US 20190204299A1 ( 19) United States ( 12) Patent Application Publication ( 10 ) Pub . No. : US 2019 /0204299 A1 Mead et al. (43 ) Pub . Date : Jul. 4 , 2019 ( 54 ) SINGLE - CELL GENOMIC METHODS TO Publication Classification GENERATE EX VIVO CELL SYSTEMS THAT (51 ) Int . Ci. RECAPITULATE IN VIVO BIOLOGY WITH GOIN 33 /50 (2006 . 01 ) IMPROVED FIDELITY A61K 35 / 17 ( 2006 .01 ) C120 1 /6869 ( 2006 .01 ) ( 71) Applicants :Massachusetts Institute of C12N 5 /071 (2006 .01 ) Technology , Cambridge, MA (US ) ; The 2 ) U . S . CI. Brigham and Women ' s Hospital, Inc. , CPC . .. .. GOIN 33 /5023 ( 2013 .01 ) ; A6IK 35 / 17 Boston ,MA (US ) (2013 .01 ) ; C12Q 1 /6869 (2013 .01 ) ; GOIN 33 /5008 ( 2013 .01 ) ; CI2N 2501 / 999 ( 2013 .01 ) ; (72 ) Inventors : Benjamin E . Mead , Cambridge, MA C12N 5 / 068 (2013 . 01 ) ; C12N 2501 / 11 (US ) ; Jose Ordovas -Montanes , ( 2013 .01 ) ; C12N 2501/ 40 ( 2013 .01 ) ; C12N Cambridge , MA (US ); Alexander K . 2501 /998 ( 2013. 01 ) ; GOIN 33 /5011 (2013 . 01 ) Shalek , Cambridge , MA (US ) ; Jeffrey (57 ) ABSTRACT Karp , Boston , MA (US ) ; Robert Disclosed here is a generally applicable framework that Langer , Cambridge , MA (US ) utilizes massively - parallel single - cell RNA - seq to compare cell types/ states found in vivo to those of in vitro models . (21 ) Appl. No. : 16 /240 , 361 Furthermore , Applicants leverage identified discrepancies to improve model fidelity . Applicants uncover fundamental gene expression differences in lineage - defining genes ( 22 ) Filed : Jan . 4 , 2019 between in vivo systems and in vitro systems. Using this information , molecular interventions are identified for ratio nally improving the physiological fidelity of the in vitro Related U . S . Application Data system . Applicants demonstrated functional (antimicrobial (60 ) Provisional application No . 62 /613 ,710 , filed on Jan . activity , niche support ) improvements in Paneth cell physi 4 , 2018 , provisional application No . 62 /702 , 168 , filed ology using the methods. on Jul. 23 , 2018 . Specification includes a Sequence Listing . murine small conventional intestine organoid (ENR ) . UM isolate crypts & enrich for ISCs (ENR + CV ) SCRNA - seg * * *2002 * * to ID Paneth *. * * B* & populations in vivo compare in vitro Paneth expression Paneth cell Patent Application Publication Jul . 4 , 2019 Sheet 1 of 41 US 2019 /0204299 A1 * 122NAN 23 WS 2017 ** * * ** * cell ** * * ** * * * conventional ' VA invitro Paneth . forISCS(ENR+CV) SCRNA-seq isolatecrypts&enrich compare expression FIG.1A *.toIDPaneth populations BB Ben murinesmall cell intestine invivo Paneth Patent Application Publication Jul . 4 , 2019 Sheet 2 of 41 US 2019 /0204299 A1 . Seosecco* WWW w R41 eeeeeee, , , , , , . ooos FIG.10 TOYS* www VVV : : : . : . : . : WM LLOW InVivoIdentificationofPanethCells ) ) 1 + UM scaled ( log( Expression Lyz1 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** ISNE1 FIG.1B Patent Application Publication Jul. 4 , 2019 Sheet 3 of 41 US 2019 / 0204299 A1 COMOO .' 772 mm .' . 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FIG.1E 2 ISNE Patent Application Publication Jul. 4 , 2019 Sheet 5 of 41 US 2019 / 0204299 A1 Score- Z Row *$29- . ce See 8920 :3283 29893 3930 in 83 2Simon EX UIOAIA XIXW I'OH 33 B . 32:3 833 v-UN3 BEI8:32 XEBA www WALIOS XXX 333 Patent Application Publication Jul. 4 , 2019 Sheet 6 of 41 US 2019 / 0204299 A1 all p « 2 . 92x10 -07 Ang4 Deta3 . (logscaledUMI+1) . GeneExpression . 3 . 4 . ENR - 4 . In . Vivo ENR - 4 In Vivo ENR - 4 in Vivo FIG . 1H < 22x10 - 16 p < 2 . 035x10 * p < 5 . 25x107 Paneth Notch obcoconut GeneSignature(PercentofTranscriptome) .. ENA - 4 In Vivo ENR - 4 In Vivo ENR - 4 In Vivo FIG . 11 Patent Application Publication Jul. 4 , 2019 Sheet 7 of 41 US 2019 / 0204299 A1 . notch(D) . -wnt(C) 9 *** * * * * W VW . - E . ** XXX . (ENR+CD) SES WWW 220V . 24 : : V conventionalchemically-induced (ENR+CV) ISCprecursor FIG.2A ROS organoid(ENR) Patent Application Publication Jul . 4 , 2019 Sheet 8 of 41 US 2019 /0204299 A1 * * * * * Lgr5 19: ** )change fold( Log Mmp7 ) change fold( Logz FIG.2B Defa1 . ) change fold( Logz Lyz1 *T UNEAO+HN3 ) change fold( Logz Patent Application Publication Jul. 4 , 2019 Sheet 9 of 41 US 2019 / 0204299 A1 whole cell cluster (6D ) ENR +CV ENR ENR + CD ?????????????????????????????????????????????????????????????????????????????????????????????????????????? ET DEFA | AC Loop 1000 LYZ LYZ&DEFA AC3 . DAPI & actin FIG . 20 Patent Application Publication Jul . 4 , 2019 Sheet 10 of 41 US 2019 /0204299 A1 single. Paneth cell in vivo in vitro ???????????????????????????????????????SI crypt ???????????????????????????????????????????????????????????????????????????? ENR +CD (6D ) . DEFA . " .. .. ! . LYZ . : : : : : : : : :: . Ciri . ' . ' . : :: : : : : : : : : : : : : : : : : : : : : :: : :: . : :: : : : : : :: : : : :: : : : : : : : : : : : : : : :: :: : . X . : . LYZ : . W . &DEFA . WW . DAPI & actin FIG , 2D Patent Application Publication Jul . 4 , 2019 Sheet 11 of 41 US 2019 /0204299 A1 ? ENR | ENR + CD CD24 + & LYZ . CD24 + & LYZ + 400 204 ENR + CV 4 6 8 10 12 ENR + CV 4 6 8 1012 %ofviablecells CD24 - & LYZ . | LYZ + & CD24 . 2 | 100 - ENR + CV 4 6 8 1012 ENR + CV 4 6 8 1012 time in culture (days ) FIG . 2E Patent Application Publication Jul . 4 , 2019 Sheet 12 of 41 US 2019 /0204299 A1 . M1-2 M2-1 M2-2 . ' . * *?? ?LLLmmmmmmm .. 200 . ** * ** * * * . WWW . X2 ENR+CVCD FIG.3A biological donor"M1 biological donor"M2 Patent Application Publication Jul . 4 , 2019 Sheet 13 of 41 US 2019 /0204299 A1 4 ChgaWif1Pla2g1b 3 Scg3Defa29Cri Mmp7 -Wnt3Ange . Refaz2 2 # # . ENR+CD/(6D) . ' ' KA FIG.3B . Log(foldchange) nr. -10 . 7 : . 3 y A . : 1 22. ** 8 -2 05.FDR<0 -3 * * * * ????????????????????????????????????????????????????????????????????????? -4 ) FDR. .( Log10- . Patent Application Publication Jul . 4 , 2019 Sheet 14 of 41 US 2019 /0204299 A1 ENR + CD / ENR (D6 ) ENR + CD / ENR (06 ) Other . Lectins GobletcellWNTOthersecreted . 4000 . .7 . " ' . eu . 2 w Gm15293 mano Enteroendocrineceli -Detensins . birinin ???????????????????????????????????????????????????????????????????????????????????? Log ( fold change) Log ; ( fold change ) FIG . 30 Patent Application Publication Jul . 4 , 2019 Sheet 15 of 41 US 2019 /0204299 A1 ENR + CD (6D ) ENR (6D ) F . enriched0 . proteins. enriched proteins . 24 M1- 1 24. M1 - 1 .. * of 75ST . % 284, 50 % 75 % * 24. M1- 2 24 M1- 2 . 22 . - . * . .. ? . het opbou 50% 75% . Log(foldchange) . 012348 50% Logz(foldchange) M2- 1 24 M2- 1 * GA 0 25* %. * * . 50 % 75 % 2 50% 75% 24I M2. -. 2 24: M2- 2 . CoV COV FIG . 3D Patent Application Publication Jul . 4 , 2019 Sheet 16 of 41 US 2019 /0204299 A1 I . Leadingedge: 58%tags 10%list63 %signal ???????????? FDRq<0.0001 Panethcellgenesetmatches 1000200030004000500060007000 Rankorder FIG.3E NES=3.11 000000 w .: : : M1-1 M1-2M2-1 M2-2 score Enrichment ) change Fold( Logz Patent Application Publication Jul . 4 , 2019 Sheet 17 of 41 US 2019 /0204299 A1 -3 . .. 0 : . 20010015 Size:gene# . 3 . Color:GSEANES . Legend . +3 . TAL1 NRSF . RP58 .... .. .. PR . **** ** * ** * * ** ** * TCF4 FIG.3F Unknown GRAR . E2F Patent Application Publication Jul . 4 , 2019 Sheet 18 of 41 US 2019 /0204299 A1 ENR+CV . .. .18 . !SY C # :: R .. .: # # WAN ** 20 - ISNE1 . P . : FIG.4A . A * ILE * # * # ti * . C 2 sta .. OPY 2 _ SNE wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww Patent Application Publication Jul . 4 , 2019 Sheet 19 of 41 US 2019 /0204299 A1 . mi . .. .. wacananianging mwingi nisingai inicial SARAN . , ISNE1 FIG.48 vingasinanananinginnanmaningmananana NSI Patent Application Publication Jul . 4 , 2019 Sheet 20 of 41 US 2019 /0204299 A1 30 . Yes 20 Paneth Proteome . 112.: GeneSignature .10 . ' W FIG.4D A . - . 9 ENR-1 EEC-2 ENR+CD-1 . ENR+CVm . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 94 . ; 8.0 * 1 ISNE1 FIG.4C * * * KUALAarbeiten in 2 ISNE Patent Application Publication Jul . 4 , 2019 Sheet 21 of 41 US 2019 /0204299 A1 ENR + CV - 1 ENR + CV - 2 ENR2009 + CV - 3 ENRICK + CV - 4 ENR - 1 ENR - 2 ENR - 3 ENR - 4 ENR + CD - 1 ENR + CD - 2 Www ENR + CD - 3 ENR + CD - 4 ENR EEC - 1 - ENR + CV EEC - 2 D ENR + CD 0 .0 0 . 1 0 . 2 0 . 3 0 . 4 0 .5 Fraction of total sample FIG . 4E Patent Application Publication Jul . 4 , 2019 Sheet 22 of 41 US 2019 /0204299 A1 . Minimi ., inirinin ,. WW ,. ,. * ** w wwwwwww 10 . * W : * . : . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * DU wiiiiiiiiiiiiiiiwiiiiiiiiiiiiiii W Critiimiiiiiiiiiii W w wwwwwwwwwwww w w w VSO 8991 Patent Application Publication Jul . 4 , 2019 Sheet 23 of 41 US 2019 /0204299 A1 an50 sposo2 . SEX ? PAN . 1998 W oox VO KUWA * * vou X . w . 33333 FIG.50 * 23* 3 3 www32 BB -PAR - -- * -22. - w UJJJJJJJJJJJJV UUUUUUUUUUUUUU Patent Application Publication Jul . 4 , 2019 Sheet 24 of 41 US 2019 /0204299