0370.007 A1 Dec. 18, 2014

markers indicative of one or more other cell types, said 9). Another combination of factors expected to be suitable for decreased or absent expression typically being evaluated rela generating neural progenitors comprises a cocktail of Leuke tive to the expression level of cells of said other type, for mia Inhibitory Factor (LIF), GSK3 inhibitor (CHIR 99021), example, decreased or absent expression of one or more vas Compound E (Y secretase inhibitor XXI) and the TGFB cular endothelial cell markers (such as VWF and/or CD31/ inhibitor SB431542 which has been previously shown to be PECAM-1), and/or decreased or absent expression of one or efficacious for generating neural crest stem cells (Li et al., more ES cell markers, and/or decreased or absent expression Proc Natl AcadSci USA. 2011 May 17: 108(20):8299-304). of neural crest stem cell markers in CEC. Cells such as hES Additional exemplary factors may include derivatives of cells or neural crest stem cells may be exposed to different factors or combinations of factors and may be monitored for SB431542, e.g., molecules that include one or more added or adoption of CEC phenotypes, optionally together with suit different Substituents, analogous functional groups, etc. and able positive and negative controls, to identify operative fac that have a similar inhibitory effect on one or more SMAD proteins. Suitable factors or combinations of factors may be tors and concentrations thereof, or to determine operative or identified, for example, by contacting hESC with said factor optimal concentrations of factors. (s) and monitoring for adoption of neural crest stem cell 0186 Dual Smad Inhibitors phenotypes, such as characteristic gene expression (including 0187 Exemplary embodiments of the presently disclosed expression of the markers described herein, expression of a method include differentiating hESC in the presence of Nog reporter gene coupled to a neural crest stem cell promoter, or gin (e.g., human Noggin polypeptide. Such as NP 005441.1 the like) or the ability to form CEC or another cell type or the mature polypeptide contained therein) and SB431542 capable of differentiating from neural crest stem cells. (collectively, “dual SMAD inhibitors'). Such cultures may produce neural crest stem cells, and further culturing (e.g., in 0189 In exemplary embodiments, hES cells, such as hES the presence of additional factors) may produce CEC or other cells undergoing neural crest induction, may be cultured in cell types. Applicants envision that alternative factors (indi the presence of SB431542, which may be present in the vidually and/or in combination) could be used in the dis culture media in a concentration as low as 10 nM, 20 nM, 50 closed methods in place of either or both of the dual SMAD nM, 0.1 uM, or lower, or as high as 20 uM, 50 uM, 100 uM, inhibitors, and/or be used in addition to one or both of these or higher, such as 10 nM to 100 uM, 0.1 uM to 50 uM, 0.1-20 factors. Though these factors are sometimes referred to as uM, or 1-20 uM, preferably about 10 uM. In exemplary “dual' SMAD inhibitors, more or fewer than two factors may embodiments, hES cells, such as hES cells undergoing neural be utilized within the scope of these methods. crest induction, may be cultured in the presence of Noggin, 0188 Noggin is a secreted BMP inhibitor that reportedly which may be present in the culture media in a concentration binds BMP2, BMP4, and BMP7 with high affinity to block as low as 10 ng, 20 ng/ml, 50 ng/ml, 100 ng/ml, or lower, or TGFB family activity. SB431542 is a small molecule that as high as 700 ng/ml, 1000 ng/ml, 1500 ng/ml, 2000 ng/ml, reportedly inhibits TGFB/Activin/Nodal by blocking phos 3000 ng/ml. 4000 ng/ml, 5000 ng/ml, or higher, such as 10 phorylation of ACTRIB, TGFBR1, and ACTRIC receptors. ng/ml to 5,000 ng/ml, 100 ng/ml to 700 ng/ml, or 400 ng/ml SB431542 is thought to destabilize the Activin- and Nanog to 600 ng/ml, preferably about 500 ng/ml. hES cells may also mediated pluripotency network as well as suppress BMP be cultured with combinations of SB431542 and Noggin, induced trophoblast, mesoderm, and endodermal cell fates by e.g., combinations of the foregoing concentrations. blocking endogenous Activin and BMP signals. It is expected 0.190 PDGFB that agents having one or more of the aforementioned activi ties could replace or augment the functions of one or both of 0191 Exemplary embodiments of the presently disclosed Noggin and SB431542, e.g., as they are used in the context of method include culturing neural crest stem cells in the pres the disclosed methods. For example, applicants envision that ence of PDGFB (e.g., human PDGFB polypeptide, such as the protein Noggin and/or the small molecule SB4312542 NP 002599.1 or the mature polypeptide contained therein), could be replaced or augmented by one or more inhibitors that and culture media comprising PDGFB (sometimes referred to affect any or all of the following three target areas: 1) pre in the literature as PDGFBB due to its existence as a dimer). venting the binding of the ligand to the receptor; 2) blocking It is envisioned that one or more additional factors may be activation of receptor (e.g., dorsomorphin), and 3) inhibition used in addition to or instead of PDGFB. For example, of SMAD intracellular proteins/transcription factors. Exem PDGFB reportedly signals through PDGFRB, which then plary potentially suitable factors include the natural secreted activates the PKC pathway. Both PDGFRC. and PDGFRB BMP inhibitors Chordin (which blocks BMP4) and Follista are expressed in the developing cornea, so PDGFAA or PDG tin (which blocks Activin), as well as analogs or mimetics FAB may be suitable for use in addition or alternative to thereof. Additional exemplary factors that may mimic the PDGFB. As an additional example, Phorbol 12-myristate effect of Noggin include use of dominant negative receptors 13-acetate (PMA) (which is thought to activate PKC) may be or blocking antibodies that would sequester BMP2, BMP4, used instead of or in addition to PDGFB. VEGF is structurally and/or BMP7. Additionally, with respect to blocking receptor related to PDGF and therefore may also be used instead of or phosphorylation, dorsomorphin (or Compound C) has been in addition to PDGFB. reported to have similar effects on stem cells. Inhibition of 0.192 In exemplary embodiments, cultures comprising SMAD proteins may also be effected using soluble inhibitors neural crest stem cells, such as in a culture comprising hES such as SIS3 (6,7-Dimethoxy-2-((2E)-3-(1-methyl-2-phe cells after commencing neural crest induction or during neu nyl-1H-pyrrolo2,3-bipyridin-3-yl-prop-2-enoyl)-1,2,3,4- ral crest induction, are cultured in the presence of PDGFB tetrahydroisoquinoline, Specific Inhibitor of Smad3, SIS3). which may be present in the culture media in a concentration overexpression of one or more of the inhibitor SMADs (e.g., as low as 0.1 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, or lower, SMAD6, SMAD7, SMAD 10) or RNAi for one of the recep or as high as 10 ng/ml, 20 ng/ml, 30 ng/ml, 50 ng/ml, 75 tor SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8/ ng/ml, 100 ng/ml, 125 ng/ml, 150 ng/ml, 200 ng/ml, 250 US 2014/0370.007 A1 Dec. 18, 2014

ng/ml, or higher, such as 0.1 ng/ml to 250 ng/ml, 0.5 ng/ml to ral crest induction, are cultured in the presence of DKK2, 150 ng/ml, 1-50 ng/ml, 2-20 ng/ml, preferably about 10 which may be present in the culture media in a concentration ng/ml. as low as 1 ng/ml, 2 ng/ml, 3 ng/ml. 4 ng/ml, 5 ng/ml, 6 ng/ml. 0193 DKK2 or lower, or as high as 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 0194 Exemplary embodiments of the presently disclosed ng/ml, 1 ug/ml, 5ug/ml, 10 ug/ml, 15 ug/ml, or higher. Such method include culturing cells in the presence of DKK2 (e.g., as 1 ng/ml-15ug/ml, 10 ng/ml-15 g/ml, 1 ng/ml-1 g/ml. 1 human DKK2 polypeptide, such as NP 055236.1 or the ng/ml-100 ng/ml, 2 ng/ml-20 ng/ml, or 5 ng/ml-20 ng/ml. mature polypeptide contained therein), and culture media preferably about 10 ng/ml. comprising DKK2. For example, neural crest stem cells (0196. Angiopoietin-Like Protein 7 (ANGPL7) (whether obtained from differentiating hES cells or other 0.197 In additional experimental results obtained by the sources) and/or differentiating hES cells cultured under con present inventors in which ANGPL7 was included in the ditions expected to produced neural crest stem cells (e.g., culture media (in combination with DKK2 and PDGFB) in culture in the presence of dual SMAD inhibitors) may be the methods disclosed in Example 1, the resulting CEC exhib cultured in the presence of DKK2. It is envisioned that one or ited tighter packing and more hexagonal morphology. These more additional factors may be used in addition to or instead results indicate that ANGPL7 may be a positive factor for of DKK2, e.g., a DKK2 agonist. DKK2 has been shown to be generating corneal endothelial cells, particularly for produc both an inhibitor and activator of the Wnt pathway in various ing CEC in a high-density sheet or layer which may be pre cell-dependent contexts. Exemplary DKK2 agonists include ferred for transplant. Accordingly, ANGPL7 may optionally any Wnt pathway activators and/or inhibitors that may func be used in the differentiation of corneal endothelial cells, for tionally replace DKK2 in the differentiation of CEC. When example by its inclusion in the culture during and/or after DKK2 acts as an inhibitor it reportedly binds to the co formation of corneal endothelial cells. ANGPL1-8 may be receptor LRP5/6 and Kremen, which then causes the receptor used in place of or in addition to ANGPL7. to be internalized and degraded. For example, RNAi that 0198 FGF2 targets and knocks down the expression of LRP5/6 or Kre 0199. In exemplary embodiments, FGF2 may be present men, may be used in addition to or instead of DKK2. Wnt in cultures of hES cells, e.g., during neural crest induction, pathway inhibitors such as DKK 1, 3, 4 and Soggy, Secreted which may be present in the culture media in a concentration frizzled related proteins (Frzb), and Wnt inhibitor factor as low as 1 ng/ml, 2 ng/ml, 3 ng/ml. 4 ng/ml, 5 ng/ml, 6 ng/ml. (WIF) may also be used in addition to or instead of DKK2. or lower, or up to 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml, Another potentially suitable Wnt pathway inhibitor is Casein 500 ng/ml, or 1 g/ml, such as 1 ng/ml-1 ug/ml, 1 ng/ml-100 Kinase 1-7 which is expected to block signal transduction. ng/ml, 2 ng/ml-10 ng/ml. 6 ng/ml-100 ng/ml, preferably Factors (such as Small molecules) affecting B catenin, for about 6 ng/ml. example factors that stabilize or destabilize B catenin, may 0200. In exemplary embodiments, FGF2 may be present also be used in addition to or instead of DKK2. Additionally, in cultures of neural crest stem cells or CEC, e.g., during CEC modulating the LEF/TCF transcription factor members may differentiation from neural crest stem cells or in cultures be used in addition to or instead of DKK2. Exemplary Wnt comprising CEC, which may be present in the culture media pathway activators include Wnt proteins, nucleic acids in a concentration as low as 0.1 ng/ml, 0.2 ng/ml, 0.5 ng/ml. encoding Wnt proteins, LiCl, inhibitors of negative regulators 1 ng/ml, 2 ng/ml, 3 ng/ml. 4 ng/ml, 5 ng/ml, 6 ng/ml, or lower, of Wnt pathway (e.g., RNAi or other inhibitors targeting Axin or up to 10 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml, and/or APC), norrin, R-spondin2. Small molecule Wnt path or 1 ug/ml. Such as 0.1 ng/ml-1 ug/ml, 0.1 ng/ml-400 ng/ml. way activators include: (hetero)arylpyrimidines, IQ1, BIO(6- 0.1 ng/ml-100 ng/ml, 0.1 ng/ml-10 ng/ml, 1 ng/ml-100 ng/ml, bromoindirubin-3'-oxime), 2-amino-4-3,4-(methylene preferably about 6 ng/ml. dioxy)benzyl-amino-6-(3-methoxyphenyl)pyrimidine, 0201 B27 WAY-316606, QS11, SB-216763, SB-216763, and DCA. (0202 B27 (B-27 Serum-Free Supplement (50x) liquid, Small molecule Wnt pathway inhibitors include: IWR, pyrv Invitrogen cat #17504-044) is a culture medium supplement inium, ICG-001, PKF115-584 (and several other com containing d-Biotin, BSA, L-Carnitine HC, Corticosterone, pounds), IWP, Ant1.4Br/Ant 1.4C1, Niclosamide, apicularen Ethanolamine HCl, D-Galactose (Anhyd.), Insulin (Human, and bafilomycin, XAV939, NSC668036, 2,4-diamino Zn), Linoleic Acid, Linolenic Acid, Progesterone, Putrescine. quinazoline, and Quercetin. Additional exemplary WNT 2HCl, Sodium Selenite (1000x), T-3/Albumin Complex, pathway inhibitors which may be utilized include ID8 (Hasa Transferrin (Human, Iron-Poor), and Vitamin A Acetate, gawa et al., Stem Cells Transl Med. 2012 January; 1(1):18 which is prepared in distilled water (Brewer et al., J. Neuro 28), Wnt C59 (Proffitt Cancer Res PublishedOnlineFirst Nov. sci. Res. 35:567-576 (1993)). In exemplary embodiments, 27, 2012: DOI:10.1158/0008-5472. CAN-12-2258), CEC may be cultured in a medium comprising B27 (such as CGK062 (Gwak et al., PLoS ONE. 2012; 7(10):e46697), after differentiation or during differentiation) in a concentra IWP2 (Blauwkamp et al., Nat Commun. 2012; 3:1070), tion as low as 0.01x, 0.02x, 0.05x, 0.1x, or lower, or as high FH535 (Iida et al., PLoS One. 2012; 7(9):e44418), and Rilu as 0.5.x, 1x, 2x, 3.x. 5x, 10x, or higher, such as 0.01x-10x. zole (Zhao et al., J Biomol Screen. 2012 October; 17(9): 1252 0.02x-5x, 0.05x-1x, or 0.1x-1x, preferably about 0.1x. 63). Combinations of the foregoing factors may also be used (0203 TGFB2 in addition to or instead of DKK2, e.g., combinations com 0204 Applicants envision that TGFB2 may be used to prising more than one Wnt pathway activator, more than one promote differentiation of neural crest stem cells into corneal Wnt pathway inhibitor, or at least one Wnt pathway activator endothelial cells. For example, in mice lacking both copies of and at least one Wnt pathway inhibitor. TGFB2, the corneal endothelium is absent. Although the neu 0.195. In exemplary embodiments, cultures comprising ral crest migrates at the correct time and place, the corneal neural crest stem cells, such as in a culture comprising hES endothelium does not form. Additionally, TGFB2 is highly cells after commencing neural crest induction or during neu expressed in the lens at the time the neural crest are adjacent US 2014/0370.007 A1 Dec. 18, 2014 to the lens. Based in part on the foregoing, Applicants envi culturing said CEC or precursors thereof on a carrier, which sion that in exemplary embodiments, TGFB2 may be used to may optionally include transferring or releasing a sheet or promote the differentiation of neural crest into corneal endot monolayer of CEC or precursors thereof onto said carrier, helial cells. wherein said composition may be suitable for transplantation. 0205 Factors that Promote Corneal Endothelial Cell Pro Additionally provided are cultures of CEC, or precursors liferation thereof, of the present disclosure, said cultures comprising 0206 Exemplary embodiments may include the use of one CEC, or precursors thereof, that are adherent to a carrier. or more factors that promote corneal endothelial cell prolif Exemplary carriers may be Suitable for transplantation. eration. For example, such factors may be included in a cul Exemplary carriers include carriers that may dissolve or oth ture of cells during and/or Subsequent to formation of corneal erwise disappear in vivo when the sheet or monolayer of endothelial cells. Particular exemplary factors include Hepa corneal endothelial cells is transplanted into a host organism, tocyte growth factor (HGF) and/or Keratinocyte growth fac Such as gelatin (see Hsiue et al., Supra). Additional exemplary tor (KGF), which have been shown to induce proliferation in carriers include fibrin-based matrixes, endothelium-denuded cultured corneal endothelial cells (Wilson et al., Invest Oph corneal buttons, denuded Descemet's membrane, directly thalmol Vis Sci. 1993 July; 34(8):2544-61). onto the Stromal layer, e.g., devitalized stromal cornea (e.g., 0207 IL1C. from a human cadaver or non-human animal), fresh corneal 0208 IL1C. has been shown to upregulate HGF and KGF stromal discs (from a human or non-human animal), and/oran in corneal stromal fibroblasts. Applicants envision that IL1C. amniotic membrane (preferably without amniotic cells). The may contribute to the formation and/or maintenance of cor dimensions and/or thickness of the carrier are preferably suit neal endothelial cells as well. Accordingly, in exemplary able for implantation into the eye, e.g., about 100 microns embodiments, IL1C. may be included in a culture of cells thick or less. during and/or Subsequent to formation of corneal endothelial 0213 For example, the CEC or precursor thereof may be cells. cultured on a Substrate from which an intact sheet or mono 0209 Sheets, Monolayers, Cultures, and Pharmaceutical layer of cells can be released, e.g., a Substrate Such as a Preparations thermoresponsive polymer Such as a thermoresponsive poly 0210 Exemplary embodiments provide a culture of cor (N-isopropylacrylamide) (PNIPA Am)-grafted surface, upon neal endothelial cells, such as a sheet or monolayer of CEC, which cells adhere and proliferate at the culture temperature, wherein the CEC are produced by differentiation of embry and then upon a temperature shift, the Surface characteristics onic stem cells. The sheet or monolayer may have a cell are altered causing release the cultured cell sheets (e.g., by density of at least 1,000 cells/mm, at least 1,500 cells/mm. cooling to below the lower critical solution temperature 2,000 cells/mm, at least 2,500 cells/mm, at least 3,000 (LCST) (see da Silva et al., Trends Biotechnol. 2007 Decem cells/mm, at least 3,500 cells/mm, at least 4,000 cells/mm. ber; 25(12):577-83; Hsiue et al., Transplantation. 2006 Feb. at least 4,500 cells/mm, at least 5,000 cells/mm, or higher. 15: 81(3):473-6: Ide, T. et al. (2006): Biomaterials 27, 607 Optionally, the sheet or monolayer of corneal endothelial 614, Sumide, T. et al. (2005), FASEB.J. 20,392–394; Nishida, cells may further comprise a Descemet's membrane or other K. etal. (2004), Transplantation 77,379-385; and Nishida, K. matrix produced by the corneal endothelial cells. For et al. (2004), N. Engl.J. Med. 351, 1187-1196 each of which example, a matrix may be prepared from media conditioned is incorporated by reference herein in its entirety). For by CEC (e.g., CEC produced from hES cells), e.g., by con example, cultured CEC may be released onto a carrier, Such as centrating proteins in said media, and optionally adding a carrier Suitable for transplantation, e.g., as described in the matrix components, growth factors, or the like. preceding paragraph. 0211 Donor-derived CEC reportedly accumulate numer 0214. As a further example, the CEC or precursor thereof ous markers and changes in gene expression that are thought may be contained in a laminate, which may comprise a trans to result from accumulated oxidative damage and other parent type I collagen sheet that optionally has been coated insults. See Joyce et al., Invest. Ophthalmol. Vis. Sci. Mar. 24. with an adhesive factor or a bioadhesive and a cultured layer 2011 Vol. 52 no. 3 1641-1649, which is incorporated by of CEC or precursor thereof provided on said transparent type reference herein in its entirety. CEC produced by the present I collagen sheet, wherein said transparent type I collagen methods are expected to exhibit one or more attributes of sheet may have a thickness ranging from 5 to 50 micrometers. “youthful' CEC. For example, as compared to donor-derived The adhesive factor or bioadhesive layer may be on the oppo CEC (especially adult-derived CEC) it is expected that the site side from the cultured layer of human corneal endothelial CEC may exhibit decreased levels of oxidative damage: cells, and/or between said transparent type I collagen sheet nuclear DNA damage foci: decreased levels of expression of and said CEC or precursor thereof. The adhesive factor may p21Cip1; decreased levels of expression of p16INK4a: be a fibronectin, e.g., human plasma fibronectin. For example, decreased levels of expression of cytoglobin protein; a laminate may be prepared by placing or releasing a sheet or decreased levels of expression of GPX-1 protein, and monolayer of CEC or precursors thereof onto a transparent decreased levels of 8-hydroxy-2-deoxyguanosine type I collagen sheet that optionally has been coated with an (8-OHdG). adhesive factor or a bioadhesive, and/or by culturing a Sus 0212. Also provided are methods of culturing CEC, or pension comprising CEC or precursors thereof in contact precursors thereof, of the present disclosure, said methods with said transparent type I collagen sheet and allowing said comprising culturing said CEC or precursors thereof on a cells to form a laminate comprising CEC or a precursor carrier, wherein said methods may optionally include trans thereof. Further exemplary compositions, Substrates, meth ferring or releasing a sheet or monolayer of CEC or precur ods, and the like, in which the cells of the present disclosure sors thereofonto said carrier. Further provided are methods of may be used, are described in U.S. Pat. No. 7.959,939 and US preparing a composition comprising CEC, or precursors 2010/0233240, each of which is incorporated by reference thereof, of the present disclosure, said methods comprising herein in its entirety. US 2014/0370.007 A1 Dec. 18, 2014

0215. In one aspect, the present disclosure provides cul growth factor may be about 0.02-3.0 mg/ml or about 0.2-2.0 tures comprising corneal endothelial cells. As further mg/ml). Further, the CEC may be cultured in a medium described herein, CEC of the present disclosure may be main comprising a serum-free cell culture medium comprising tained in culture for prolonged periods of time and may form insulin, transferrin, and selenium; fibroblast growth factor a population that is non-dividing. (pituitary); epidermal growth factor, nerve growth factor, a 0216 Exemplary compositions may include an inhibitor calcium chelator, and optionally an antibiotic antimycotic of Rho-associated kinase (ROCK), such as Y-27632 (also solution. See U.S. Pat. No. 6,548,059 and Senoo et al. Inves referred to as (+)-trans-4-(1-aminoethyl)-1-(4-pyridyl car tigative Ophthalmology & Visual Science, September 2000, bamoyl)cyclohexane or by its IUPAC name (1R,4r)-4-((R)- Vol. 41, No. 10, pg. 2930-2935, each of which is incorporated 1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide; by reference herein in its entirety. see Watanabe et al., Nat Biotechnol. 2007 June; 25(6):681 0219. As a further example, the CEC may be cultured in a 6)), preferably in an amount sufficient to promote survival of medium comprising insulin; transferrin; selenium: 1-100 the cells. See, e.g., Okumura et al., Invest OphthalmolVis Sci. ng/ml nerve growth factor; 5-400 ng/ml fibroblast growth 2009:50:3680-3687, Okumura et al., BrJOphthalmol. 2011 factor (pituitary): 1-200 ng/ml epidermal growth factor; July:95(7):1006-9, published U.S. patent applications 2010/ 1-25% fetal bovine serum; 10-50 micrograms/ml ascorbic 02094.02 and 2010/0233240, each of which is incorporated by acid; 0.001-0.01% human lipids; 0.01-0.12% chondroitin reference herein in its entirety. sulfate; 100-300 micrograms/ml calcium chloride. Option 0217. In a further aspect the CEC may be cultured in the ally, the medium may further comprise one or more of 10-100 presence of Y-27632 and/or another ROCK inhibitor, for micrograms/ml gentamycin; RPMI-1640 multiple vitamin example before, during and/or after passaging, which may solution (1/50-1/200); and antibiotic antimycotic solution improve cell viability. Additional exemplary ROCK inhibi (1/50-1/200). For example, the CEC may be cultured in tors include small molecules, siRNAs, miRNAs, antisense growth medium may comprise 20 ng/ml nerve growth factor; RNA, or the like, that may target a rho-associated kinase or 40 ng/ml fibroblast growth factor (pituitary); 5 ng/ml epider member of the ROCK signaling pathway. Exemplary ROCK mal growth factor; 8% fetal bovine serum; 20 micrograms/ml inhibitors include H-1152, Y-301.41, Wf-536, HA-1077, ascorbic acid; 0.005% human lipids; 0.08% chondroitin sul hydroxyl-HA-1077, GSK269962A and SB-772077-B, 1-(5- fate; 200 micrograms/ml calcium chloride. Optionally, the isoquinolinesulfonyl)homopiperazine (fasudil), (+)-trans-4- medium may further comprise one or more of 50 micro (1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane grams/ml gentamycin; RPMI-1640 multiple vitamin solution (Y-27632), as well as salts thereof, preferably pharmaceuti (1/100); and antibiotic antimycotic solution (1/100). See U.S. cally acceptable salts such as hydrochloride salts. In exem Pat. No. 6,541.256, which is incorporated by reference herein plary embodiments, the ROCK inhibitor may have a concen in its entirety. tration of about 0.05 to about 50 microM, for example, at least 0220. As another example, the CEC may be placed or or about 0.05, 0.1, 0.2,0.5,0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, cultured on an amniotic membrane. Such as the basement 25, 30, 35, 40, 45, or 50 microM, including any range deriv membrane side of the amniotic membrane, e.g., with or with able therein, or any concentration effective for promoting cell out amniotic cells, wherein the amniotic membrane may have growth and/or Survival. an extracellular matrix. The CEC may be placed on the amni 0218. The CEC of the present disclosure may also be otic membrane as a sheet or applied thereto as a individual cultured under conditions that promote proliferation. For cells, such as cells in a Suspension, which may be allowed to example, the CEC may be contacted with an effective amount settle by gravity and/or centrifugation. The CEC may be of at least one growth factor that promotes proliferation of cultured on the amniotic membrane and allowed to prolifer corneal endothelial cells (e.g., an antibody that specifically ate. The CEC placed or cultured on an amniotic membrane binds to a cell Surface protein on the corneal adult human may also be used as a Surgical graft (or used in the manufac corneal endothelial cell that is involved in cell-cell adhesion, ture of a medicament), e.g., for the treatment of a disease of Such as antibody that specifically binds to a protein selected corneal endothelial cells. See, e.g., U.S. Pub. No. 2007/ from the group consisting of a cadherin, ZO-1 protein, and 0254361, which is incorporated by reference herein in its connexin-43, which may results in interruption in cell-cell entirety. contacts in at least 15%, at least 50%, or at least 80% of the 0221) Therapeutic Methods CEC, wherein the concentration of said growth factor may be 0222. In another aspect, the present disclosure provides about 0.02-3.0 mg/ml or about 0.2-2.0 mg/ml); and subse therapeutic methods for the prevention and/or treatment of quently exposed to an effective amount of at least one agent disease, preferably diseases affecting corneal endothelial that promotes interruption of cell-cell contacts between adja cells or amenable to treatment by the transplantation or cent corneal endothelial cells (such as a calcium chelator, e.g., administration thereof, including, for example, primary dis ethylenediaminetetraacetic acid (EDTA) or ethylene glycol eases such as Fuchs dystrophy, iridocorneal endothelial Syn bisbeta-aminoethylether-N.N.N',N'-tetraacetic acid drome, posterior polymorphous dystrophy, and congenital (EGTA)), and, optionally, Subsequently contacting the CEC hereditary endothelial dystrophy, and secondary diseases for with an effective amount of at least one growth factor that which an effective treatment is replacement of the corneal promotes proliferation of corneal endothelial cells (e.g., an endothelium including corneal dystrophies, contact lens antibody that specifically binds to a cell surface protein on the usage, cataract Surgery, and late endothelial failure in cornea corneal adult human corneal endothelial cell that is involved transplantation. in cell-cell adhesion, such as antibody that specifically binds 0223 Exemplary therapeutic methods may further to a protein selected from the group consisting of a cadherin, include administration of an immunosuppressive agent. ZO-1 protein, and connexin-43, which may results in inter Immunosuppressants that may be used include but are not ruption in cell-cell contacts in at least 15%, at least 50%, or at limited to anti-lymphocyte globulin (ALG) polyclonal anti least 80% of the CEC, wherein the concentration of said body, anti-thymocyte globulin (ATG) polyclonal antibody, US 2014/0370.007 A1 Dec. 18, 2014

azathioprine, BASILIXIMAB(R) (anti-IL-2RO. receptor anti each donor. These and additional therapeutic methods are body), cyclosporin (cyclosporin A), DACLIZUMABOR (anti further described in Thomas John, Corneal Endothelial IL-2RC. receptor antibody), everolimus, mycophenolic acid, Transplant: DSAEK, DMEK & DLEK (JP Medical Ltd, RITUXIMAB(R) (anti-CD20 antibody), sirolimus, tacroli 2010), which is incorporated by reference herein in its mus, mycophemolate mofetil, corticosteroids and mesenchy entirety. mal stem cells. The immunosuppressants may be dosed at 0226 Exemplary compositions of the present disclosure least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg/kg. When immuno may be formulation Suitable for use in treating a human Suppressants are used, they may be administered systemically patient, such as pyrogen-free or essentially pyrogen-free, and or locally, and they may be administered prior to, concomi pathogen-free. When administered, the pharmaceutical tantly with, or following administration of the CEC. Immu preparations for use in this disclosure may be in a pyrogen nosuppressive therapy may continue for weeks, months, free, pathogen-free, physiologically acceptable form. years, or indefinitely following administration of cells. For 0227. In certain embodiments, the preparation is suitable example, the patient may be administered 5 mg/kg for administration to a human patient, and more preferably cyclosporin for 6 weeks following administration of the CEC. pyrogen free and/or free of non-human animal products. Furthermore, a composition of CEC may comprise an immu 0228. In other embodiments, the preparation is suitable for nosuppressive agent, e.g., any of the foregoing. administration to a non-human veterinarian mammal. Such as 0224. In one aspect, the present disclosure provides thera a dog, cat or horse. peutic methods comprising transplantation of a cultured sheet or monolayer of CEC or precursors thereof into the eye of a DEFINITIONS Subject in need thereof, e.g., an individual Suffering from a 0229. Unless defined otherwise, all technical and scien disease of corneal endothelial cells. For example, the eye of tific terms used herein have the same meaning as those com the subject may be prepared by removal of the Descemet's monly understood by one of ordinary skill in the art to which membrane, and said cultured sheet or monolayer of CEC may this invention belongs. Although methods and materials simi be placed into the anterior chamber of said eye, e.g., in contact lar or equivalent to those described herein can be used in the with (and preferably attached or affixed to) the posterior invention or testing of the present invention, Suitable methods corneal stroma. Optionally, the sheet or monolayer of CEC or and materials are described below. The materials, methods precursors thereof may be provided on a carrier, e.g., as and examples are illustrative only, and are not intended to be described above, and administered to an eye of a patient. limiting. 0225. One exemplary treatment which may be clinically 0230. In order to further define the invention, the follow preferred when only the corneal endothelium is compromised ing terms and definitions are provided herein. is Descemet's stripping with endothelial keratoplasty 0231. As used in the description herein and throughout the (DSEK), which includes the removal of diseased Descemet's claims that follow, the meaning of “a,” “an and “the membrane and the corneal endothelium, and Subsequent includes plural reference unless the context clearly dictates transplantation of donor tissue. Procedures have been devel otherwise. Also, as used in the description herein, the mean oped to replace the entire cornea (penetrating keratoplasty or ing of “in” includes “in” and “on” unless the context clearly PK) or leave the patient’s Descemet's membrane and endot dictates otherwise. helium and replace the remaining layers with donated tissue 0232 Throughout this specification, the word “comprise' (lamellar keratoplasty). See, generally, U.S. Pat. No. 5,755, or variations such as “comprises” or “comprising will be 785, U.S. Pat. No. 5,649,944, U.S. Pat. No. 7,147,648, U.S. understood to imply the inclusion of a stated integer or groups Pat. No. 7,300,653, U.S. Pat. No. 5,584.881, U.S. Pat. No. of integers but not the exclusion of any other integer or group 5,686,414, U.S. Pat. No. 7,300,654, U.S. patent application of integers. Ser. No. 10/525,391, each of which is incorporated by refer 0233 “Agonist” (such as “DKK2 agonist,” “PDGFBago ence in its entirety. Additional methods of corneal endothelial nist,” “SB431542 agonist.” “Noggin agonist, etc.) as used Surgical replacement are under development, including herein, refers to the named agent and any others that may Descemet's Membrane Endothelial Keratoplasty (DMEK), function in the place thereof. In the context of cell differen in which the donor tissue consists only of Descemet's mem tiation, an agonist of a given factor may be recognized by brane and corneal endothelium. Another potentially promis similar differentiation result (such as formation of NCSCs or ing therapeutic avenue is corneal endothelial reconstruction, CEC) being obtained in the presence of the agonist and the in which corneal endothelial cells are cultured in vitro prior to absence (or decreased concentration or duration of exposure) transplantation. For example, donated human corneal cells of said agent. An agonist may also be recognized, for were cultured on a polymer, released onto a bioadhesive example, by its having a similar effect on a process affected gelatin disc, and then Successfully integrated into denuded by the Subject agent (e.g., similar degree of activation or rabbit corneas, with the gelatin disc dissolving after trans inhibition of the Wnt pathway). plantation (Hsiue et al., Transplantation. 2006 Feb. 15: 81 (3): 0234 “Effective amount, as used herein, refers broadly to 473-6, which is incorporated by reference herein in its the amount of a compound or cells that, when administered to entirety). However, methods utilizing culture cells presup a patient for treating a disease, is sufficient to effect Such pose a source of said cells, and thus are affected by the treatment for the disease. The effective amount may be an shortage of suitable donated tissues as described above. Addi amount effective for prophylaxis, and/or an amount effective tionally, due to differences among donated cells, it may prove for prevention. The effective amount may be an amount effec difficult to produce corneal endothelial cell cultures of con tive to reduce, an amount effective to prevent the incidence of sistent quality and efficacy. Regulatory hurdles may also signs/symptoms, to reduce the severity of the incidence of make Such methods logistically difficult to perform on a large signs/symptoms, to eliminate the incidence of signs/symp scale, due to the possibility that extensive testing for safety toms, to slow the development of the incidence of signs/ and/or efficacy may be required for the cells obtained from symptoms, to prevent the development of the incidence of US 2014/0370.007 A1 Dec. 18, 2014

signs/symptoms, and/or effect prophylaxis of the incidence of 0237 "Embryonic stem cells' (ES cells or ESC) encom signs/symptoms. The “effective amount may vary depend passes pluripotent cells produced from embryonic cells (such ing on the disease and its severity and the age, weight, medical as from cultured inner cell mass cells or cultured blastomeres) history, Susceptibility, and preexisting conditions, of the as well as induced pluripotent cells (further described below). patient to be treated. The term “effective amount” is synony ES cells typically include at least mammalian ES cells, such mous with “therapeutically effective amount for purposes of as human ES cells (“hES cells” or “hESC) as well as murine, this invention. primate, non-human primate, bovine, porcine, etc. Fre 0235 “Pluripotent cells” and “pluripotent stem cells” as quently such cells are or have been serially passaged as cell used herein, refers broadly to a cell capable of prolonged or lines. Embryonic stem cells may be used as a pluripotent stem virtually indefinite proliferation in vitro while retaining their cell in the processes of producing hemangioblasts as undifferentiated state, exhibiting a stable (preferably normal) described herein. For example, ES cells may be produced by karyotype, and having the capacity to differentiate into all methods known in the art including derivation from an three germ layers (i.e., ectoderm, mesoderm and endoderm) embryo produced by any method (including by sexual or under the appropriate conditions. Typically pluripotent cells asexual means) such as fertilization of an egg cell with sperm (a) are capable of inducing teratomas when transplanted in or sperm DNA, nuclear transfer (including somatic cell immunodeficient (SCID) mice; (b) are capable of differenti nuclear transfer), or parthenogenesis. As a further example, ating to cell types of all three germ layers (e.g., ectodermal, embryonic stem cells also include cells produced by Somatic mesodermal, and endodermal cell types); and (c) express at cell nuclear transfer, even when non-embryonic cells are used least one hES cell marker (such as Oct-4, alkaline phos in the process. For example, ES cells may be derived from the phatase, SSEA 3 surface antigen, SSEA 4 surface antigen, ICM of blastocyst stage embryos, as well as embryonic stem NANOG, TRA 160, TRA 181, SOX2, REX1). Exemplary cells derived from one or more blastomeres. Such embryonic pluripotent cells may express Oct-4, alkaline phosphatase, stem cells can be generated from embryonic material pro SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 160, duced by fertilization or by asexual means, including Somatic and/or TRA 1 81. Additional exemplary pluripotent cells cell nuclear transfer (SCNT), parthenogenesis, and androgen include but are not limited to embryonic stem cells, induced esis. As further discussed above (see “pluripotent cells), ES pluripotent cells (iPS) cells, embryo-derived cells, pluripo cells may be genetically modified or otherwise modified to tent cells produced from embryonic germ (EG) cells (e.g., by increase longevity, potency, homing, or to deliver a desired culturing in the presence of FGF-2, LIF and SCF), partheno factor in cells that are differentiated from such pluripotent genetic ES cells, ES cells produced from cultured inner cell cells (for example, MSCs, and hemangioblasts). mass cells, ES cells produced from a blastomere, and ES cells 0238 ES cells may be generated with homozygosity or produced by nuclear transfer (e.g., a Somatic cell nucleus hemizygosity in one or more HLA genes, e.g., through transferred into a recipient oocyte). Exemplary pluripotent genetic manipulation, screening for spontaneous loss of het cells may be produced without destruction of an embryo. For erozygosity, etc. ES cells may be genetically modified or example, induced pluripotent cells may be produced from otherwise modified to increase longevity, potency, homing, or cells obtained without embryo destruction. As a further to deliver a desired factor in cells that are differentiated from example, pluripotent cells may be produced from a biopsied Such pluripotent cells (for example, MSCs and hemangio blastomere (which can be accomplished without harm to the blasts). Embryonic stem cells, regardless of their source or the remaining embryo); optionally, the remaining embryo may particular method used to produce them, typically possess be cryopreserved, cultured, and/or implanted into a Suitable one or more of the following attributes: (i) the ability to host. Pluripotent cells (from whatever source) may be geneti differentiate into cells of all three germ layers, (ii) expression cally modified or otherwise modified to increase longevity, of at least Oct-4 and alkaline phosphatase, and (iii) the ability potency, homing, or to deliver a desired factor in cells that are to produce teratomas when transplanted into immunocom differentiated from such pluripotent cells (for example, promised animals. Embryonic stem cells that may be used in MSCs, and hemangioblasts). As non-limiting examples embodiments of the present invention include, but are not thereof, the pluripotent cells may be genetically modified to limited to, human ES cells (“ESC” or “hES cells”) such as express Sirt1 (thereby increasing longevity), express one or MA01, MAO9, ACT-4, No. 3, H1, H7, H9, H14 and ACT30 more telomerase Subunit genes optionally under the control embryonic stem cells. Additional exemplary cell lines include of an inducible or repressible promoter, incorporate a fluo NED1, NED2, NED3, NED4, NED5, and NED7. See also rescent label, incorporate iron oxide particles or other Such NIH Human Embryonic Stem Cell Registry. An exemplary reagent (which could be used for cell tracking via in vivo human embryonic stem cell line that may be used is MA09 imaging, MRI, etc., see Thu et al., Nat Med. 2012 Feb. 26: cells. The isolation and preparation of MAO9 cells was pre 18(3):463-7), express bFGF which may improve longevity viously described in Klimanskaya, et al. (2006) “Human (see Go et al., J. Biochem. 142, 741-748 (2007)), express Embryonic Stem Cell lines Derived from Single Blas CXCR4 for homing (see Shietal. Haematologica. 2007 July; tomeres.” Nature 444: 481-485. The human ES cells used in 92(7):897-904), express recombinant TRAIL to induce accordance with exemplary embodiments of the present caspase-mediatedX apoptosis in cancer cells like Gliomas invention may be derived and maintained in accordance with (see Sasportas et al., Proc Natl AcadSci USA. 2009 Mar. 24: GMP Standards. 106(12):4822-7), etc. 0239 Exemplary hES cell markers include but are not 0236 “Embryo” or "embryonic,” as used herein refers limited to: Such as alkaline phosphatase, Oct-4, Nanog, broadly to a developing cell mass that has not implanted into Stage-specific embryonic antigen-3 (SSEA-3), Stage-spe the uterine membrane of a maternal host. An "embryonic cific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, cell' is a cell isolated from or contained in an embryo. This TRA-2-49/6E, Sox2, growth and differentiation factor 3 also includes blastomeres, obtained as early as the two-cell (GDF3), reduced expression 1 (REX1), fibroblast growth stage, and aggregated blastomeres. factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), US 2014/0370.007 A1 Dec. 18, 2014

developmental pluripotency-associated 2 (DPPA2), DPPA4, Sancho-Martinez et al., Journal of Molecular Cell Biology telomerase reverse transcriptase (hTERT), SALL4, E-CAD (2011) 1-3: Anokye-Danso et al., Cell StemCell 8,376-388, HERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), Apr. 8, 2011: Orkin and Hochedlinger, Cell 145,835-850, GCTM-2, Genesis, Germ cell nuclear factor, and Stem cell Jun. 10, 2011, each of which is incorporated by reference factor (SCF or c-Kit ligand). As an addition example, embry herein in its entirety). Reprogramming factors may be pro onic stem cells may express Oct-4, alkaline phosphatase, vided from an exogenous source, e.g., by being added to the SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 160, culture media, and may be introduced into cells by methods and/or TRA 1 81. known in the art Such as through coupling to cell entry pep 0240. The ESCs may be initially co-cultivated with tides, protein or nucleic acid transfection agents, lipofection, murine embryonic feeder cells (MEF) cells. The MEF cells electroporation, biolistic particle delivery system (gene gun), may be mitotically inactivated by exposure to mitomycin C microinjection, and the like. iPS cells can be generated using prior to seeding ESCs in co culture, and thus the MEF's do not fetal, postnatal, newborn, juvenile, or adult somatic cells. In propagate in culture. Additionally, ESC cell cultures may be certain embodiments, factors that can be used to reprogram examined microscopically and colonies containing non ESC Somatic cells to pluripotent stem cells include, for example, a cell morphology may be picked and discarded, e.g., using a combination of Oct4 (sometimes referred to as Oct 3/4), stem cell cutting tool, by laser ablation, or other means. Sox2, c-Myc, and Klf4. In other embodiments, factors that Typically, after the point of harvest of the ESCs for seeding can be used to reprogram Somatic cells to pluripotent stem for embryoid body formation no additional MEF cells are cells include, for example, a combination of Oct-4, SoX2, used. Nanog, and Lin28. In other embodiments, Somatic cells are 0241 Exemplary ESC cell markers may also include, but reprogrammed by expressing at least 2 reprogramming fac are not limited to: alkaline phosphatase, Oct-4, Nanog, Stage tors, at least three reprogramming factors, or four reprogram specific embryonic antigen-3 (SSEA-3), Stage-specific ming factors. In other embodiments, additional reprogram embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, ming factors are identified and used alone or in combination TRA-2-49/6E, Sox2, growth and differentiation factor 3 with one or more known reprogramming factors to reprogram (GDF3), reduced expression 1 (REX 1), fibroblast growth a somatic cell to a pluripotent stem cell. iPS cells typically can factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), be identified by expression of the same markers as embryonic developmental pluripotency-associated 2 (DPPA2), DPPA4, stem cells, though a particular iPS cell line may vary in its telomerase reverse transcriptase (hTERT), SALL4, E-CAD expression profile. HERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), 0244. The induced pluripotent stem cell may be produced GCTM-2, Genesis, Germ cell nuclear factor, and Stem cell by expressing or inducing the expression of one or more factor (SCF or c-Kit ligand). reprogramming factors in a Somatic cell. The Somatic cell is a 0242 “Induced pluripotent stem cells” or “iPS cells” fibroblast, such as a dermal fibroblast, synovial fibroblast, or refers to a further exemplary type of pluripotent stem cells lung fibroblast, or a non-fibroblastic somatic cell. The generated by reprogramming a Somatic cell by expressing or Somatic cell is reprogrammed by expressing at least 1, 2, 3, 4, inducing expression of a combination of factors (“reprogram 5 reprogramming factors. The reprogramming factors may be ming factors'). iPS cells may be generated using cells from a selected from Oct 3/4, Sox2, NANOG, Lin28, c Myc, and variety of sources such as fetal, postnatal, newborn, juvenile, Klf4. Expression of the reprogramming factors may be or adult somatic cells. iPS cells may be obtained from a cell induced by contacting the Somatic cells with at least one bank. Alternatively, iPS cells may be newly generated (e.g., agent, Such as a small organic molecule agents, that induce by processes known in the art) prior to commencing differ expression of reprogramming factors. entiation to CEC, NCSC, or cells or another cell type. The 0245. The Somatic cell may also be reprogrammed using a making of iPS cells may be an initial step in the production of combinatorial approach wherein the reprogramming factor is differentiated cells. iPS cells may be specifically generated expressed (e.g., using a viral vector, plasmid, and the like) and using material from a particular patient or matched donor the expression of the reprogramming factor is induced (e.g., with the goal of generating tissue-matched cells. iPS cells can using a small organic molecule.) For example, reprogram be produced from cells that are not substantially immuno ming factors may be expressed in the Somatic cell by infection genic in an intended recipient, e.g., produced from autolo using a viral vector. Such as a retroviral vector or a lentiviral gous cells or from cells histocompatible to an intended recipi vector. Also, reprogramming factors may be expressed in the ent. As further discussed above (see “pluripotent cells”). Somatic cell using a non-integrative vector, such as an episo pluripotent cells including iPS cells may be genetically modi mal plasmid. See, e.g., Yu et al., Science. 2009 May 8: 324 fied or otherwise modified to increase longevity, potency, (5928):797-801, which is hereby incorporated by reference in homing, or to deliver a desired factor in cells that are differ its entirety. When reprogramming factors are expressed using entiated from such pluripotent cells (for example, MSCs and non-integrative vectors, the factors may be expressed in the hemangioblasts). cells using electroporation, transfection, or transformation of 0243 As a further example, induced pluripotent stem cells the Somatic cells with the vectors. For example, in mouse may be generated by reprogramming a Somatic or other cell cells, expression of four factors (Oct3/4, Sox2, c myc, and by contacting the cell with one or more reprogramming fac Klf4) using integrative viral vectors is Sufficient to reprogram tors. For example, the reprogramming factor(s) may be a Somatic cell. In human cells, expression of four factors expressed by the cell, e.g., from an exogenous nucleic acid (Oct3/4, Sox2, NANOG, and Lin28) using integrative viral added to the cell, or from an endogenous gene in response to vectors is sufficient to reprogram a Somatic cell. a factor such as a small molecule, microRNA, or the like that 0246. Once the reprogramming factors are expressed in promotes or induces expression of that gene (see Suh and the cells, the cells may be cultured. Over time, cells with ES Blelloch, Development 138, 1653-1661 (2011); Miyosh et characteristics appear in the culture dish. The cells may be al., Cell Stem Cell (2011), doi:10.1016/j.stem.2011.05.001; chosen and subcultured based on, for example, ES morphol US 2014/0370.007 A1 Dec. 18, 2014

ogy, or based on expression of a selectable or detectable (Current Protocols, 1988); Ausubel et al., Short Protocols in marker. The cells may be cultured to produce a culture of cells Molecular Biology (Current Protocols: 5th Edition, 2002); that resemble ES cells—these are putative iPS cells. iPS cells Sambrook et al., Molecular Cloning: A Laboratory Manual typically can be identified by expression of the same markers (Cold Spring Harbor Laboratory Press, 3rd edition, 2001); as other embryonic stem cells, though a particular iPS cell Sambrook et al., The Condensed Protocols from Molecular line may vary in its expression profile. Exemplary iPS cells Cloning: A Laboratory Manual (Cold Spring Harbor Labora may express Oct-4, alkaline phosphatase, SSEA 3 surface tory Press, 2006), each of which is incorporated by reference antigen, SSEA4 surface antigen, TRA 160, and/or TRA 181. herein in its entirety. 0247 To confirm the pluripotency of the iPS cells, the cells (0250 “Corneal endothelial cells” or “CEC refers gener may be tested in one or more assays of pluripotency. For ally to the mitochondria-rich cells that (in a living organism) example, the cells may be tested for expression of ES cell line the posterior surface of the cornea and faces the anterior markers; the cells may be evaluated for ability to produce chamber of the eye. CEC may also be produced from another teratomas when transplanted into SCID mice; the cells may cell type, e.g., by differentiation of neural crest stem cells or be evaluated for ability to differentiate to produce cell types of ES cells, using the methods described herein. CEC differen all three germ layers. Once a pluripotent iPS cell is obtained tiated from NCSCs or ES cells may be identified or recog it may be used to produce hemangioblast and MSC cells. nized by their exhibition of one or more of the attributes of 0248 “Embryo-derived cells” (EDC), as used herein, endogenous CEC, such as expression of CEC markers, ability refers broadly to pluripotent morula-derived cells, blastocyst to form a monolayer of uniformly sized cells with a predomi derived cells including those of the inner cell mass, embry nantly hexagonal shape, ability to form a "leaky pump' which onic shield, or epiblast, or other pluripotent stem cells of the allows leakage of Solutes and nutrients from the aqueous early embryo, including primitive endoderm, ectoderm, and humor to the more superficial layers of the cornea while at the mesoderm and their derivatives. “EDC also including blas same time actively pumping water in the opposite direction, tomeres and cell masses from aggregated single blastomeres from the stroma to the aqueous. Exemplary CEC markers or embryos from varying stages of development, but excludes includebutare not limited to: Na+/K+ ATPase, ZO-1, KLF13, human embryonic stem cells that have been passaged as cell AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2 lines. SCL4A2, SCL16A1, CA12, CA4, FoxC1). For example, 0249. As used herein, the term “marker or “cell marker CEC typically express Collagen VIII, Na+K+-ATPase pump, refers to a gene (e.g., as an RNA) or protein whose presence and ZO-1, and do not express VWF and CD31 (the latter being identifies a particular cell or cell type. A marker for a cell may present in vascular endothelial cells). In addition CEC may not be limited to one marker, markers may refer to a “pattern express one or more corneal endothelial pump markers of markers such that a designated group of markers may (which include: AQP1, CA2, CA4, CA12, SCL14A2, identity a cell or cell type from another cell or cell type, e.g., SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, a pattern including expression of Some markers and absence voltage-dependent anion channels VDAC2 and VDAC3, or low expression of other markers indicative of other cell chloride channel proteins CLCN2 and CLC), periocular neu types. For example, a population of CEC may be positive for ral crest markers (which include: PITX2, and FOXC1), and/ markers of CEC and negative for markers indicative of other or cell adhesion and matrix proteins (which include: Occlu cell types, such as absence of markers that are expressed on din, Connexin 43, 9.3E antigen, Collagen III, Collagen IV, N other endothelial cell types, absence of markers expressed by cadherin, VE cadherin, E cadherin, beta catenin, p120, p.190 hES cells, and/or absence of markers expressed by neural Laminin alpha 4, Nidogen-2, and Netrin 4). For example, crest stem cells. Additionally, when marker expression is CEC may express at least one corneal endothelial pump detected by cell staining methods (e.g., immunofluorescence marker, at least one periocular neural crest marker, and at least and the like) a cell may be identified as positive for a particu one cell adhesion and matrix protein. lar marker given an expected Staining pattern, Such as tight 0251 “Passaging refers generally to the removal of cells junction localization of the marker ZO-1. Expression of the from a culture substrate, followed by plating of the cells to markers may be detected by any method known in the art, grow on a culture Substrate. Optionally passaging may effect including but not limited to: Western Blotting, mRNA ampli a change in culture density, e.g., an increase or decrease the fication-based methods (e.g., PCR, isothermal amplification, number of cells per unit area. In exemplary embodiments the etc., which may include reverse transcription and may be cells may be passaged 1:1, i.e., removed from and replated on applied to detect expression from single cells or multiple culture Substrates having equal or approximately equal cul cells), Northern blotting, immunostaining, etc. Additionally, ture area per cell. The cells may also be passaged at a different expression of said markers may be inferred by expression of ratio, e.g., 1:2 (i.e., having twice the culture areaper cell), 1:3. a reporter construct (such as a fluorescent protein whose 1:4, etc., or 2:1 (i.e., having half the culture area per cell), 3:1 expression may be visually detected, an antibiotic resistance (i.e., having three times the culture area per cell), or higher or gene whose expression may be detected by cell survival in the lower ratios or non-whole number ratios. Passaging may be presence of the antibiotic, etc.) under the control of a genetic effected using a variety of different methods to remove the element that confers cell type specific expression, such as the cells from the culture substrate. Exemplary methods include promoter of one of the foregoing markers or a fragment use of chemical agents, enzymatic agents, and/or mechanical thereof. Exemplary reporter constructs from the literature is steps, e.g., use of ethylenediaminetetraacetic acid solution the pCCT4-GFP and pCCT4-LUC genes which drive expres (EDTA), Cell Dissociation buffer (Invitrogen catil 13151 sion of GFP and luciferase, respectively, in ES cells, expres 014) Gentle Cell Dissociation Buffer (Stemcell Technologies sion of either of which is readily detectable using conven cat #07174), Enzyme Free Cell Dissociation Solution PBS tional methodologies. Further methods of detecting marker Based (Millipore catfiS-014-C), Cell Dissociation Solution expression that may be used are known in the art. See, gen Non-enzymatic (Sigma catfiC5789), Cellstripper (Cellgro erally, Ausubel, Current Protocols in Molecular Biology catfi25-056-CI), mechanical scraping, optical tweezers, laser US 2014/0370.007 A1 Dec. 18, 2014

catapulting, tituration, circulation of a fluid (e.g., by orbital SMAD inhibitors (such as SB431542 and/or Noggin); see shaking or rocking), trypsin, Accutase, a collagenase such as Menendez et al., PNAS Nov. 29, 2011 vol. 108 no. 48 19240 Collagenase B. Combinations may be used, e.g., one or more 19245. For example, efficient induction of NCSCs was chemical or enzymatic agent in combination with tituration, reported after contacting hESCs with SB431542, and BIO rocking, shaking, or other mechanical dissociation. A pre (with or without Noggin), or with Wnt3a and SB431542. ferred exemplary cell dissociation buffers is EDTA 0.02% in NCSCs may also be obtainable from cultures of neural DPBS (0.5 mM) (Sigma CathE8008-100ML), Cell Dissocia rosettes, for example by culturing hES cells on MS5 stromal tion buffer (Invitrogen catii 13151-014) and Gentle Cell Dis feeder cells (see Lee, et al., Stem Cells 25 (8), 1931-1393 sociation Buffer (Stemcell Technologies cat #07174). Addi (2007), which is incorporated by reference herein in its tional exemplary cell dissociation buffers that may be used entirety. NCSCs are also obtainable from numerous tissues, include: Enzyme Free Cell Dissociation Solution PBS Based including in developing embryos, in the neural tube, Sciatic (Millipore cathS-014-C), Cell Dissociation Solution Non nerve, gut, and dorsal root ganglia; and in the juvenile and enzymatic (Sigma cathic5789), Cellstripper (Cellgro cati 25 adult, in the dorsal root ganglia, bone marrow, skin, heart, 056-CI). cornea, teeth, and caratoid body. See Nagoshi et al., Journal of 0252. As used herein, the term “anaplastic lymphoma Cellular Biochemistry 107:1046-1052 (2009); Crane and kinase' refers to family of membrane-associated membrane Trainor, Annu. Rev. Cell Dev. Biol. 2006. 22:267-86; and associated tyrosine kinase receptors that includes ALK4 (an Blum, Brain Research Bulletin 83 (2010) 189-193, each of activin receptor), ALK5 (a TGF-31 receptor), ALK7 (a Nodal which is incorporated by reference herein in its entirety. and Nodal-related protein receptor). 0257 Neural crest stem cells may be identified by expres 0253 “Signs of disease, as used herein, refers broadly to sion of markers identified herein and known in the art. Exem any abnormality indicative of disease, discoverable on exami plary neural crest stem cell markers include but are not lim nation of the patient; an objective indication of disease, in ited to: Sox10, AP2, HNK1, Pax3, PAX7, and p75 (NGFR), as contrast to a symptom, which is a subjective indication of well as low or absent Pax6 expression. disease. 0258 “Disease of corneal endothelial cells” or “diseases 0254 “Symptoms” of disease as used herein, refers of corneal endothelial cells includes any disease or condition broadly to any morbid phenomenon or departure from the amenable to treatment by administration of CEC, including normal instructure, function, or sensation, experienced by the diseases in which a subject's CEC decrease in numbers or die, patient and indicative of disease. decrease in density, or otherwise become dysfunctional. Pri 0255 “Therapy,”“therapeutic.”“treating,” or “treatment', mary diseases that affect the corneal endothelium include as used herein, refers broadly to treating a disease, arresting or Fuchs dystrophy, iridocorneal endothelial syndrome, poste reducing the development of the disease or condition or its rior polymorphous dystrophy, and congenital hereditary clinical symptoms, and/or relieving the disease or condition, endothelial dystrophy. Secondary diseases or conditions for causing regression of the disease or condition or its clinical which an effective treatment may include replacement of the symptoms. Therapy encompasses prophylaxis, prevention, corneal endothelium include corneal dystrophies, contact treatment, cure, remedy, reduction, alleviation, and/or pro lens usage, cataract Surgery, and late endothelial failure in viding relief from a disease, signs, and/or symptoms of a cornea transplantation. Diseases of corneal endothelial cells disease. Therapy encompasses an alleviation of signs and/or additionally include any injury to the cornea, e.g., caused by symptoms in patients with ongoing disease signs and/or chemical irritation, injury due to contact lens use, reaction or symptoms Therapy also encompasses prophylaxis and pre sensitivity (e.g., to contact lens solutions, cosmetics, eye vention. Prophylaxis includes preventing disease occurring drops, medications, Smoke, etc.), scratches, scrapes, abra Subsequent to treatment of a disease in a patient or reducing sions, bruising, a foreign object in the eye (e.g., sand or dust), the incidence or severity of the disease in a patient. The term or exposure to ultraviolet light (from e.g., Sunlight, Sun lamps, “reduced, for purpose of therapy, refers broadly to the clini Snow reflections, water reflections, or arc-welding or other cal significant reduction in signs and/or symptoms. Therapy exposure). includes treating relapses or recurrent signs and/or symp 0259 “Transdifferentiation' In the present disclosure, toms. Therapy encompasses but is not limited to precluding transdifferentiation refers to conversion of one differentiated the appearance of signs and/or symptoms anytime as well as cell type to another desired cell type. An example of transdif reducing existing signs and/or symptoms and eliminating ferentiation is the changes in a differentiated cell (e.g., human existing signs and/or symptoms. Therapy includes treating Somatic cell in tissue culture), that result upon introduction of chronic disease (“maintenance') and acute disease. For one or more transcription factors, growth factors, microR example, treatment includes treating or preventing relapses or NAS, Small molecules, and/or a component of a cell of a the recurrence of signs and/or symptoms. Exemplary treat different cell type than said differentiated cell (e.g., cyto ments that may be efficacious for diseases of corneal endot plasm, nucleoplasm, whole cell extract, or a fraction or com helial cells are described in the section “Therapeutic Meth ponent thereof). ods. Supra. 0260 “ROCK inhibitors' refer to any substance that 0256 “Neural crest stem cells” or “NCSCs' generally inhibits or reduces the function of Rho-associated kinase or refer to a neural progenitor cell having the developmental its signaling pathway in a cell. Such as a small molecule, an potential to produce pigmented cells co-expressing the mel siRNA, a miRNA, an antisense RNA, or the like. “ROCK anosome marker, HMB45. Neural crest stem cells may be signaling pathway,” as used herein, may include any signal differentiated from hES cells, e.g., using dual SMAD inhibi processors involved in the ROCK-related signaling pathway, tors as described herein or as described in WO/2010/096496. such as the Rho-ROCK-Myosin II signaling pathway, its Neural crest stem cells may be differentiated from hES cells upstream signaling pathway, or its downstream signaling using a combination of Wnt agonists (such as e.g., Wnt3a pathway in a cell. An exemplary ROCK inhibitor that may be and/or (2Z.3E)-6-bromoindirubin-3'-oxime (BIO)) and used is Stemgent's Stemolecule Y-27632, a rho-associated US 2014/0370.007 A1 Dec. 18, 2014

protein kinase (ROCK) inhibitor (see Watanabe et al., Nat Example 1 Biotechnol. 2007 June; 25(6):681-6) Other ROCK inhibitors include, e.g., H-1152, Y-301.41, Wf-536, HA-1077, hydroxyl Derivation of Corneal Endothelial Cells from Human HA-1077, GSK269962A and SB-772077-B. Doe et al., J. Embryonic Stem Cells (without Passaging) Pharmacol. Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. 0263 Step 1. Human embryonic stem cells (hESC) were Pharmacol., 57:976-983, 2000; Nakajima et al., Cancer cultured in the absence offeeder cells (specifically, on Matri Chemother. Pharmacol., 52:319-324, 2003; and Sasaki et al., gel with mTESR1 media). hESC were passaged as clumps Pharmacol. Ther., 93:225-232, 2002, each of which is incor (using EDTA or dispase). hESC passaged with dispase were porated herein by reference as if set forth in its entirety. routinely split at a ratio of 1:6 to 1:10 every 5-7 day when ROCK inhibitors may be utilized with concentrations and/or large colonies were beginning to touch or about 75% conflu culture conditions as known in the art, for example as ency. Typical colony size is significantly larger than with described in US PGPub No. 2012/0276063 which is hereby EDTA method, with the colony number around 150 per well incorporated by reference in its entirety. Additional examples in a 6 well culture dish. Passaging was essentially as outlined of the Rho-associated kinase inhibitors include compounds by manufacturer (Stem Cell Technologies). hESC passaged disclosed in the following references: U.S. Pat. No. 4,678, with EDTA were routinely passaged at a ratio of 1:8-1: 12 every 3-5 days when the colonies are beginning to touch or 783, U.S. Pat. No. 3,421,217, WO99/20620, WO99/61403, about 75% confluency. Colony size was significantly smaller WO02076976, WO02/076977, WO02/100833, WO03/ than the Dispase method, with the colony number around 300 059913, WO03/062227, WO2004/009555, WO2004/ per well in a 6 well culture dish. 022541, WO2004/108724, WO2005/003101, WO2005/ 0264 mTeSR1 media was prepared as directed by the 039564, WO2005/034.866, WO2005/037197, WO2005/ manufacture, optionally with the addition of antibiotics (e.g., 037198, WO2005/035501, WO2005/035503, WO2005/ pen-strep at 1x concentration). It is expected that other cul 035506, WO2005/080394, WO2005/103050, WO2006/ ture conditions could also be used (which may or may not 057270, WO2007/026664 and the like. Such compounds can include use of murine embryonic fibroblasts (MEFs) or other be produced according to the method described in each of the feeder cells), however, it may be preferred to avoid feeder respective references. Specific examples include 1-(5-iso cells (particularly Xenogeneic feeder cells) when the resulting quinolinesulfonyl)homopiperazine (fasudil), (+)-trans-4-(1- cells are intended for clinical use. It is expected that other aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane (Y-27632) passaging methodologies could be used (e.g., manually, or and the like, as well as salts thereof, preferably pharmaceu using other enzymes Such as trypsin, optionally in combina tically acceptable salts such as hydrochloride salts. In exem tion with an inhibitor of Rho-associated kinase (ROCK), such plary embodiments, the ROCK inhibitor may have a concen as Y-27632 (Watanabe et al., Nat Biotechnol. 2007 June; tration of about 0.05 to about 50 microM, for example, at least 25(6):681-6)). or about 0.05, 0.1, 0.2,0.5,0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, 0265 Step 2. Cells were grown to approximately the den 25, 30, 35, 40, 45, or 50 microM, including any range deriv sity achieved 1-2 days before they would be ready to be split able therein, or any concentration effective for promoting cell (timing was determined during routine passaging as growth or Survival. described in step 1). 0266 Step 3. Day 0: Medium was changed to dual SMAD medium (see formulation below) containing the dual SMAD EXAMPLES inhibitors, Noggin (500 ng/mL human recombinant Noggin, obtained from Peprotech (cat #120-10C)) and SB431542 (10 0261 Examples 1 and 2 illustrate directed differentiation micromolar SB431542, obtained from Tocris (catil 1614) or of corneal endothelial cells from embryonic stem cells. Cor Stemgent (catio4-0010)). As discussed above, Noggin is neal endothelial cells were produced from feeder-free cul thought to bind BMP2, BMP4, and BMP7, and SB431542 is tures of embryonic stem cells, without the need for embryoid thought to block phosphorylation of ACTRIB, TGFBR1, and bodies, neural rosettes, or stromal inducer cells. The resulting ACTRIC receptors, and it is envisioned that one or more of corneal endothelial cells are expected to be particularly suit the alternative factors discussed above may be used in place able for clinical use because Xenogeneic cells were not used in of or in addition to Noggin, SB431542, or both. Optionally, their derivation. formation of neural crest may be monitored, e.g., by detecting the level of mRNA for neural crest markers such as Sox10 and 0262 Morphologically, the resulting cells exhibited the NGFR over time. Elevated expression of these markers was hexagonal or polygonal shape and tight adherence to one detected about one day after commencing exposure to the another that are characteristic of naturally occurring human dual SMAD inhibitors. In addition, the corneal endothelium corneal endothelial cells. Further, analysis by qPCR and marker ColVIII was detected to be upregulated, while expres immunostaining revealed that these cells express markers sion of Nanog (a marker of pluripotency) was decreased (see indicative of corneal endothelial cells including the Na+K+ FIG.9). ATPase pump, ZO-1, and KLF13. Further, these cells were 0267 Step 4. Day 2: The culture medium was changed to distinguished from vascular endothelial cells (whose marker Cornea medium, which included PDGFB and DKK2 (see expression has some overlap with corneal endothelial cells) formulation below). At this point the cells exhausted the by the lack of expression of the vascular endothelial cell culture medium quickly, and freshCornea medium was added markers v WF and CD31 (assayed by qPCR and immun daily. This was apparently due to high metabolic activity of ostaining), which are typical vascular endothelium proteins, the cells, which is an expected CEC phenotype and provides but are not expressed in human corneal endothelial cells. further confirmation of the cells identity. It is envisioned that Based on our observations, we concluded that the resulting after some number of days in culture (e.g., after 2, 3, 4, 5, or hESC-derived cells were corneal endothelial cells. 6 days, or up to one or two weeks) the cells could be main US 2014/0370.007 A1 Dec. 18, 2014 20 tained in culture in the absence of DKK2, which may option described in WO/1998/030679; see Amit et al., Devel ally be gradually decreased in concentration and then omitted opmental Biology 227, 271-278 (2000)). from the culture medium. 0279 1% Non-essential Amino Acids Invitrogen 0268. It is envisioned that alternatives to PDGF and/or cati 11140-050 DKK2, including those further described above, could be 0280 1 mM L-glutamine Invitrogen cath250300-81 used instead of, or in addition to, those respective factors. 0281 0.1 mM b-mercaptoethanol Sigma cat #M7522 Other components of the media may also include: Angiopoi 0282 6 ng/ml human recombinant FGF2, Invitrogen etin like protein 7, Transforming growth factor 32, Hepato catii 13256-O29 cyte growth factor, Keratinocyte growth factor, and Interleu (0283 10,000 units of penicillin (base) and 10,000 ug of kin 1C. (again, it is envisioned that potential alternatives, streptomycin Invitrogen catil 1514.0122 including those identified above, may be used instead of or in 0284. Dual Smad Medium addition to one or both of these factors). Though this step was 0285) 80% DMEM-F12 Invitrogen cat #11330-032 performed at day 2, additional time exposure of cells to the 0286 20%. Knock Out Serum Replacer Invitrogen dual SMAD inhibitors did not significantly change the cati 10828-O28 expression of the neural crest genes, NGFR and Sox. 10 (FIG. 0287 1% Non-essential Amino Acids Invitrogen 8); therefore, it is expected that exposure of the cells to the cati 11140-050 dual SMAD inhibitors for longer or shorter durations (e.g., 0288) 1 mM L-glutamine Invitrogen cath250300-81 between 1-4 days, up to 6 days, or longer) would likely be 0289 0.1 mM b-mercaptoethanol Sigma cat #M7522 acceptable. 0290 6 ng/ml human recombinant FGF2, Invitrogen 0269 Step 5. Cultures become confluent. At the edges of catii 13256-O29 the colonies, the morphology is altered, cells become larger 0291 10,000 units of penicillin (base) and 10,000 ug of and shape is more polygonal or hexagonal. streptomycin Invitrogen catil 1514.0122 0270 Step 6. By Day 7: Hexagonal cells can be seen at the 0292 500 ng/ml human recombinant Noggin Peprotech contact points of the colonies as well as circular colonies of cati 120-10C hexagonal cells Scattered throughout the dish. 0293 10 micromolar SB431542 Tocris cati 1614 or Stemgent cath()4-0010 (0271 Step 7. At day 9, the cultures expressed mRNA for 0294 Cornea Media (Modified WiMedium Containing collagen 8 (Col8al gene), a major component of Descemet's PDGFB, DKK2, B27) membrane, (FIG. 7A), Na+K+-ATPase pump (ATPa1 gene), a 0295) 80% DMEM-F12 Invitrogen cat #11330-032 major component of endothelium pump function (FIG. 4), as 0296 20% Knock Out Serum Replacer Invitrogen well as the following potential components of the endothe cati 10828-O28 lium pump function: Carbonic anhydrase II (CA2), Anion 0297 1% Non-essential Amino Acids exchanger 2 (AE2/SCL4A2), Solute Carrier Family 16 A1 0298] 1 mM L-glutamine (see recipe) Invitrogen (SCL16A1), SCL16A3, Carbonic anhydrase 12 (CA 12), cati250300-81 Carbonic Anhydrase 4 (CA4), cystic fibrosis transmembrane 0299 0.1 mM beta-mercaptoethanol Sigma cat conductance regulator (CFTR). The cells also expressed the HM7522 transcription factors FoxC1 and Pitx2 which are expressed by 0300 6 ng/ml human recombinant FGF2, Invitrogen ocular neural crest during development and mutations in catii 13256-O29 which can result in corneal abnormalities (FIG. 10). By 0301 10,000 units of penicillin (base) and 10,000 ug of immunostaining, the cells were ZO-1 (FIG. 3) and Na+K+ streptomycin Invitrogen catil 1514.0122 ATPase (FIG. 4) positive and expressed at the cell junction. 0302) 10 ng/ml human recombinant PDGFB, Peprotech The cells were negative for vascular endothelium cell mark cathi AF-100-14B ers, von Wildebrand factor (VWF) and CD31/PECAM-1 (FIG. 6). 0303 10 ng/ml mouse recombinant DKK2 R&D Sys 0272 Step 8. At Day 11 and Day 17, cells surrounding the tems cat #2435-DK-010 (or human recombinant DKK2) hexagonal colonies are optionally removed (e.g., manually or 0304 0.1xB27 Invitrogen cat #17504-044 by laser ablation) to increase room for hexagonal colonies to Example 2 expand. 0273 Step 9. At Day 19, hexagonal cell colonies were Derivation of Corneal Endothelial Cells from Human dissected in solid sheets and transferred to Matrigel coated Embryonic Stem Cells (Replating Method) glass coverslips for immunostaining and grown to expand colonies. 0305 Steps 1 to 4 were performed as in Example 1, above: 0274 Step 10. At Day 25, hexagonal cell colonies were it is expected that the alternative methodologies, factors, and collected for analysis, e.g., immunostaining or detection of timing discussed therein would be similarly suitable for use expressed mRNAs. In addition to the markers described with this method. above, cells expressed KLF13 mRNA and protein (a tran (0306 Step 5. At Day 3, cells were passaged 1:1. Preferred scription factor expressed in corneal endothelial cells, FIG. cell dissociation buffers include ethylenediaminetetraacetic acid solution (EDTA) 0.02% in DPBS (0.5 mM) (Sigma 5). CathE8008-100ML), Cell Dissociation buffer (Invitrogen 0275 Media Formulations Used in this Example are as cati13151-014) and Gentle Cell Dissociation Buffer (Stem Follows: cell Technologies cat #07174). Additional exemplary cell 0276 Modified WiMedium dissociation buffers that may be used include: Enzyme Free (0277 80% DMEM-F12 Invitrogen cat #11330-032 Cell Dissociation Solution PBS Based (Millipore catfiS-014 (0278. 20% Knock Out Serum Replacer Invitrogen C), Cell Dissociation Solution Non-enzymatic (Sigma cati 10828-028 (the composition of which is reportedly cathic5789), Cellstripper (Cellgro cathi 25-056-CI). US 2014/0370.007 A1 Dec. 18, 2014 21

0307 Passaging was effected using EDTA as follows: 1 ml Example 3 of buffer was added and the plate was left undisturbed for 9-12 minutes. Cells were checked for signs of detachment by Cultured CEC Produce a Matrix Similar to light microscopy. Cell dissociation buffer was then aspirated. Descemet's Membrane Cornea cell medium was then added in a dropwise fashion to gently lift the cells from the plate. No agitation was needed to 0312 This example describes production of a gel-like remove cells. The dissociated cells were then transferred to a matrix from media conditioned by a culture of hES-derived 15 ml falcon tube. Remaining cells in the dish were rinsed CEC. These results indicate that the CEC secrete one or more with additional corneal cell medium and added to the 15 ml factors that can form a gel, which Applicants hypothesize falcon tube. Cells are then appropriated diluted at a range of include components of Descemet's membrane (which is pro 1:1-1:5 and transferred to new wells of Matrigel coated duced by CEC in vivo). Particularly, as shown above, the CEC plates. strongly upregulate expression of ColVIII (a component of 0308 Though this step was performed two days after addi Descemet's membrane), Suggesting that secreted ColVIII is a tion of Dual SMAD inhibitors and one day after addition of component of the observed matrix. Western blotting was per PDGFB and DKK2, it is expected that the timing of this step formed to confirm that the matrix indeed contained COL8A1 could be altered. Some cells remained adherent and produced and COL8A2 proteins. Moreover, a matrix produced from corneal endothelial cells with comparable efficiency as in factors secreted by the CEC may be used as a CEC culture Example 1 (when further cultured as from steps 5 onward of Substrate and/or as a carrier to be used during cell transplan Example 1). The removed cells, however, were plated (in the tation. same medium as in Step 4) and formed corneal endothelial 0313 Corneal endothelial cells were produced from hES cells more quickly and uniformly than the cultures of cells as described in the preceding examples and cultured. Example 1: properties and characterization of these cells are Conditioned cell medium was removed from corneal endot the Subject of the remaining steps. helial cultures after at least one day of culture from two wells 0309 Step 6. At Day 8, cultures contained large numbers in a 6-well plate. The combined volume was 8 mLs. The of hexagonal cells with Smaller patches of putative progenitor conditioned cell medium was centrifuged to remove cell cells (distinguished based on non-polygonal morphology) debris and other large fragments. Medium was centrifuged interspersed. for 5 min at 1000xg and 10°C. to remove cell debris. Super 0310 Step 7. At Day 10, the plate contained mostly hex natant was added to an Amicon Ultra 15 with a 10,000 Da agonal cells with very small ball-like colonies of putative molecular weight cut-off (MWCO). The medium was con progenitor cells. Cell expressed the same markers as centrated in this device by centrifugation in a Beckman described above in Example 1 (including CA2, SCL4A2, Coulter Allegra X-15R with a swinging bucket rotor (two SLC16A1, SLC16A3, CA12, CA4, and CFTR). Expression spins at 4000xg for 15 min at 4°C.), and the volume was of additional pump mRNAs were also detected: Aquaporin 1 reduced from 8 mLs to -500 uL. For SDS-PAGE analysis, 32 (AQP1), Sodium Hydrogen exchanger (NHE1) and Sodium LL of the concentrated medium was removed and added to an Bicarbonate transporter (NBC-1) (Table 1, FIG. 7B, C). SDS-containing solution with a final concentration of 30 mM 0311. The resulting hESC-derived CEC were then evalu Tris-HCl, 1% SDS, and 10% glycerol. Upon heating this ated by qPCR for expression of pump genes indicative of solution to 95°C. for 2 min, the solution appeared brown in CEC identity. The cultures were positive for expression of all color and had polymerized into a gel-like semi-solid. 12 pump genes tested. In initial experiments the expression of 0314. It is thought that the SDS solution was not necessary only 10 out of the 12 pump genes was detected, however, use for polymerization; rather, concentrated media may option of different PCR primers permitted detection of expression of ally be polymerized with or without additional components the remaining two pump genes. Such as the cornea media, a basal culture medium, additional TABLE 1 hESC derived corneal endothelial cells expressed 12 out of 12 corneal pump markers (detected by qPCR). Expression levels are denoted “high, “medium. or “low” based on the Ct value (the cycle number at which abundance of the RT-PCR product exceeded a threshold level that is within the exponential phase of amplification; lower values indicate greater abundance). Relative Expression levels Pump markers CtWalue High AQP1 26 CA2 29 SCL4A2 29 SLC16A1 26 SLC16A3 28 Medium ADCY1O 33 CA12 33 CA4 32 CFTR 34 NBC1 33 NHE1 31 SCL16A7 32 US 2014/0370.007 A1 Dec. 18, 2014 22 growth factors or other matrix proteins, etc. Moreover, it is Accutase, Collagenase B, Collagenase B in combination with expected that similar results may be achieved using other cell dissociation buffer, or cell dissociation buffer alone. Cells concentrators (e.g., 10K Spin Column, BioVision; Vivaspin, were replated for one week and then analyzed microscopi Sartorius Stedim; and 10K Spin Column, MBL International, cally for morphology and yield (indicated by whether conflu etc.) or other concentration method (e.g., pressure ultrafiltra ent cultures were Subsequently obtained). Additionally, tion, evaporation, dialysis against glycerol, and others). Incu expression of the markers COL8A1 and AQP1 was deter bation time and temperature may be varied to permit the gel to mined by qPCR. Results are summarized in Table 2. TABLE 2 Influence of dissociation methods on CEC yield, morphology, and marker expression. CEC were produced as described in Example 2 and harvested using the method indicated in each row. Under “Morphology, '+' indicates polygonal or hexagonal cell shape, '-' indicates fibroblastic morphology, and +/- indicates a mixture of polygonal and fibroblastic morphology. “Confluent indicates that the culture exhibited an uninterrupted solid sheet of cells ("+"), or did not (-) after one week in culture Subsequent to plating. The columns COL8A1 and AQP1 indicate the level of mRNA expression of these respective genes as detected by QPCR relative to PD1T cells; "+" indicates comparable levels and "-j- indicates reduced mRNA levels. Dissociation Method Morphology Confluent COL8A1 AQP1

Manual -- +f- 0.05% Trypsin ------+f- 0.25% Trypsin 5 min ------0.25% Trypsin 10 min ------0.25% Trypsin: Cell Diss. Buffer 1:1 ------Accutase -- -- Collagenase Bo?n -- +f- Collagenase B 1 hr + Cell Diss. +f------Buffer Cell Diss. Buffer +f- -- -- form, for example attemperatures ranging from 37°C. to 95° 0318 AQP1 and COL8A1 mRNA levels were also tested C. and durations between 0.5 minutes to overnight. After by qPCR (FIG. 14A-C). AQP1 and COL8A1 levels were polymerization, liquid may be removed. CEC may be cul elevated in each instance relative to hESC. However, com tured on the resulting matrix, e.g., seeded directly on the pared to cells which had not been harvested (produced as in matrix, which (prior to, during, or Subsequent to polymeriza Example 2), all harvesting methods resulted in decreased tion) may be supplemented with DKK2, PDGFB, and/or expression levels of AQP1 and increased expression levels of other factors that contribute to growth. COL8A1 0315. The protein constituents of the matrix were further 0319 Based on these results, use of trypsin (alone or in confirmed by Western blotting to detect the presence of combination with cell dissociation buffer) can produce high COL8A1 and COL8A2 proteins. Cells were removed and quality CEC potentially suitable for transplantation. Other lysed from the subcellular ECM using Ammonium Hydrox methodologies may potentially affect cell morphology after ide as described previously (Sugino et al., 2010). This one week of culturing, however, this may or may not allowed removal of the CEC without damaging the extracel adversely affect cell function or suitability for transplanta lular matrix that was secreted below the cells. After exposure tion. to the ammonium hydroxide, the plate was washed three 0320 Methods times with PBS to dislodge any remaining cells. Reducing 0321 CEC were derived as described in Example 2 and sample buffer was then added to collect the extracellular then harvested under different experimental conditions fur matrix protein. Standard SDS PAGE and Western blotting ther described below. All enzymatic harvesting methods was performed using antibodies to COL8A1 (FIG. 19A) and included a rinse with Phosphate buffered saline (Life Tech COL8A2 (FIG. 19B), and both proteins were detected, indi nologies cati 14190-250) before enzymatic digestion. Cells cating that these proteins were secreted by the hESC-derived were then harvested by the following methods and then plated CEC to form an extracellular-like matrix. onto a Matrigel plate and cultured for an additional week. Cells were analyzed for their morphology: polygonal, a mix Example 4 ture of polygonal and fibroblastic, and fibroblastic. The cells were analyzed to see if they were able to return to 100% Influence of Harvest Methods on CECYield, confluency after harvesting and replating. After the week of Morphology, and Marker Expression culture, the RNA was collected and analyzed for the expres 0316. This example tests the effect of various harvest sion of COL8A1 and AQP1 by QPCR. methods on CEC morphology, yield, and marker expression, 0322 For “Manual dissociation, a fine glass dissecting which are potentially indicative of the suitability of the cells tool was made from a Pasteur pipet under a flame. Under a for transplantation. dissecting scope and sterile hood, sections of corneal endot 0317 CEC were produced as described in Example 2 and helial cells were gently peeled from the plate. Cells were then harvested using each of the methods enumerated in Table 2, transferred to a new plate to test for viability after removal. i.e., manually, using trypsin (with differing time and concen 0323 For “0.05% Trypsin' dissociation, 0.05% Trypsin tration), trypsin in combination with cell dissociation buffer, EDTA (1x), Phenol Red Life technologies cat #25300-054 US 2014/0370.007 A1 Dec. 18, 2014

was used. Cells were treated with 0.05% trypsin at 37 degrees 0331 Methods C for 10 minutes. Cells were then triturated with the trypsin 0332 hESC corneal endothelial cells were grown on solution to remove cells from well. The enzymatic digestion matrigel coated coverslips for 1 week. Immunostaining was was stopped with the addition of cornea media. Cells were performed as previously described with an antibody to ZO-1. then centrifuged to pellet cells, resuspended, and plated onto After immunostaining, nuclei were detected by exposing the a fresh matrigel plate. coverslips to DAPI. ZO-1 staining was used to identify cor 0324 For “0.25% Trypsin' dissociation, 0.25% Trypsin neal endothelial cells, and pictures of the DAPI staining for EDTA (1x), Phenol Red Life technologies cat #252.00056 the same field were taken and counted. Cell number was was used. Cells were treated with 0.25% trypsin at 37 degrees C for 5 or 10 minutes, and treated as described above for manually counted in the program Image J 1.45s (NIH). The 0.05% Trypsin. area of the 40x field was calculated by measuring the size of 0325 For “0.25% Trypsin: Cell dissociation buffer 1:1 a 0.1 mm reference and the area of the 40x field of view was 0.25% Trypsin-EDTA (1x), Phenol Red Life technologies cat calculated to by 0.0767 mm. Cell counts were divided by #252.00056 and Cell Dissociation Buffer, enzyme-free, PBS 0.0767 mm to obtain the number of cells/mm. Life technologies cat #13151-014 were used. Cells were Example 6 treated with 1:1 dilution of 0.25% trypsin and Cell dissocia tion buffer at 37 degrees C for 15 minutes, and treated as hESC-Derived CEC Gene Expression and described above for 0.05% Trypsin. Morphology Over Prolonged Culture 0326 For “Accutase' treatment, ACCUTASETM Cell 0333. This example further characterizes the morphology Detachment Solution, Stem cell technologies cat #07920 was and gene expression of CEC cultures produced from hESC as used. Cells were treated with Accutase at 37 degrees C for 25 described in Example 2. minutes, and treated as described above for 0.05% Trypsin. 0327. For “Collagenase B' treatment, cells were treated 0334 Cells morphology was monitored over 4 weeks after with collagenase B (2 mg/ml) at 37 degrees C for overnight, differentiation (FIG. 11). Hexagonal cell shape continued to and treated as described above for 0.05% Trypsin. develop and improve over the four week course, with a high 0328. For “Collagenase B+Cell dissociation buffer” cells degree of uniformity of cell shape being present at week 4. were treated with collagenase B (1 mg/ml) at 37 overnight C 0335| Additionally, CEC cultures were stained for expres for 1 hr. The collagenase B was then replaced with Cell sion of the CEC protein markers ZO-1 and AQP1 at each dissociation buffer (see above) at 37 degrees C for 10 minutes week between weeks 1 and 4. As expected, ZO-1 expression and treated as described above for 0.05% Trypsin. was detected at the tightjunctions between CEC (FIG. 12). In 0329. For “Cell Dissociation Buffer, Cell Dissociation parallel with the adoption of highly uniform hexagonal mor Buffer, enzyme-free, PBS Life technologies cat #13151-014 phology observable in FIG. 11, the tight junctions became was used. Cells were treated with cell dissociation buffer at 37 more uniform in appearance over the four weeks depicted. degrees C for 1 hr., and treated as described above for 0.05% AQP1 expression was detected at the cell borders as expected Trypsin. (FIG. 13) at each time point. Example 5 0336 CEC cultures were also tested for the level of AQP1 and COL8A1 mRNA expression at each week between weeks CEC Cell Density 1 and 4. Both COL8A1 (FIG. 15A) and AQP1 (FIG. 15B) 0330. This example quantifies the density of CEC cultures were highly expressed at all time points (normalized to produced from hESC as described in Example 2. CEC were hESC). AQP1 expression increased monotonically over the stained with ZO-1 to confirm identity and manually counted four week course of the experiment and paralleled the time in random fields. From two separate experiments, three ran course of adoption of hexagonal morphology as illustrated in dom 40x fields were counted. The density of the cultured FIG. 11. COL8A1 was highly expressed at all time-points corneal endothelial cells was 7605+379 cells/mm. tested. The temporary increase in COL8A1 expression (meant-SEM). This is higher than the reported CEC cell den detected at week 2 coincided with the transfer to a new tissue sity even for infants (i.e., 5624 cells/mm) and well in excess culture well and is thought to reflect the production of extra of the low density levels associated with dysfunction (e.g., cellular matrix. Many pumps mRNA increased expression lower than about 500 to 1000 cells/mm). See Peh et al., relative to hESC. However, the expression of some pumps Transplantation. 2011 Apr. 27; 91 (8):811-9). These results remained similar to hESC, indicating that these pumps may indicate that the Subject methods can be used to produce be expressed in hESC and/or may be subject to post-transcrip sheets of cells having a density well above the level associated tional regulation. Table 3 summarizes the CEC pump expres with in vivo corneal function. sion results. TABLE 3 Summary of Cornea gene expression levels detected by qPCR. The three groups of genes indicate increased pump expression versus hESC (Pumps Upregulated'), pumps expressed at similar levels in hESC and CEC (“Pumps Present), and expression of Collagen8 genes. High (24-28), medium (29-33), and low (34-36) are indicative of raw Ctvalues, with lower the Ctvalue reflecting a greater amount of mRNA. Pumps present in CEC and upregulated relative to hESC

Gene High Medium Low

AQP1 -- -- CA2 -- CA4 -- US 2014/0370.007 A1 Dec. 18, 2014 24

TABLE 3-continued Summary of Cornea gene expression levels detected by qPCR. The three groups of genes indicate increased pump expression versus hESC (Pumps Upregulated'), pumps expressed at similar levels in hESC and CEC (“Pumps Present), and expression of Collagen8 genes. High (24-28), medium (29-33), and low (34-36) are indicative of raw Ctvalues, with lower the Ctvalue reflecting a greater amount of mRNA.

SLC16A3 -- CFTR -- SLC16A7 -- SLC4A4 -- Pumps present in CEC and hESC

Gene High Medium Low

AQP1 -- -- CA2 -- CA4 -- SLC16A3 -- CFTR -- SLC16A7 -- SLC4A4 -- Collagen 8 Genes

Gene High Medium Low

COL8A1 -- COL8A2 --

Example 7 centrations up to 20 micromolar (FIG.17A-D). Expression of COL8A1 and SLC16A3 were elevated relative to hESC (FIG. 17E-F). Effect of ROCK Inhibitor 0338. The harvested cells were further examined for mor phology, ability to reestablished a confluent culture, and 0337 This example demonstrates that addition of a rho retaining expression of the CEC markers COL8A1, AQP1, associated protein kinase (“ROCK) inhibitor can improve and SLC16A by qPCR. The results demonstrate that treat post-harvest plating cell quality. hESC-derived CEC were ment with ROCK inhibitor can help maintain cell morphol harvested with or without the addition of a ROCK inhibitor ogy, particularly when ROCK inhibitor is present when the and/or ASC2P vitamin C isoform (ASC) and examined for cells are replated after harvesting. Moreover, Maintenance morphology and gene expression after plating. A dose-re with media containing ROCK inhibitor can help the cells sponsive improvement in cell uniformity and polygonal/hex maintain a high quality CEC morphology after harvesting and agonal morphology was observed for ROCK inhibitor con replating. TABLE 4

Rock inhibitor can improve cell Survival after harvesting. The ROCK inhibitor Y-27632 and/or ASC2P vitamin C isoform (ASC) was added to hESC derived CEC as indicated under “Treatment, and harvested cells were evaluated based on their morphology and gene expression after replating. “O/N confluent indicates the confluence of cultured cells after overnight plating, reflecting the degree of cell Survival and/or attachment. Morphology indicates the appearance of the cultured cells after 1 week in culture which was recorded as a high quality polygonal hexagonal cell shape (++), a polygonal/hexagonal shape ("+"), or fibroblastic morphology ("-). COL8A, AQP1, SLC16A3 indicate expression ("+") or non-expression (“- ) of the respective mRNAs, as detected by QPCR.

ON Treatment Confluent Morphology COL8A1 AQP1 SLC16A3

Rock Pretreat ------Rock + ASC Pretreat ------Rock on ------Rock + ASC on ------Rock 1 microM (maint.) ------Rock 5 microM (maint.) ------Rock 10 microM (maint.) ------US 2014/0370.007 A1 Dec. 18, 2014 25

TABLE 4-continued Rock inhibitor can improve cell Survival after harvesting. The ROCK inhibitor Y-27632 and/or ASC2P vitamin C isoform (ASC) was added to hESC derived CEC as indicated under “Treatment, and harvested cells were evaluated based on their morphology and gene expression after replating. “O/N confluent indicates the confluence of cultured cells after overnight plating, reflecting the degree of cell Survival and/or attachment. Morphology indicates the appearance of the cultured cells after 1 week in culture which was recorded as a high quality polygonal hexagonal cell shape (++), a polygonal/hexagonal shape ("+"), or fibroblastic morphology (-). COL8A, AQP1, SLC16A3 indicate expression ("+") or non-expression (“-”) of the respective mRNAs, as detected by QPCR. ON Treatment Confluent Morphology COL8A1 SLC16A3

Rock 20 microM (maint.) ------Rock 10 microM+ASC ------(maint.) Rock 20 microM+ASC ------(maint.) Rock, Rock + ASC (D1) -- ND ND ND

ND = not determined. Abbreviations: of n = overnight, ASC = ASC2P vitamin C isoform added to culture at 0.3 mM, maint, = maintaining (i.e., the indicated factors were included in culture media on each day after plating), D1 = indicated factors were included in the culture media for the first week after plating and were thereafter omitted.

0339 Methods described above, and cultured in presence of Rock inhibitor 0340 Rock inhibitor Y-27362 (Wako, cat #253-00513) for 1 week. Cornea cells were then grown for additional week was dissolved in distilled H20 to make a 10 mM stock solu in the presence or absence of Rock inhibitor, to determine tion and sterile filtered and aliquoted. Rock inhibitor aliquot whether the ROCK inhibitor contributed to the maintenance was thawed and added fresh to media daily. of their morphology. 0341 Cornea cells were grown as described in Example 2 and harvested by treatment with 0.25% Trypsin as described Example 8 in Example 4 and replated, with variations (i.e., inclusion of ROCK inhibitor and/or ASC as described below). hESC-Derived CEC Maintain Structural Integrity 0342. After plating, cells were viewed to identify any mor when Harvested in Sheets phological changes, and specifically whether they regained 0348. To obtain and test sheets of CEC for potential trans their polygonal/hexagonal shape, were a mixed population of plantation, hESC-derived CEC were produced as described in polygonal and fibroblastic shape, or if they had turned fibro Example 2 and cultured on UPCELL tissue culture plates. blastic. These culture plates include a temperature sensitive cell cul 0343 Media was changed daily with fresh Rock inhibitor ture Surface that can release cells as sheet of cells in response except for “Pretreatment” and “D1' paradigms. After 1 week to a temperature shift. of culture, RNA was collected and analyzed by QPCR for (0349. After release from the UPCELL plates, CEC were changes in COL8A1, AQP1, and SLC16A3 mRNA levels. stained for ZO-1 to test physical integrity of the cell sheet, The efficacy of the Rock inhibitor for harvesting cells were particularly whether tight junctions were maintained. As tested between 1-20 microM. shown in FIG. 18, ZO-1 expression indicated that tight junc 0344) For the “Pretreatment paradigm, CEC were pro tions were maintained after removal of cell sheet. These duced as described in Example 2. The cornea media was results indicate that the CEC can maintain structural integrity removed and replaced with either 10 microM Rock or 10 when removed as sheets, suggesting that CEC function can be microM Rock with 0.3 mM ASC-P2 (Lascorbic acid phos maintained for transplantation of sheets of cells. phate magnesium salt n-hydrate (Wako, catio13-19641)) for 0350 Methods 1 hour before harvesting. Cells were harvested using 0.25% 0351 UPCELL tissue culture plates contain a temperature trypsin as described above, however, no Rock inhibitor was sensitive cell culture surface that release cells as sheet of cells added during harvesting. without requiring enzymatic treatment. The manufacturer 0345 For the “Rock overnight paradigm, CEC were pro Suggests optimizing the amount of matrix to coat plates and duced as described in Example 2, harvested with 0.25% coating at 37 degrees C even if normally done at room tem trypsin as described above, and then plated overnight in the perature. All solutions were equilibrated at 37 degrees C prior presence of 10 microM Rock inhibitor. The following morn to contact with the plates to prevent premature cell detach ing, the media was removed, and no further Rock inhibitor ment. was added to the media. 0352 UPCELL tissue culture dishes (10 cm; (Nunc cat 0346 For the “Rock maintenance' paradigm, CEC were #14902) were coated with 2.5x-5x the amount of Matrigel produced as described in Example 2, harvested using 0.25% recommended by the manufacturer of Matrigel (BD, cat trypsin as described above, and were maintained in Rock 354277) at 37 degrees C for at least one hour. inhibitor added daily to fresh media. 0353 Cornea cells were produced as described in (0347 For the “D1' paradigm, CEC were produced as Example 2 and harvested using 0.25% trypsin for 10 minutes. described in Example 2, harvested using 0.25% trypsin as Cells were then triturated with the trypsin solution to remove US 2014/0370.007 A1 Dec. 18, 2014 26 cells from well. The enzymatic digestion was stopped with 0357. It was predicted that the hESC CEC would be more the addition of cornea media. Cells were then centrifuged to youthful than the primary adult CEC, as reflected by indica pellet cells and resuspended. tions of decreased level of accumulated oxidative stress and/ 0354) Matrigel was quickly removed and cells were plated or DNA damage, as well as increased ability to recapture at the normal density in the presence of 10 microM Rock glutathione compared to CEC isolated from a donor corneal inhibitor Y-27362 (Wako, cat #253-00513). For all media endothelium. Genes exhibiting more than a 3-fold difference changes, media was pre-warmed to 37 degrees C. Cells were expression between hESC-derived CEC and donated CECare grown up to two weeks. To detach the cells, incubation at listed in FIG. 20. Among these genes are genes indicative of room temperature for 30 minutes (in accord with the manu a more youthful phenotype of the hESC-derived CEC as facturer's directions) was insufficient to achieve full release. further described below. However, incubation for 1 hour at 4 degrees C was sufficient 0358. The ability to repair DNA damage decreases with to detach cells as a sheet. After detachment, cells were stained age in older corneas. Indications of this reduced ability can be for ZO-1 and microscopically examined. seen in lower levels of DNA repair pathway molecules such as Example 9 ATM and OGG1. ATM and OGG1 proteins levels are 1.5 and 1.2 fold lower in older adult corneas (Joyce et al., Invest Ophthalmol Vis Sci. 2011 Mar. 1; 52(3):1641-9). The Characterization of Global Gene Expression by CEC microarray revealed results similar to youthful corneas, with and Detection of a “Youthful” Cellular Phenotype an approximate 1.9 and 2.8 fold decrease for ATM and OGG1 0355 This example describes comparison of global gene (for adult CEC compared to hESC-derived CEC), respec expression by hESC-derived CEC to donated CEC. The high tively. degree of shared gene expression further confirm the identity of the hESC-derived cells as CEC. Further, the differences in 0359 A further gene, the enzyme gamma-glutamyl gene expression indicate that the hESC-derived CEC exhibit transpeptidase (GGT), is an ectoenzyme important to main gene expression characteristic of more “youthful” cells, Sug taining corneal dehydration as well recapturing the anti-oxi gesting that the cells may be even more efficacious for trans dant glutathione. Redmond et al. (Cornea. 2012 Sep. 27. plantation than donor-derived tissues. Epub ahead of print. doi: 10.1097AICO. 0356. When comparing hESC CEC to adult primary cor Ob013e3182656881) reported that old corneas exhibit 1.7 neal endothelial cells (donated cells), only 792/28374 genes fold less GGTactivity than younger corneas. Consistent with were changed more than 3 fold, indicating the cell types this literature report for youthful CEC, the microarray express 97% of all genes at similar levels. This indicates the revealed that GGT7 was downregulated 1.8 fold compared to hESC derived corneal endothelial cells are highly similar to hESC CEC. Adult corneal endothelial cells. Additionally, we found that 0360 Yet another gene shown in the literature to be the following corneal endothelial genes were expressed by reduced in older corneas, BMI1 polycomb ring finger onco both populations including but not limited to: ADCY10, gene (BMI1), specifically by 2.5 fold (Wang et al., Mol Vis. ATP1A1, CA2, CFTR, COL8A2, FOXC1, KLF13, PITX2, 2012:18:803-15). In the microarray, the adult HCEC had 1.6 SLC16A1 SLC16A3, SLC16A7, SLC4A2, SLC9A1, ZO-1. fold less BMI1 compared to hESC CEC. TABLE 5

Genes found by microarray that indicate that hESC CEC are more youthful than Adult primary CEC. A negative value indicates that the expression is lower in the adult CEC compared to the hESC CEC. Literature-reported changes in gene expression for “youthful' CEC as compared to “old” CEC are shown for reference (negative values in this column indicate that expression is lower in the old CEC as compared to young CEC).

Fold Change Literature Adult primary reported Gene CEC vs. hESC expression Symbol Gene name Accession number CEC fold change

ATM ataxia telangiectasia NM 000051 -1.87 -1.5 (Joyce et mutated al., 2011) BMI1 BMI1 polycomb NM OO518O -1.62 -2.5 (Wang et ring finger al., 2012) Oncogene GGT5 gamma- NM OO1099781 -1.86 -1.7 glutamyltransferase (Redmond et 5 al., 2012) OGG1 8-oxoguanine DNA NM OO3656 -2.80 -1.2 (Joyce et glycosylase al., 2011) US 2014/0370.007 A1 Dec. 18, 2014 27

0361 Methods remove any cell clumps. A portion of the unsorted cells were 0362 hESC corneal endothelium was grown in three sepa saved for Flow Cytometry analysis. Cells were subsequently rate experiments as described in Example 2 but with an extra incubated with the CD271 microbeads and then depleted week of growth (PD1T 2 week). Adult Human Corneal using the LD Columns (Militenyi Biotec, #130-042-901). A Endothelium was isolated and expanded in culture as previ portion of the sorted cells were analyzed by Flow Cytometry, ously described (Joyce et al., Molecular Vision 2010; 16:897 with the remainder being immediately plated. There are two 906) and collected at Passage 0. RNA was isolated using RNA controls for the growth of the cells, first untouched hESC easy RNA isolation kit (Qiagen, cati74104) along with CEC were used as well as hESC CEC that were trypsinized DNAase treatment (Qiagen, cati79254) and processed by and replated (Passage 1). manufacturer's directions. RNA samples were further pro 0368 Flow Cytometry analysis was performed comparing cessed for hybridization to Affymetrix Human Exome ST unsorted and Sorted cells. arrays at the University of Miami Genomics Core Facility. 0369 CD271-APC antibody (Militenyi Biotec, cath 130 Raw data were normalized and analyzed using Genespring 091-884) at 1:11 was utilized to detect expression of CD271 GX12 and GeneGo Metacore software applications. After in unsorted and sorted cells. The events of unlabeled cells for normalization, probes were filtered using 2 different criteria: expression values between 20 and 99" percentile and at least unsorted and sorted cells were used to gate at 99.5%. 2 out of the 3 samples in at least one condition passed these criteria. 28374 out of 28869 total probes (98%) were used for Example 11 Subsequent analysis. Genes were compared using an unpaired TTest with Asymptotic p-value comparison. Multiple testing Clinical Testing of CEC correction was done with Benjamini-Hochberg. 0370. The example describes experiments to establish the safety of transplanted hES-derived corneal endothelial cells. Example 10 Clinical grade corneal endothelial (CE) cells are generated at a GMP-compliant clinical production facility. CE cells are Improved CEC Purity. Using Magnetic Bead Subjected to strict validation and quality controls prior to final Subtraction of Other Cell Types release of the CE cell suspension for transplantation. Each lot 0363 This example demonstrates that cell subtraction of CE cells undergoes a battery of quality control safety using microbeads may increase the purity of CEC derived testing including testing for sterility, presence of myco from hESC or NCSC. Magneticbead subtraction was utilized plasma, presence of endotoxins, absence of pluripotent stem to remove non-CEC cells from culture and thereby improve cells, and karyotyping. Identity is confirmed by DNA finger purity of CEC cultures. printing, appropriate endothelial morphology, and marker 0364 Antibody-coupled magnetic beads were used to expression consistent with CE cells. Purity is determined by preferentially capture non-CEC from the culture (see, e.g., immunohistochemical staining for the acceptable levels and Peh et al., Int J Biomater. 2012: 2012:601302). Specifically, distribution of CE-specific proteins including Na+K+-ATPase microbeads coupled to an anti-CD271 antibody were utilized. pump, ZO-1, and KLF13. In addition, each lot is character CEC cultures were produced without passaging (FIG. 21A), ized by qRT-PCR to demonstrate downregulation of hESC with passaging at day 10 (FIG. 21B), or with passaging and markers (OCT-4, NANOG, and Sox-2) and upregulation of removal of CD271 + cells by magnetic bead separation which CE cell specific genes in accordance with validated specifi produced cultures that appeared to be somewhat less uniform cations. in hexagonal/polygonal shape (FIG. 21C). Flow cytometry 0371 Corneal endothelial cells produced according to the confirmed that cells expressing higher levels of CD271 were methods described herein are used in non-human animal successfully removed, with 23% of cells being positive for models to establish potential safety and/or efficacy for human CD271 expression before subtraction (FIG. 21D) but only 2% use. Pharmaceutical compositions of cells are used in non of cells being positive for CD271 after subtraction (FIG. human animal models that assess CEC function. Cells (e.g., 21E). as a sheets or Suspension) may be Surgically administered to 0365. Applicants also observed that some hESC-derived the eye of a non-human animal. For example, CEC composi CEC expressed low levels of CD271 (detected by both flow tions may be used in rabbit models as described in one or cytometry and from the microarray data) and based thereon it more of Honda et al., Arch Ophthalmol. 2009 October; 127 is thought that other markers may be even more effective for (10): 1321-6: Hitani et al., Mol Vis. 2008 Jan. 3; 14:1-9: improving purity. Nonetheless, the results above generally Mimura et al., (Invest Ophthalmol Vis Sci. 2005; 46:3637 validate the use of magnetic bead subtraction with hESC 3644; Hsuie et al., Transplantation 2006; 81: 473–476; Lai et derived CEC and indicate that the method would be similarly al., Transplantation 2007; 84: 1222-1232; Shimmura et al., Br effective in removing cells that express other markers, such as J Ophthalmol 2005: 89:134-137; Chen et al., Molecular pluripotent cell markers (e.g., SSEA-1, TRA-1-60, and/or Vision 2011; 17:2148-2156; and Gospodrowicz et al., Proc. SSEA-4, etc.), epithelial cell markers (e.g., CD326), or Natl. Acad. Sci. USA, Vol. 76, No. 1, pp. 464–468, January NCSC markers, whether individually or in combination. 1979; and/or may be used in rodent (e.g., mouse/rat) models 0366 Methods as described in Hayashi et al., Investigative Ophthalmology & 0367 CD271+(nerve growth factor receptor) cells were Visual Science, July 2009, Vol. 50, No. 7, pg. 3151-3158: depleted from hESC CEC cultures using the CD271 micro Mimura et al., Experimental Eye Research 79 (2004) 231 beads (Militenyi Biotec, catil 130-097-128) according to the 237: Tchah, J Korean MedSci, 1992 December;7(4):337-42: manufacturer's directions. Briefly, hESC CEC were and/or may be used in non-human primate models as trypsinized to remove cells from plate. After centrifugation to described in Koizumi et al., Invest Ophthalmol Vis Sci. 2007; remove trypsin, cells were resuspended and put through the 48:4519-4526); Koizumi et al., Cornea 2008: 27(Suppl. Pre-separation Filter (Militenyi Biotec, Cat 130-041–407) to 1):S48-S55; and/or in a human or non-human as described in US 2014/0370.007 A1 Dec. 18, 2014 28

Peh et al., Transplantation. 2011 Apr. 27; 91 (8):811-9. Each 6. The method of any one of claims 2 to 5, wherein said at foregoing publication is incorporated by reference herein in least one factor that induces differentiation of said neural its entirety. crest cells into CEC comprises at least one DKK2 agonistand 0372 Corneal endothelial cells produced according to the at least one PDGFB agonist. methods described herein are used for patient therapy as 7. The method of any one of claims 2 to 6, wherein said at follows: (i) patients initially receive an immunosuppressive least one DKK2 agonist comprises DKK2 polypeptide. treatment (e.g., Steroids); (ii) patients are optionally assigned 8. The method of any one of claims 2 to 7, wherein said at to a treatment cohort (e.g., four cohorts of three patients least one PDGFBagonist comprises PDGFB polypeptide. each); (iii) escalating doses of cells are administered to the 9. The method of claim 8, wherein said at least one DKK2 cohorts (preferably unilaterally, i.e., to one of each patients agonist comprises DKK2 polypeptide and said at least one eyes). Each patient’s clinical course is monitored post-trans PDGFBagonist comprises PDGFB polypeptide. plant, e.g., over the first 6 weeks post-transplant, and option 10. The method of claim 7 or 9, wherein the concentration ally at further (prior or subsequent) timepoints, preferably for of said DKK2 polypeptide is between 1 ng/ml and 15 lug/ml, at least one year. Primary evaluation of patients includes between 10 ng/ml and 15 lug/ml, between 1 ng/ml and 1 monitoring for adverse events (AE) and dose-limiting toxici ug/ml, between 1 ng/ml and 100 ng/ml, between 2 ng/ml and ties (DTL) including assays for detection of immune-medi 20 ng/ml, between 5 ng/ml and 20 ng/ml, or about 10 ng/ml. ated pathology, teratoma formation, and/or abnormal blood 11. The method of claim 8 or 9, wherein the concentration vessel growth. Patients are additionally assessed for second of said PDGFB polypeptide is between 0.1 ng/ml and 250 ary endpoints including efficacy with regard to intraocular ng/ml, between 0.5 ng/ml and 150 ng/ml, between 1 ng/ml pressure (IOP), visual acuity, and/or endothelial cell count of and 50 ng/ml, between 2 ng/ml and 20 ng/ml, or about 10 the graft. Long term follow-up preferably continues for up to ng/ml. 15 years or more to evaluate long term affects. AS satisfactory 12. The method of any foregoing claim, further comprising safety data are obtained from the initial patient cohorts, uni culturing said CEC on a matrix. lateral or bilateral treatment of additional patients is under 13. The method of claim 12, wherein said matrix is selected taken. Additionally, patients in the initial unilateral cohorts from the group consisting of laminin, fibronectin, vitronec may be offered the opportunity to receive therapy in the tin, proteoglycan, entactin, collagen, collagen I, collagen IV. previously untreated cornea. collagen VIII, heparan Sulfate, Matrigel (a soluble prepara tion from Engelbreth-Holm-Swarm (EHS) mouse sarcoma 1. A method of producing corneal endothelial cells (CEC), cells), a human basement membrane extract, and any combi comprising (a) contacting neural crest stem cells (NCSCs) nation thereof. with at least one factor that induces differentiation of said 14. The method of any one of claims 12 to 13, wherein said NCSCS into CEC. matrix is of human or non-human animal origin. 2. The method of claim 1, wherein said at least one factor 15. The method of any one of claims 12 to 13, wherein said that induces differentiation of said neural crest cells into CEC matrix is of bovine, mouse or rat origin. comprises at least one DKK2 agonist and/or at least one 16. The method of claim 12, wherein said matrix comprises PDGFBagonist. Matrigel (a soluble preparation from Engelbreth-Holm Swarm (EHS) mouse sarcoma cells). 3. The method of claim 2, wherein said at least one DKK2 17. The method of any foregoing claim, wherein, subse agonist includes a factor selected from the group consisting quent to commencement of step (a), said differentiating of LRP5/6 antagonists; Kremen antagonists; Dkk 1: Dkk 3; NCSCs are passaged. Dkk 4: Soggy; secreted frizzled related proteins (Frzb); Wnt 18. The method of claim 17, wherein said passaging is inhibitor factor (WIF); a Wnt modulator; Casein Kinase 1-7 B effected between 1 hour and 5 days, between 2 hours and 4 catenin antagonists; LEF/TCF transcription factor members days, between 3 hours and 3 days, between 4 hours and 2 modulators; IWR: pyrvinium; ICG-001; PKF115-584; IWP: days, or about 1 day Subsequent to commencement of step (a). Ant1.4Br/Ant 1.4Cl; Niclosamide: apicularen; bafilomycin; 19. The method of claim 17, wherein said passaging is XAV939; NSC668036; 2,4-diamino-quinazoline; Quercetin: effected about 3 days Subsequent to commencement of step and any combination thereof. (a). 4. The method of claim 2, wherein said at least one DKK2 20. The method of any one of claims 17 to 19, wherein said agonist includes a factor selected from the group consisting passaging is effected by a method comprising contacting the of Wnt proteins, nucleic acids encoding Wnt proteins, LiCl, cells with a cell dissociation buffer, or by a method compris AXinantagonists; APC antagonists; norrin; R-spondin2; (het ing mechanical dissociation of the cells or a Subset thereof, or ero)arylpyrimidines; IQ1: BIO(6-bromoindirubin-3'-oxime); by a method comprising optical isolation of the cells or a 2-amino-4-3,4-(methylenedioxy)benzyl-amino-6-(3- subset thereof. methoxyphenyl)pyrimidine; a Wnt modulator; WAY-316606: 21. The method of claim 20, wherein said cell dissociation QS11; SB-216763; SB-216763; DCA; and any combination buffer is non-enzymatic. thereof. 22. The method of claim 20, wherein said cell dissociation 5. The method of any one of claims 2 to 4, wherein said at buffer comprises ethylenediaminetetraacetic acid (EDTA). least one PDGFBagonist includes a factor selected from the 23. The method of any foregoing claim, wherein the dura group consisting of: a PDGFRB agonist, a PKC pathway tion of said contacting NCSCs with a factor that induces agonist, PDGFAA polypeptide, a nucleic acid encoding differentiation of said neural crest cells into CEC is at least 2 PDGFAA, PDGFAB polypeptide, a nucleic acid encoding days, between 2 and 25 days, or is between 2 and 10 days. PDGFAB, Phorbol 12-myristate 13-acetate (PMA), VEGF, 24. The method of any foregoing claim, wherein step (a) and any combination thereof. further comprises culturing said NCSCs on a matrix. US 2014/0370.007 A1 Dec. 18, 2014 29

25. The method of claim 24, wherein said matrix is selected somorphin (or Compound C), SIS3 (6,7-Dimethoxy-2-((2E)- from the group consisting of laminin, fibronectin, vitronec 3-(1-methyl-2-phenyl-1H-pyrrolo2,3-bipyridin-3-yl-prop tin, proteoglycan, entactin, collagen, collagen I, collagen IV. 2-enoyl))-1,2,3,4-tetrahydroisoquinoline, Specific Inhibitor collagen VIII, heparan Sulfate, Matrigel (a soluble prepara of Smad3 (SIS3), an inhibitor SMAD, SMAD6, SMAD7, tion from Engelbreth-Holm-Swarm (EHS) mouse sarcoma SMAD10, an antagonist of a receptor SMAD, an antagonist cells), a human basement membrane extract, and any combi of SMAD1, an antagonist of SMAD2, an antagonist of nation thereof. SMAD3, an antagonist of SMAD5, an antagonist of SMAD8/ 26. The method of any one of claims 24 to 25, wherein said 9, or any combination thereof. matrix is of human or non-human animal origin. 44. The method of any one of claims 36 to 43, wherein the 27. The method of any one of claims 24 to 25, wherein said duration of said contacting ES cells with one or more inhibi matrix is of bovine, mouse or rat origin. tors of SMAD protein signaling is at least 2 days, between 1 28. The method of claim 24, wherein said matrix comprises Matrigel (a soluble preparation from Engelbreth-Holm and 10 days, or is between 2 and 6 days. Swarm (EHS) mouse sarcoma cells). 45. The method of claim 44, wherein the duration of said 29. The method of any foregoing claim, wherein said contacting ES cells with one or more inhibitors of SMAD NCSCs are obtained from an animal tissue. protein signaling is between about 1 and 10 days or no more 30. The method of claim 29, wherein said animal tissue is than 10,9,8,7,6, 5, 4, 3, or 2 days. selected from the group consisting of the gut, dorsal root 46. The method of claim 44, wherein the duration of said ganglia, bone marrow, skin, heart, cornea, caratoid body, contacting ES cells with one or more inhibitors of SMAD neural tube, teeth, and Sciatic nerve. protein signaling is between about 1-3 days or about 2 days. 31. The method of claim 29 or 30, wherein said animal 47. The method of any one of claims 36 to 46, wherein said tissue is human tissue. neural crest stem cells are produced from ES cells by a 32. The method of any one of claims 1 to 28, wherein said method further comprising contacting ES cells with at least NCSCs are produced by transdifferentiation of a somatic cell. one Wntagonist. 33. The method of any one of claims 1 to 28, wherein said 48. The method of any one of claims 36 to 46, wherein said NCSCs are obtained from cultures of neural rosettes. neural crest stem cells are produced from ES cells by a 34. The method of any one of claims 1 to 28, wherein said method further comprising contacting ES cells with at least neural crest stem cells are produced from ES cells by a one Wntagonist comprising (2Z.3E)-6-bromoindirubin-3'- method comprising culturing ES cells with MS5 stromal feeder cells. oxime (BIO) and/or Wnt3a. 35. The method of any one of claims 1 to 28, wherein said 49. The method of any one of claims 36 to 46, wherein said NCSCs are produced from ES cells. neural crest stem cells are produced from ES cells by a 36. The method of claim35, wherein said neural crest stem method further comprising contacting ES cells with at least cells are produced from ES cells by a method comprising one Wntagonist selected from the group consisting of Wnt contacting ES cells with one or more inhibitors of SMA/ proteins, nucleic acids encoding Wnt proteins, LiCl, AXin Mothers Against Decapentaplegic (SMAD) protein signal antagonists; APC antagonists; norrin; R-spondin2: (hetero) arylpyrimidines; IQ1; BIO(6-bromoindirubin-3'-oxime); 1ng. 2-amino-4-3,4-(methylenedioxy)benzyl-amino-6-(3- 37. The method of claim 36, wherein ES cells are cultured with said one or more inhibitors of SMAD protein signaling methoxyphenyl)pyrimidine; WAY-316606: QS11; for between about 1 and 10 days or for no more than 10,9,8, SB-216763; SB-216763; DCA; and any combination thereof. 7, 6, 5, 4, 3, or 2 days. 50. The method of claim35, wherein said neural crest stem 38. The method of claim 36, wherein ES cells are cultured cells are produced from ES cells by a method comprising with said one or more inhibitors of SMAD protein signaling contacting ES cells with a first inhibitor of SMAD protein for 1-3 days or for about 2 days. signaling and a second inhibitor of SMAD protein signaling. 39. The method of any one of claims 36 to 38, wherein said 51. The method of claim 50, wherein said first inhibitor of one or more inhibitors of SMAD protein signaling prevent the SMAD protein signaling is selected from the group consist binding of a TGF-B family ligands to its corresponding recep ing of Noggin polypeptide, dorsomorphin, LDN-193189, tOr. and any combination thereof. 40. The method of any one of claims 36 to 39, wherein said 52. The method of any one of claims 50 to 51, wherein said one or more inhibitors of SMAD protein signaling prevent the first inhibitor of SMAD protein comprises Noggin polypep activation of a TGF-B receptor. tide. 41. The method of any one of claims 36 to 40, wherein said one or more inhibitors of SMAD protein signaling inhibits 53. The method of any one of claims 50 to 52, wherein said one or more SMAD intracellular proteins/transcription fac Noggin polypeptide is present in a concentration between 10 tOrS. ng/ml and 5,000 ng/ml, between 100 ng/ml and 700 ng/ml, 42. The method of any one of claims 36 to 41, wherein said between 400 ng/ml and 600 ng/ml, or about 500 ng/ml. one or more inhibitors of SMAD protein signaling comprise 54. The method of claim 50, wherein said first inhibitor of Leukemia Inhibitory Factor (LIF), GSK3 inhibitor (CHIR SMAD protein signaling is selected from the group consist 99021), Compound E(Y secretase inhibitor XXI), SB431542, ing of antagonists of BMP2; antagonists of BMP4; antago or any combination thereof. nists of BMP7; and antagonists of TGFB; 43. The method of any one of claims 36 to 42, wherein said 55. The method of any one of claims 50 to 54, wherein said one or more inhibitors of SMAD protein signaling comprise second inhibitor of SMAD protein signaling comprises an Chordin, Follistatin, dominant negative receptors or blocking inhibitor of an anaplastic lymphoma kinase signaling path antibodies that sequester BMP2, BMP4, and/or BMP7, dor way. US 2014/0370.007 A1 Dec. 18, 2014 30

56. The method of any one of claims 50 to 55, wherein said 71. The method of any one of claims 35 to 70, wherein said second inhibitor of SMAD protein signaling inhibits a signal ES cells are human ES cells. ing pathway selected from the group consisting of Lefty, 72. The method of any one of claims 35 to 71, wherein said Activin, and TGFbeta. ES cells are iPS cells. 57. The method of any one of claims 50 to 56, wherein said 73. The method of any one of claims 35 to 72, wherein said second inhibitor of SMAD protein signaling inhibits both ES cells do not exhibit changes or mutations in genes asso activin and nodal signaling. ciated with a disease of corneal endothelial cells. 58. The method of any one of claims 50 to 56, wherein said 74. The method of any one of claims 35 to 73, wherein said second inhibitor of SMAD protein signaling inhibits the ES cells exhibit a normal karyotype. Lefty/Activin/TGFbeta pathways by blocking phosphoryla 75. The methodofany one of claims 35 to 74, whereinprior tion of the ALK4, ALK5 and ALK7 receptors. to differentiation into NCSCs, said ES cells are maintained in 59. The method of any one of claims 50 to 56, wherein said culture in the absence of feeder cells. second inhibitor of SMAD protein signaling is an ALK4 76. The methodofany one of claims 35 to 75, whereinprior receptor inhibitor. to differentiation into NCSCs said ES cells are cultured on a 60. The method of any one of claims 50 to 59, wherein said matrix. second inhibitor of SMAD protein signaling is selected from 77. The method of claim 76, wherein said matrix is selected the group consisting of 4-4-(1,3-benzodioxol-5-yl)-5-(2- from the group consisting of laminin, fibronectin, vitronec pyridinyl)-1H-imidazol-2-ylbenzamide (SB431542) and tin, proteoglycan, entactin, collagen, collagen I, collagen IV. derivatives thereof. heparan Sulfate, Matrigel (a soluble preparation from Engel breth-Holm-Swarm (EHS) mouse sarcoma cells), a human 61. The method of any one of claims 50 to 56, wherein said basement membrane extract, and any combination thereof. second inhibitor of SMAD protein signaling comprises 78. The method of any one of claims 76 to 77, wherein said SB431542. matrix is of human or non-human animal origin. 62. The method of any one of claims 60 to 61, wherein said 79. The method of any one of claims 76 to 77, wherein said SB431542 is present in a concentration between 10 nM and matrix is of bovine, mouse or rat origin. 100 uM, between 0.1 uMand50 uM, between 0.1 and 20 uM, 80. The method of claim 76, wherein said matrix comprises between 1 and 20 uM, or about 10 uM. Matrigel (a soluble preparation from Engelbreth-Holm 63. The method of any one of claims 50 to 56, wherein said Swarm (EHS) mouse sarcoma cells). second inhibitor of SMAD protein signaling blocks phospho 81. The method of any foregoing claim, wherein said rylation of ACTRIB, TGFBR1, and ACTRIC receptors. NCSCs express one or more markers selected from the group 64. The method of any one of claims 50 to 56, wherein said consisting of Sox10, AP2, HNK1, PAX7, p75 (NGFR), and second inhibitor of SMAD protein signaling inhibits TGFB/ any combination thereof. Activin/Nodal signaling. 82. The method of any foregoing claim, wherein said CEC 65. The method of claim 64, wherein said second inhibitor express one or more markers selected from the group consist of SMAD protein signaling blocks endogenous Activin and ing of Na+/K+ ATPase, ZO-1, KLF13, AQP1, Collagen VIII, BMP signals. SLC16A3, CFTR, NBC1, CA2, AE2/SCL4A2, SCL16A1, 66. The method of claim 50, wherein said first inhibitor of CA12, CA4, FoxC1, and any combination thereof. SMAD protein signaling and/or said second inhibitor of 83. The method of any foregoing claim, wherein said CEC SMAD protein signaling are each selected from the group express the markers CollagenVIII, Na+K+-ATPase pump, and consisting of Chordin, Follistatin, dominant negative recep ZO-1, and do not express the markers VWF and CD31. tors or blocking antibodies that sequester BMP2, BMP4, 84. The method of any foregoing claim, wherein said CEC and/or BMP7, dorsomorphin (or Compound C), SIS3 (6.7- express one or more corneal endothelial pump markers. Dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo2,3- 85. The method of any foregoing claim, wherein said CEC bipyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquino express one or more periocular neural crest markers. line, Specific Inhibitor of Smad3 (SIS3), an inhibitor SMAD, 86. The method of any foregoing claim, wherein said CEC SMAD6, SMAD7, SMAD10, an antagonist of a receptor express one or more cell adhesion and matrix proteins. SMAD, an antagonist of SMAD1, an antagonist of SMAD2, 87. The method of any foregoing claim, wherein said CEC an antagonist of SMAD3, an antagonist of SMAD5, and an express at least one corneal endothelial pump marker, at least antagonist of SMAD8/9. one periocular neural crest marker, and at least one cell adhe 67. The method of claim 50, wherein said first inhibitor of sion and matrix protein. SMAD protein signaling comprises Noggin and said second 88. The method of claim 84 or 87, wherein said one or more inhibitor of SMAD protein signaling comprises SB431542. corneal endothelial pump markers are selected from the 68. The method of any one of claims 50 to 67, wherein the group consisting of AQP1, CA2, CA4, CA12, SCL14A2, duration of said contacting ES cells with said first inhibitor of SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, SMAD protein signaling and said second inhibitor of SMAD voltage-dependent anion channels VDAC2 and VDAC3, protein signaling is at least 2 days, between 1 and 10 days, or chloride channel proteins CLCN2 and CLC. is between 2 and 6 days. 89. The method of claim 85 or 87, wherein said periocular 69. The method of claim 68, wherein ES cells are cultured neural crest markers are selected from the group consisting with said one or more inhibitors of SMAD protein signaling of PITX2, and FOXC1. for between about 1 and 10 days or for no more than 10,9,8, 90. The method of claim 86 or 87, wherein said cell adhe 7, 6, 5, 4, 3, or 2 days. sion and matrix proteins are selected from the group consist 70. The method of claim 68, wherein ES cells are cultured ing of Occludin, Connexin 43, 9.3E antigen, Collagen III, with said one or more inhibitors of SMAD protein signaling Collagen IV, N cadherin, VE cadherin, E cadherin, beta cate for 1-3 days or for about 2 days. nin, p120, p.190 Laminin alpha 4, Nidogen-2, and Netrin 4. US 2014/0370.007 A1 Dec. 18, 2014

91. The method of any foregoing claim, wherein said CEC 107. The method of claim 104, wherein said harvesting express the markers Collagen VIII, Na+K+-ATPase pump, comprises contacting said CEC with trypsin, EDTA, AQP1, CA2, CA4, CA12, SCL14A2, SLC16A1, SLC16A3, accutase, collagenase, collagenase B, or a combination SLC16A7, CFTR, NHE1, ADCY10, PITX2, and FOXC1, thereof, or comprises manual dissociation of said CEC. and do not express the markers VWF and CD31. 108. The method of any one of claims 104-107, wherein 92. The method of any foregoing claim, wherein said CEC Subsequent to said harvesting, said CEC are cultured in the form a monolayer of uniformly sized cells with a predomi presence of an inhibitor of rho-associated protein kinase nantly polygonal shape. (“ROCK inhibitor”). 93. The method of claim 92, wherein at least 50%, at least 109. The method of claim 108, wherein said ROCK inhibi 60%, at least 70%, at least 80% at least 85%, at least 90%, or tor is Y-27362, optionally in a concentration of at least 1, 2, 3, at least 95% of said CEC exhibit said polygonal shape. 4, 5, 10, 15, or 20 microM. 94. The method of any foregoing claim, wherein said CEC 110. The method of any one of claims 1-103, further com form a monolayer of uniformly sized cells with a predomi prising harvesting said CEC as a sheet of cells. nantly hexagonal shape. 111. The method of claim 110, wherein said harvesting 95. The method of claim 94, wherein at least 50%, at least comprises culturing said CEC on a temperature sensitive cell 60%, at least 70%, at least 80% at least 85%, at least 90%, or culture surface that release cells as sheet of cells without at least 95% of said CEC exhibit said hexagonal shape. requiring enzymatic treatment. 96. The method of any foregoing claim, wherein said CEC 112. The method of claim 110, wherein said harvesting allows unidirectional leakage of Solutes and nutrients. comprises culturing said CEC on a carrier and removing said 97. The method of claim 96, wherein said CEC actively carrier comprising said CEC from a culture Substrate. pump water in the opposite direction of said unidirectional 113. The method of claim 112, wherein said carrier com leakage. prises gelatin, fibrin-based matrixes, endothelium-denuded 98. The method of any foregoing claim, wherein said CEC corneal buttons, denuded Descemet's membrane, devitalized exhibit a high level of metabolic activity that is comparable to stromal cornea, fresh corneal stromal discs, and/or an amni animal-derived CEC. otic membrane, and is optionally biocompatible. 99. The method of any foregoing claim, wherein said CEC 114. The method of any foregoing claim, wherein said are human CEC. CEC comprise at least 50%, at least 60%, at least 70%, at least 100. The method of any foregoing claim, wherein said 80%, or at least 90% of the cells in the resulting culture. CEC exhibit a culture density of at least 1000 cells/mm, at 115. A composition comprising corneal endothelial cells least 2000 cells/mm, between 2000 and 9000 cells/mm. (CEC) produced according to the method of any foregoing between 3000 and 8,000 cells/mm, between 4000 and 7,000 claim. cells/mm, between 2000 and 4000 cells/mm, between 2000 116. A composition comprising sheet of CEC having a and 3500 cells/mm, or about 2500 cells/mm. culture density of at least 1000 cells/mm. at least 2000 cells/ 101. The method of any foregoing claim, wherein said mm, between 2000 and 9000 cells/mm, between 3000 and CEC exhibit a culture density of at least 4000 cells/mm, at 8,000 cells/mm, between 4000 and 7,000 cells/mm. least 5000 cells/mm, at least 6000 cells/mm, at least 7000 between 2000 and 4000 cells/mm, between 2000 and 3500 cells/mm, at least 8000 cells/mm, or, at least 9000 cells/ cells/mm, or about 2500 cells/mm. mm and/or the average size of the CEC in the culture is less 117. A composition comprising sheet of CEC having a than 1/9,000 mm, less than 1/8,000 mm, less than 1/7,000 culture density of at least 4000 cells/mm, at least 5000 cells/ mm, less than 1/6,000 mm, less than 1/5,000 mm, or less mm, at least 6000 cells/mm. at least 7000 cells/mm, at least than 1/4,000 mm. 8000 cells/mm, or, at least 9000 cells/mm and/or the aver 102. The method of any foregoing claim, wherein said age size of the CEC in the culture is less than 1/9,000 mm. CEC exhibit a statistically significantly decreased level of less than 1/8,000 mm, less than 1/7,000 mm, less than accumulated oxidative stress and/or DNA damage compared 1/6,000 mm, less than 1/5,000 mm, or less than 1/4,000 to CEC isolated from a host. mm. 103. The method of claim 102, wherein said level of oxi dative stress and/or DNA damage is detected by measuring 118. A composition comprising CEC that exhibit a the quantity of one or more of nuclear DNA damage foci: decreased level of accumulated oxidative stress and/or DNA level of expression of p21(Cip1, level of expression of damage compared to CEC isolated from a living host. p16INK4a: level of expression of cytoglobin protein, level of 119. A composition comprising corneal endothelial cells expression of GPX-1 protein, and level of 8-hydroxy-2 - (CEC) produced from neural crest stem cells (NCSCs). deoxyguanosine (8-OHdG). 120. A composition comprising corneal endothelial cells 104. The method of any foregoing claim, further compris (CEC), wherein when transplanted into a human patient or ing harvesting said CEC as a Suspension of cells optionally non-human animal said CEC exhibit a statistically signifi comprising individual cells and/or clusters or clumps of cells. cantly improved rate of Survival after two years compared to 105. The method of claim 104, wherein said harvesting transplantation of donated CEC. comprises contacting said CEC with a cell dissociation 121. A composition comprising a pharmaceutical prepara buffer, or by a method comprising mechanical dissociation of tion of corneal endothelial cells (CEC) produced according to the cells or a Subset thereof, or by a method comprising the method of any one of claims 1-114. optical isolation of the cells or a subset thereof 122. A composition comprising a pharmaceutical prepara 106. The method of claim 104, wherein said harvesting tion comprising a sheet of CEC having a culture density of at comprises contacting said CEC with Trypsin, optionally in a least 1000 cells/mm, at least 2000 cells/mm, between 2000 concentration of 0.25%. and 9000 cells/mm, between 3000 and 8,000 cells/mm. US 2014/0370.007 A1 Dec. 18, 2014 32 between 4000 and 7,000 cells/mm, between 2000 and 4000 135. The composition according to claim 119 or 134, cells/mm, between 2000 and 3500 cells/mm, or about 2500 wherein said NCSCs are produced from embryonic stem (ES) cells/mm. cells. 123. A composition comprising a pharmaceutical prepara 136. The composition according to claim 135, wherein said tion comprising a sheet of CEC having a culture density of at NCSCs are produced from ES cells by a method comprising least 4000 cells/mm, at least 5000 cells/mm, at least 6000 contacting said ES cells with one or more inhibitors of SMAD cells/mm, at least 7000 cells/mm, at least 8000 cells/mm. signaling. or, at least 9000 cells/mm and/or the average size of the CEC 137. A composition comprising corneal endothelial cells in the culture is less than 1/9,000 mm less than 1/8,000 mm, (CEC) produced from embryonic stem (ES) cells. less than 1/7,000 mm, less than 1/6,000 mm, less than 138. The composition of claim 137, which is produced by 1/5,000 mm, or less than 1/4,000 mm. the method of any one of claims 35-80. 124. A composition comprising a pharmaceutical prepara 139. The composition of any one of claims 137 to 138, tion of CEC that exhibit a decreased level of accumulated wherein said ES cells are human ES cells. oxidative stress and/or DNA damage compared to CEC iso 140. The composition any one of claims 137 to 139, lated from a living host. wherein said ES cells are iPS cells. 125. A composition comprising a pharmaceutical prepara 141. The composition of any one of claims 137 to 140, tion of corneal endothelial cells (CEC) produced from neural wherein said ES cells do not exhibit changes or mutations in crest stem cells (NCSCs). genes associated with a disease of corneal endothelial cells. 126. A composition comprising a pharmaceutical prepara 142. The composition of any one of claims 137 to 141, tion of corneal endothelial cells (CEC), wherein when trans wherein said ES cells exhibit a normal karyotype. planted into a human patient or non-human animal said CEC 143. The composition of any one of claims 137 to 142, exhibit a statistically significantly improved rate of survival wherein prior to differentiation into NCSCs, said ES cells are after two years compared to transplantation of donated CEC. maintained in culture in the absence of feeder cells. 127. A composition comprising a pharmaceutical prepara 144. The composition of any one of claims 137 to 143, tion of corneal endothelial cells (CEC) that differ from CEC wherein prior to differentiation into NCSCs said ES cells are derived from a host in the expression of one or more genes. cultured on a matrix. 145. The composition of claim 144, wherein said matrix is 128. The composition of claim 127 wherein said one or selected from the group consisting of laminin, fibronectin, more genes comprise upregulated expression of one or more vitronectin, proteoglycan, entactin, collagen, collagen I, col of PRKCZ, GUCY1B3, CCBE1, COL3A1, and/or FLRT3, lagen IV, heparan Sulfate, Matrigel (a soluble preparation and/or downregulated expression of one or more of ABCC4, from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CTSF, SMAD9, PLOD2, PITX2, and/or PDCD1 LG2. a human basement membrane extract, and any combination 129. The composition of claim 127 wherein said one or thereof. more genes comprise transporter genes, pump genes, cell 146. The composition of any one of claims 144 to 145, adhesion genes, extracellular matrix genes, growth factors wherein said matrix is of human or non-human animal origin. genes, BMP/TGF/Activin related genes, transmembrane pro 147. The composition of any one of claims 144 to 145, teins, and/or genes associated with WNT signaling. wherein said matrix is of bovine, mouse or rat origin. 130. The composition of claim 129 wherein said trans 148. The composition of claim 144, wherein said matrix porter genes comprise upregulated expression of one or more comprises Matrigel (a soluble preparation from Engelbreth of NKAIN1, SLC5A12, SLC2A3, SLC7A1 SLC39A2, Holm-Swarm (EHS) mouse sarcoma cells). SLC7A5, SLC6A4, SLC16A6, ATP8B1, SLC39A10, 149. The composition of any one of claims 115, 119 to 136 SLC8A1, SLC19A3, SLC15A2, and/or AQP1, and/or down or 138 to 148, wherein said NCSCs express one or more regulated expression of one or more of TAP2, SLCO3A1, markers selected from the group consisting of Sox10, AP2, SLC9A7, SLC12A2, TAP2, SLC9A3R2, SLC7A8, SLC6A6, HNK1, PAX7, p75 (NGFR), and any combination thereof. NHEDC2, and/or CA2. 150. The composition of any one of claims 115 to 149, 131. The composition of claim 129 wherein said cell adhe wherein said CEC express one or more markers selected from sion genes comprise upregulated expression of one or more of the group consisting of Na+/K+ ATPase, ZO-1, KLF13, ITGA8, PCDH9, CDH8, ITGB4, TJP2, PCDH11XIY, and/or AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2/ PCDH11Y|X and/or downregulated expression of one or SCL4A2, SCL16A1, CA12, CA4, FoxC1, and any combina more of PCDHB13, ICAM1, ASAM, MCAM, and/or tion thereof. NCAM1. 151. The composition of any one of claims 115 to 150, 132. The composition of claim 129 wherein said genes wherein said CEC express the markers Collagen VIII, Na+K+ associated with WNT signaling comprise upregulated expres ATPase pump, and ZO-1, and do not express the markers sion of one or more of DACT1, WNT3, WNT2, and/or SFRP1 WWF and CD31. and/or downregulated expression of one or more of WNT5A 152. The composition of any one of claims 115 to 151, and/or DKK1. wherein said CEC express one or more corneal endothelial 133. The composition of claim 129 wherein said pump pump markers. genes comprise upregulated expression of one or more of 153. The composition of any one of claims 115 to 152, AQP1, CA2, SCL4A2, SLC16A1, SLC16A3, ADCY10, wherein said CEC express one or more periocular neural crest CA12, CA4, CFTR, NBC1, NHE1, and/or SCL16A7. markers. 134. The composition of any one of claims 118 to 133, 154. The composition of any one of claims 115 to 153, wherein said CEC are produced the method of any one of wherein said CEC express one or more cell adhesion and claims 1 to 28. matrix proteins. US 2014/0370.007 A1 Dec. 18, 2014

155. The composition of any one of claims 115 to 154, level of expression of p21(Cip1, level of expression of wherein said CEC express at least one corneal endothelial p16INK4a: level of expression of cytoglobin protein, level of pump marker, at least one periocular neural crest marker, and expression of GPX-1 protein, and level of 8-hydroxy-2 - at least one cell adhesion and matrix protein. deoxyguanosine (8-OHdG). 156. The composition of claim 152 or 155, wherein said 170. The composition of any one of claims 115 to 169, one or more corneal endothelial pump markers are selected wherein said CEC comprise at least 50%, at least 60%, at least from the group consisting of: AQP1, CA2, CA4, CA12, 70%, at least 80%, or at least 90% of the cells in the resulting SCL14A2, SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, culture. ADCY10, voltage-dependent anion channels VDAC2 and 171. The composition of any one of claims 115 to 170, VDAC3, chloride channel proteins CLCN2 and CLC. wherein said CEC are contained in a sheet of CEC. 157. The composition of claim 153 or 155, wherein said periocular neural crest markers are selected from the group 172. The composition of claim 171, wherein said sheet of consisting of PITX2, and FOXC1. cells comprises or consists essentially of an approximately 158. The composition of claim 154 or 155, wherein said circular disc of cells having a diameter of at least 1, 2, 3, 4, 5, cell adhesion and matrix proteins are selected from the group 6, 7, 8, 9, 10, 11, or 12 mm. consisting of Occludin, Connexin 43,9.3E antigen, Collagen 173. The composition according to claim 171 or 172, wherein said CEC exhibit a culture density of at least 1000 III, Collagen IV, N cadherin, VE cadherin, E cadherin, beta cells/mm, at least 2000 cells/mm, between 2000 and 9000 catenin, p120, p.190 Laminin alpha 4, Nidogen-2, and Netrin cells/mm, between 3000 and 8,000 cells/mm, between 4. 4000 and 7,000 cells/mm, between 2000 and 4000 cells/ 159. The composition of any one of claims 115 to 158, mm, between 2000 and 3500 cells/mm, or about 2500 cells/ wherein said CEC express the markers Collagen VIII, Na+K+ mm. ATPase pump, AQP1, CA2, CA4, CA12, SCL14A2, SLC16A1 SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, 174. The composition according to claim 171 or 172, PITX2, and FOXC1, and do not express the markers v WF and wherein said CEC exhibit a culture density of at least 4000 CD31. cells/mm, at least 5000 cells/mm. at least 6000 cells/mm. 160. The composition of any one of claims 115 to 159, at least 7000 cells/mm, at least 8000 cells/mm, or, at least wherein said CEC form a monolayer of uniformly sized cells 9000 cells/mm and/or the average size of the CEC in the with a predominantly hexagonal shape. culture is less than 1/9,000 mm, less than 1/8,000 mm, less 161. The composition of claim 160, wherein at least 50%, than 1/7,000 mm, less than 1/6,000 mm, less than 1/5,000 at least 60%, at least 70%, at least 80%, or at least 90% of said mm, or less than 1/4,000 mm. CEC exhibit said hexagonal shape. 175. The composition of any one of claims 171 to 174, 162. The composition of any one of claims 115 to 161, wherein said CEC are situated on a carrier. wherein said CEC allows unidirectional leakage of solutes 176. The composition of any one of claims 171 to 175, and nutrients. wherein said CEC are cultured on a substrate and released 163. The composition of claim 162, wherein said CEC onto a carrier. actively pump water in the opposite direction of said unidi 177. The composition of claims 175 or 176, wherein said rectional leakage. Substrate comprises athermoresponsive polymer orathermo 164. The composition of any one of claims 115 to 163, responsive poly(N-isopropylacrylamide) (PNIPAAm)- wherein said CEC exhibit a high level of metabolic activity grafted Surface. that is comparable to animal-derived CEC. 178. The composition of any one of claims 175 to 177, 165. The composition of any one of claims 115 to 164, wherein said carrier comprises gelatin, fibrin-based matrixes, wherein said CEC are human CEC. endothelium-denuded corneal buttons, denuded Descemet's 166. The composition of any one of claims 115 to 165, membrane, devitalized stromal cornea, fresh corneal stromal wherein said CEC exhibit a culture density of at least 1000 discs, and/or an amniotic membrane. cells/mm, at least 2000 cells/mm, between 2000 and 9000 cells/mm, between 3000 and 8,000 cells/mm, between 179. The composition of any one of claims 115 to 170, 4000 and 7,000 cells/mm, between 2000 and 4000 cells/ wherein said CEC are in suspension. mm, between 2000 and 3500 cells/mm, or about 2500 cells/ 180. The composition of claim 179, further comprising an mm inhibitor of Rho-associated kinase (ROCK). 167. The composition of any one of claims 115 to 165, 181. The composition of claim 180, wherein said inhibitor wherein said CEC exhibit a culture density of at least 4000 of Rho-associated kinase comprises Y-27632. cells/mm, at least 5000 cells/mm. at least 6000 cells/mm. 182. The composition of any one of claims 115 to 181, at least 7000 cells/mm. at least 8000 cells/mm, or, at least which comprises at least 2000, at least 3000, at least 8000, at 9000 cells/mm and/or the average size of the CEC in the least 11000, at least 18000, at least 25000, at least 31000, at culture is less than 1/9,000 mm, less than 1/8,000 mm, less least 44000, at least 49000, at least 69000, at least 71000, at than 1/7,000 mm, less than 1/6,000 mm, less than 1/5,000 least 96000, at least 99000, at least 126000, at least 135000, at mm, or less than 1/4,000 mm. least 159000, at least 176000, at least 196000, at least 223000, 168. The composition of any one of claims 115 to 167, at least 275000, at least 237000, at least 283000, at least wherein said CEC exhibit a decreased level of accumulated 332000, or at least 396000 CEC. oxidative stress and/or DNA damage compared to CEC iso 183. The composition of any one of claims 115 to 181, lated from a living host. which comprises at least 300,000, at least 350,000, at least 169. The composition of claim 168, wherein said level of 400,000, at least 450,000, at least 500,000, at least 550,000, at oxidative stress and/or DNA damage is detected by measur least 600,000, at least 650,000, at least 700,000, at least ing the quantity of one or more of nuclear DNA damage foci: 750,000, at least 800,000, or at least 850,000 CEC. US 2014/0370.007 A1 Dec. 18, 2014 34

184. The composition of any one of claims 115 to 183, endothelium including corneal dystrophies, contact lens which comprises at least 50%, at least 60%, at least 70%, at usage, cataract Surgery, and late endothelial failure in cornea least 80%, or at least 90% CEC. transplantation. 185. The composition of any one of claims 115 to 184, 199. The method of any one of claims 196 to 198, further further comprising a pharmaceutically acceptable excipient. comprising administering an immunosuppressive agent or 186. The composition of any one of claims 115 to 185, immune tolerizing agent to said patient. further comprising an immunosuppressive or immune toler 200. The method of claim 199, wherein said immunosup izing agent. pressive agent or immune tolerizing agent is administered in 187. The composition of claim 186, wherein said immu an amount Sufficient to reduce the risk of rejection of said nosuppressive or immune tolerizing agent comprises one or CEC. more of mesenchymal stem cells, anti-lymphocyte globulin 201. The method of claim 199 or 200, wherein said immu (ALG) polyclonal antibody, anti-thymocyte globulin (ATG) nosuppressive agent or immune tolerizing agent is adminis polyclonal antibody, azathioprine, BASILIXIMAB(R) (anti tered prior to, concurrently with, and/or Subsequent to admin IL-2RC. receptor antibody), cyclosporin (cyclosporin A), istration of said CEC to said patient. DACLIZUMAB(R) (anti-IL-2RO. receptor antibody), everoli 202. The method of any one of claims 196 to 201, wherein mus, mycophenolic acid, RITUXIMAB(R) (anti-CD20 anti said composition comprising CEC further comprises an body), sirolimus, tacrolimus, mycophemolate mofetil, and immunosuppressive agent or immune tolerizing agent. corticosteroids. 203. The method of any one of claims 199 to 202, wherein said immunosuppressive or immune tolerizing agent com 188. The composition of any one of claims 115 to 187. prises one or more of mesenchymal stem cells, anti-lympho which is free of detectable pyrogens, bacterial contaminants, cyte globulin (ALG) polyclonal antibody, anti-thymocyte mycoplasmal contaminants, and viruses. globulin (ATG) polyclonal antibody, azathioprine, BASIL 189. The composition of any one of claims 115 to 188 IXIMAB(R) (anti-IL-2RC. receptor antibody), cyclosporin which comprises human CEC. (cyclosporin A), DACLIZUMABOR (anti-IL-2RC. receptor 190. The composition of any one of claims 115 to 189 antibody), everolimus, mycophenolic acid, RITUXIMABR) which is suitable for transplantation into the eye of apatient in (anti-CD20 antibody), Sirolimus, tacrolimus, mycophemo need thereof. late mofetil, and corticosteroids. 191. The composition of claim 190, wherein said patient is 204. The method of any one of claims 196 to 203, wherein human. said CEC exhibit a statistically significantly improved rate of 192. Use of a composition according to any one of claims Survival after two years compared to transplantation of 115 to 191 in the manufacture of a medicament. donated CEC, wherein said rate of survival is determined in a 193. The use of claim 192, wherein said medicament is for human patient or a non-human animal. the treatment of a disease of corneal endothelial cells. 205. A method of producing neural crest stem cells (NC SCs) or NCSC progenitors comprising contacting ES cells 194. The use of claim 193, wherein said disease of corneal endothelial cells comprises Fuchs dystrophy, iridocorneal with one or more inhibitors of SMA/Mothers Against Deca endothelial syndrome, posterior polymorphous dystrophy, pentaplegic (SMAD) protein signaling. and congenital hereditary endothelial dystrophy, and second 206. The method of claim 205, wherein ES cells are cul ary diseases for which an effective treatment is replacement tured with said one or more inhibitors of SMAD protein of the corneal endothelium including corneal dystrophies, signaling for between about 1 and 10 days or for no more than contact lens usage, cataract Surgery, and late endothelial fail 10,9,8,7,6, 5, 4, 3, or 2 days. ure in cornea transplantation. 207. The method of claim 205, wherein ES cells are cul tured with said one or more inhibitors of SMAD protein 195. The use of any one of claims 192 to 194, wherein said signaling for 1-3 days or for about 2 days. medicament is adapted for administration by a method com 208. The method of claim 205, wherein said one or more prising Descemet's stripping with endothelial keratoplasty inhibitors of SMAD protein signaling prevent the binding of (DSEK), Penetrating Keratoplasty (PKP), lamellar kerato a TGF-3 family ligands to its corresponding receptor. plasty, Descemet's Membrane Endothelial Keratoplasty 209. The method of any one of claims 205 to 208, wherein (DMEK), DSAEK, and DLEK. said one or more inhibitors of SMAD protein signaling pre 196. A method of treatment of a disease of corneal endot vent the activation of a TGF-B receptor. helial cells or injured corneal endothelial cells, comprising 210. The method of any one of claims 205 to 209, wherein administering a composition comprising CEC according to said one or more inhibitors of SMAD protein signaling inhib any one of claims 115 to 191 to a patient in need thereof. its one or more SMAD intracellular proteins/transcription 197. The method of claim 196, wherein said CEC are factors. administered by a method comprising Descemet's stripping 211. The method of any one of claims 205 to 210, wherein with endothelial keratoplasty (DSEK), Penetrating Kerato said one or more inhibitors of SMAD protein signaling com plasty (PKP), lamellar keratoplasty, Descemet's Membrane prise Leukemia Inhibitory Factor (LIF), GSK3 inhibitor Endothelial Keratoplasty (DMEK), DSAEK, DMEK and (CHIR 99021), Compound E (Y secretase inhibitor XXI), DLEK. SB431542, or any combination thereof. 198. The method of any one of claims 196 to 197, wherein 212. The method of any one of claims 205 to 210, wherein disease of corneal endothelial cells is selected from the group said one or more inhibitors of SMAD protein signaling com consisting of Fuchs dystrophy, iridocorneal endothelial Syn prise Chordin, Follistatin, dominant negative receptors or drome, posterior polymorphous dystrophy, and congenital blocking antibodies that sequester BMP2, BMP4, and/or hereditary endothelial dystrophy, and secondary diseases for BMP7, dorsomorphin (or Compound C), SIS3 (6,7- which an effective treatment is replacement of the corneal Dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo2,3- US 2014/0370.007 A1 Dec. 18, 2014

bipyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquino 226. The method of any one of claims 219 to 225, wherein line, Specific Inhibitor of Smad3 (SIS3), an inhibitor SMAD, said second inhibitor of SMAD protein signaling inhibits SMAD6, SMAD7, SMAD10, an antagonist of a receptor both activin and nodal signaling. SMAD, an antagonist of SMAD1, an antagonist of SMAD2, 227. The method of any one of claims 219 to 225, wherein an antagonist of SMAD3, an antagonist of SMAD5, an said second inhibitor of SMAD protein signaling inhibits the antagonist of SMAD8/9, or any combination thereof. Lefty/Activin/TGFbeta pathways by blocking phosphoryla 213. The method of any one of claims 205 to 212, wherein tion of the ALK4, ALK5 and ALK7 receptors. the duration of said contacting ES cells with one or more 228. The method of any one of claims 219 to 225, wherein inhibitors of SMAD protein signaling is at least 2 days, said second inhibitor of SMAD protein signaling is an ALK4 between 1 and 10 days, or is between 2 and 6 days. receptor inhibitor. 214. The method of claim 213, wherein the duration of said 229. The method of any one of claims 219 to 229, wherein contacting ES cells with one or more inhibitors of SMAD said second inhibitor of SMAD protein signaling is selected protein signaling is between about 1 and 10 days or no more from the group consisting of 4-4-(1,3-benzodioxol-5-yl)-5- than 10,9,8,7,6, 5, 4, 3, or 2 days. (2-pyridinyl)-1H-imidazol-2-ylbenzamide (SB431542) and 215. The method of claim 213, wherein the duration of said derivatives thereof. contacting ES cells with one or more inhibitors of SMAD 230. The method of any one of claims 219 to 225, wherein protein signaling is between about 1-3 days or about 2 days. said second inhibitor of SMAD protein signaling comprises 216. The method of any one of claims 205 to 215, wherein SB431542. said neural crest stem cells are produced from ES cells by a 231. The method of any one of claims 229 to 230, wherein method further comprising contacting ES cells with at least said SB431542 is present in a concentration between 10 nM one Wntagonist. and 100 uM, between 0.1 uM and 50 uM, between 0.1 and 20 217. The method of any one of claims 205 to 215, wherein uM, between 1 and 20 LM, or about 10 uM. said neural crest stem cells are produced from ES cells by a 232. The method of any one of claims 219 to 225, wherein method further comprising contacting ES cells with at least said second inhibitor of SMAD protein signaling blocks one Wntagonist comprising (2Z.3E)-6-bromoindirubin-3'- phosphorylation of ACTRIB, TGFBR1, and ACTRIC recep oxime (BIO) and/or Wnt3a. tOrS. 218. The method of any one of claims 205 to 215, wherein said neural crest stem cells are produced from ES cells by a 233. The method of any one of claims 219 to 225, wherein method further comprising contacting ES cells with at least said second inhibitor of SMAD protein signaling inhibits one Wntagonist selected from the group consisting of Wnt TGFB/Activin/Nodal signaling. proteins, nucleic acids encoding Wnt proteins, LiCl, AXin 234. The method of claim 223, wherein said second inhibi antagonists; APC antagonists; norrin; R-spondin2; (hetero) tor of SMAD protein signaling blocks endogenous Activin arylpyrimidines; IQ1; BIO(6-bromoindirubin-3'-oxime); and BMP signals. 2-amino-4-3,4-(methylenedioxy)benzyl-amino-6-(3- 235. The method of claim 219, wherein said first inhibitor methoxyphenyl)pyrimidine; WAY-316606: QS11; of SMAD protein signaling and/or said second inhibitor of SMAD protein signaling are each selected from the group SB-216763; SB-216763; DCA; and any combination thereof. consisting of Chordin, Follistatin, dominant negative recep 219. The method of claim 205, wherein said neural crest tors or blocking antibodies that sequester BMP2, BMP4, stem cells are produced from ES cells by a method compris and/or BMP7, dorsomorphin (or Compound C), SIS3 (6.7- ing contacting ES cells with a first inhibitor of SMAD protein Dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo2,3- signaling and a second inhibitor of SMAD protein signaling. bipyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquino 220. The method of claim 219, wherein said first inhibitor of SMAD protein signaling is selected from the group con line, Specific Inhibitor of Smad3 (SIS3), an inhibitor SMAD, sisting of: Noggin polypeptide, dorsomorphin, LDN-193189, SMAD6, SMAD7, SMAD10, an antagonist of a receptor and any combination thereof. SMAD, an antagonist of SMAD1, an antagonist of SMAD2, 221. The method of any one of claims 219 to 220, wherein an antagonist of SMAD3, an antagonist of SMAD5, and an said first inhibitor of SMAD protein comprises Noggin antagonist of SMAD8/9. polypeptide. 236. The method of claim 219, wherein said first inhibitor 222. The method of any one of claims 220 to 221, wherein of SMAD protein signaling comprises Noggin and said sec said Noggin polypeptide is present in a concentration ond inhibitor of SMAD protein signaling comprises between 10 ng/ml and 5,000 ng/ml, between 100 ng/ml and SB431542. 700 ng/ml, between 400 ng/ml and 600 ng/ml, or about 500 237. The method of any one of claims 219 to 236, wherein ng/ml. the duration of said contacting ES cells with said first inhibi 223. The method of claim 219, wherein said first inhibitor tor of SMAD protein signaling and said second inhibitor of of SMAD protein signaling is selected from the group con SMAD protein signaling is at least 2 days, between 1 and 10 sisting of: antagonists of BMP2; antagonists of BMP4; days, or is between 2 and 6 days. antagonists of BMP7; and antagonists of TGFB; 238. The method of claim 237, wherein ES cells are cul 224. The method of any one of claims 219 to 223, wherein tured with said one or more inhibitors of SMAD protein said second inhibitor of SMAD protein signaling comprises signaling for between about 1 and 10 days or for no more than an inhibitor of an anaplastic lymphoma kinase signaling path 10,9,8,7,6, 5, 4, 3, or 2 days. way. 239. The method of claim 237, wherein ES cells are cul 225. The method of any one of claims 219 to 224, wherein tured with said one or more inhibitors of SMAD protein said second inhibitor of SMAD protein signaling inhibits a signaling for 1-3 days or for about 2 days. signaling pathway selected from the group consisting of 240. The method of any one of claims 205 to 239, wherein Lefty, Activin, and TGFbeta. said ES cells are human ES cells. US 2014/0370.007 A1 Dec. 18, 2014 36

241. The method of any one of claims 205 to 239, wherein said ES cells are iPS cells. 242. A composition comprising NCSC or NCSC progeni tors produced by the method of any one of claims 205 to 241. k k k k k