US 2007 O155661A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0155661 A1 Kim (43) Pub. Date: Jul. 5, 2007

(54) METHODS AND COMPOSITIONS FOR Related U.S. Application Data MODULATING THE DEVELOPMENT OF STEM CELLS (60) Provisional application No. 60/447,673, filed on Feb. 14, 2003. (75) Inventor: Seung Kim, Stanford, CA (US) Publication Classification Correspondence Address: FSH & NEAVE P GROUP (51) Int. Cl. ROPES & GRAY LLP A6II 38/18 (2006.01) ONE INTERNATIONAL PLACE A6II 38/17 (2006.01) BOSTON, MA 02110-2624 (US) GOIN 33/53 (2006.01) CI2P 2/06 (2006.01) (73) Assignee: THE BOARD of TRUSTEES of the A6II 38/28 (2006.01) LELAND STANDORD JUNOR UNI- (52) U.S. Cl...... 514/12: 435/7.1: 435/69.1; VERSITY, PALO ALTO, CA (US) 530/303 (21) Appl. No.: 10/545,582 (57) ABSTRACT (22) PCT Filed: Feb. 17, 2004 The application provides, among other things, methods for increasing production of insulin in cells by contacting the (86). PCT No.: PCT/USO4/O4639 cells with a BMP family member and/or an activator of a BMP pathway. The application also provides methods for S 371(c)(1), increasing the production of beta cell precursor cells by (2), (4) Date: Nov. 28, 2006 contacting suitable cells with an inhibitor of a BMP pathway. Patent Application Publication Jul. 5, 2007 Sheet 1 of 7 US 2007/0155661 A1

FIGURE I Patent Application Publication Jul. 5, 2007 Sheet 2 of 7 US 2007/0155661 A1

ad C s O S 9 O C e Patent Application Publication Jul. 5, 2007 Sheet 3 of 7 US 2007/0155661 A1 |------

FIGURE 3

Patent Application Publication Jul. 5, 2007 Sheet 4 of 7 US 2007/0155661 A1

a . . . . b A wW-.. ; s

r w -A Y as:

y s 16 IJ l Patent Application Publication Jul. 5, 2007 Sheet 5 of 7 US 2007/O155661 A1

Patent Application Publication Jul. 5, 2007 Sheet 6 of 7 US 2007/0155661 A1

• ~~~~-:::::::---~~~ .**

FIGURE 6 Patent Application Publication Jul. 5, 2007 Sheet 7 of 7 US 2007/0155661 A1

14000

1OOOO

8000

4000

2000

2 3IN AT (s

2uMRA 100M RA Condition f 2 3 8 9 10 1 2 3 4 5 16 TT 8 FIGURE 7 US 2007/O 155661 A1 Jul. 5, 2007

METHODS AND COMPOSITIONS FOR ence of a BMP family member, such as a GDF8/GDF11 MODULATING THE DEVELOPMENT OF STEM subfamily member, or an activator of a BMP signaling CELLS pathway. Optionally, the BMP family member has one or more of the following characteristics: an amino acid CROSS-REFERENCE TO RELATED sequence that is at least 80% identical to a human GDF 11 APPLICATIONS or GDF8; ability to bind to an ActRIIA and/or ActRIIB receptor; an ability to increase Smad2 and/or Smad3 phos 0001. This application claims the benefit of the filing date phorylation in a stem cell; an ability to increases expression of U.S. Provisional Patent Application Ser. No. 60/447,673 of a gene that is positively regulated by Smada. In certain entitled “Methods and Compositions for Modulating the embodiments, the application provides methods for increas Development of Stem Cells' and listing Seung Kim as ing insulin production in a cell composition comprising stem inventor. The aforementioned provisional application is cells, the method comprising contacting the cell composition incorporated herein in its entirety. with a substance that stimulates an ActRIIA and/or ActRIIB signaling pathway. Activators of an ActRIIA and/or ActRIIB BACKGROUND signaling pathway may, for example, cause an increase in 0002 Diabetes mellitus (DM) is a major cause of mor Smad2 and/or Smad3 function or phosphorylation, or bidity and mortality worldwide, and incidence rates of type increase expression of a gene that is positively regulated by I and type II DM are increasing. In type I DM, destruction Smada. A BMP signaling pathway may be activated by of insulin-producing pancreatic islets leads to a prolonged causing overexpression of one or more positive regulators, illness often culminating in devastating multisystem organ such as Smad3, Smad3, or a BMP family member. A BMP failure and early mortality. Clinical trials demonstrate that signaling pathway may also be activated by inhibiting an tight glucose regulation can prevent the development of inhibitor, Such as noggin or chordin. Optionally, the cell diabetic complications, but attempts to achieve this regula composition comprises beta cell precursor cells. In certain tion by exogenous insulin administration are only partially embodiments, the cell composition is derived from embry Successful. onic stem cells that have been cultured in the presence of a 0003 Recent evidence suggests that islet cell transplan retinoid. tation with improved systemic immunosuppression may 0008. In certain embodiments, the application provides a provide a short-term durable remission in insulin require method for promoting the maturation of beta cell precursor ments in type I diabetics (Shapiro et al., 2000, NEngl J Med. cells, the method comprising contacting the beta cell pre 343: 230-238; Ryan et al., 2001, Diabetes 50: 710-719). cursor cells with a BMP family member. However, in DM and the vast majority of other human 0009. In certain aspects the application provides methods diseases amenable to treatment by tissue replacement, there for increasing the number of beta cell precursor cells in a cell is an extreme shortage of engraftable donor tissues. An composition by culturing the cell composition in the pres expandable source of tissues like human stem cells may ence of an antagonist of a BMP signaling pathway. The provide the best promise for tissue replacement strategies for antagonist of a BMP signaling pathway may be a secreted human diseases. polypeptide. Such as noggin, chordin or follistatin that binds 0004 Stem cells, including embryonic stem (ES) cells directly to a BMP family member. and various adult stem cells provide a promising potential 0010. In certain aspects, the application provides cell means for cell-replacement therapy in human diseases. Stem culture methods that employ sequential inhibition and acti cells may provide serve as an inexhaustible source for the vation of a BMP signaling pathway to obtain insulin production of replacement islets for transplantation in dia producing cells. For example, the cells may be cultured in betic humans. However, conditions to produce stably-dif the presence of a noggin, chordin or follistatin and then ferentiated functional insulin-producing cell compositions cultured in the presence of a BMP family member, such as (ICCs) with stem cells generally, and particularly ES stem cells, have not been developed to a clinically satisfactory a member of the GDF8/GDF11 subfamily. Optionally, the level. stem cells are islet precursor cells. 0011. In certain aspects, the application provides methods 0005 Methods to provide a renewable source of replace for ameliorating a condition associated with insufficient ment islets from stem cells could transform therapeutics in pancreatic function by administering a BMP family member, DM. Likewise, methods for stimulating the production of such as a GDF8/GDF 11 subfamily member, or an activator insulin producing cells in patients could also have significant of a BMP signaling pathway. Suitable subjects include those therapeutic effects. that have been diagnosed with type I or type II diabetes. SUMMARY 0012. In certain aspects, the application provides meth ods for increasing the production of beta cell precursor cells 0006. In certain aspects, the application provides meth in a subject, the method comprising administering to the ods for manipulating the development of insulin producing subject a substance that inhibits a BMP signaling pathway, cells and beta cell precursor cells by activating or antago Such as a noggin, chordin or follistatin. In some instances, nizing a BMP signaling pathway. Such methods may be sequential use of BMP pathway antagonists and activators applied, for example, to cultured cells or to subjects in need may be desirable. of improved pancreatic function. 0013 Insulin production in a subject may also be 0007. In certain embodiments, the application provides enhanced by administering a composition comprising insu methods for increasing the production of insulin in a cell lin producing cells produced according to a method of the composition by culturing the cell composition in the pres application. US 2007/O 155661 A1 Jul. 5, 2007

0014. In certain aspects, the application provides meth acinar cell volume in Gdf11 mice. Original magnification ods for assessing the effectiveness of a test agent for 4x(a,b), 16x(de) and 10x(gh) modulating the development of insulin producing cells. For example, a method may comprise: forming a mixture com 0018 FIG. 3. Defective pancreatic islet B-cell and C.-cell prising the test agent and a BMP pathway polypeptide; and numbers in Gdfl 1 deficient mice. (a-c) Immunohistochemi detecting binding between the test agent and the polypep cal detection of insulin (brown staining) at E17.5 in mice tide, wherein a test agent that binds the BMP pathway with the indicated genotypes. (d) Quantification of E17.5 polypeptide specifically and with an affinity of at least f-cell mass by point-counting morphometry in Gdf11" ", 10M has an increased likelihood of being effective for Gdf11", and Gdf11 pancreata. Data are shown as the modulating the development of insulin producing cells. In a average measurements from at least 4 mice of indicated further method embodiment, a cell is cultured with the test genotypeststandard error of the mean. (e-g) Immunohis agent; and an activity of a BMP signaling pathway is tochemical detection of glucagon (brown staining) at E17.5 detected, wherein a test agent that increases the activity of in mice with the indicated genotypes. (h-i) Pancreatic islet a BMP signaling pathway has an increased likelihood of cell expression of insulin (FITC, green) and glucagon (Cy3, being effective for increasing insulin production in a cell. red) at postnatal day 1 (n=3 mice per genotype). Represen Test agents for promoting formation of beta cell precursor tative samples are shown. (k) Ratio of B-cell to C.-cell cells may similarly be tested by determining whether they Volume in mice with indicated genotypes. Data are shown as cause a decreases in the activity of a BMP signaling path the average measurements:tstandard error of the mean (n=3 way. Cells for use in Such assays preferably express an mice per genotype). Original magnification 4x(a-c, e-g) and ActRIIA and/or ActRIIB receptor. Optionally, assay cells 63x(h-j). comprise a reporter gene, wherein expression of the reporter 0019 FIG. 4. Premature expansion and increased num gene is positively or negatively regulated by a BMP signal bers of ngn3 pancreatic cells in Gdfl 1-deficient mice. ing pathway. The activity of a BMP signaling pathway may Pancreatic ngn3 expression (blue staining) detected by anti be assessed in a variety of ways, including measuring sense RNA in situ hybridization in Gdf11"" (a-c), Gdfl1" expression of a reporter gene regulated by a BMP signaling (d-f) and Gdf11 embryos (g-i) at the indicated embryonic pathway or by measuring a change in one or more compo stages. (j) Quantification of ngna" pancreatic cells/mm. nents of the BMP signaling pathway. For example, phos Data from 3-4 embryos per genotype are presented as the phorylation of one or more pathway proteins, such as averagetstandard error of the mean. Original magnification ActRIIA, ActRIIB, Smad2 and Smad3, may be evaluated. 10x(a-i). 0.015 The embodiments and practices of the present 0020 FIG. 5. Pancreatic defects in E17.5 Smad3 mutant application, other embodiments, and their features and char embryos. (a,b) Neurogenin-3 expression (blue staining) acteristics, will be apparent from the description, figures and detected by antisense RNA in situ hybridization. By E17.5 claims that follow, with all of the claims hereby being in wildtype pancreas, only small clusters of ngn3" cells are incorporated by this reference into this Summary. detected. In Smad3 heterozygous mutants, ngn3 expression is abnormally persistent in clustered periductal epithelial BRIEF DESCRIPTION OF THE DRAWINGS cells. (c,d) Nkx2.2 expression (brown nuclei) adjacent to ductal epithelium. (e.f) Nkx6.1 expression (brown nuclei 0016 FIG. 1. Gdf11 expression in the pancreatic islet marked by arrows). (gh) Insulin expression (green) detected progenitor cell niche. E13.5 pancreatic cells expressing ngn3 by IHC and confocal microscopy. Inset, h: ngn3" nuclei ((blue staining) are detected by antisense RNA in situ (Cy3, red) adjacent to insulin cells (FITC, green). These hybridization. ngn3"cells are found adjacent and contiguous images are representative of 5 or more animals per genotype. to Gdfl 1-expressing epithelial cells stained brown with an Original magnification 4x(a-f), 25x(gh). antiserum specific for Gdfl 1. Original magnification 16x. 0021 FIG. 6. Defects of B-cell maturation in Gdf11 0017 FIG. 2. Defective pancreatic development in mice. Measurements from E17.5 pancreata in the indicated embryos deficient for Gdf11. Gdfl 1 indicates that the genotypes. (a-b) Accumulation of cells expressing NkX6.1 observed phenotype is indistinguishable in Gdf11" and (darkly stained nuclei) in Gdfl1 mice. (c) Quantification Gdf11" embryos. (a,b) Whole mounted preparations of of NkX6.1" nuclei per mm tissue. Data here and in panels pancreas from Gdf11" and Gdf11 mice at postnatal day f and i are shown as the average measurements itstandard 1. (c) Pancreatic mass unaffected in Gdf11 mice. Data are error of the mean (n=3 mice per genotype). (d-e) Accumu shown as the average measurements from at least 4 mice of lation of cells expressing Nkx2.2 (darkly stained nuclei) in indicated genotypesistandard error of the mean. (de) Gdf11 mice. (f) Quantification of Nkx2.2"nucleiper mm Reduced branching of pancreatic epithelium in E13.5 tissue. (gh) Reduction of Is11 pancreatic ?cells in Gdf11 mice. Dark epithelium is stained using antiserum Gdf11-/- mice. (i) Quantification of Isl1" nuclei per mm specific for PDX1. (f) Quantification of branching defect in tissue. Original magnification 16x, (a-b; d-e; g,h). Gdfl 1-deficient mice. Arbitrary units measure individual epithelial clusters with a central lumen. Data are shown as 0022 FIG. 7. Insulin yields from embryoid bodies the average measurements from at least 4 mice of indicated derived from human embryonic stem cells (UC06) following genotypeststandard error of the mean. (gh) Reduced exo treatment with the sequence of indicated conditions. Con crine acinar cell development in Gdf11-deficient mice at ditions #1-9: exposure of human embryoid bodies to 2 E17.5. Dark epithelium is stained using antiserum specific micromolar retinoic acid (RA) for 7 days, followed by for carboxypeptidase A (carbA). Intralobular septae indi exposure for 7 days to (1) two micromolar RA, (2) 10 mM cated by the double-headed arrow. These septae are reduced Nicotinamide, (3) 10 micromolar LY294.002, (4) Both 10 in number in Gdfl 1-deficient mice. (i) Quantification of mM Nicotinamide and 10 micromolar LY294.002, (5) 10 morphometry showing significant reduction of exocrine ng/mL GDF8, (6) 10 nM Nicotinamide and 10 ng/mL GDF8, US 2007/O 155661 A1 Jul. 5, 2007

(7) 10 mM Nicotinamide and 10 micromolar LY294.002 and to be some variation in certain characteristics even within a 10 ng/mL GDF8, (8) 2 nM activin A, or (9) 10 mM recognizable cell type. A cell composition may comprise, in Nicotinamide and 2 nM activin A. Conditions #10-18: addition to cells, essentially any component(s) that are exposure of human embryoid bodies to 100 nM retinoic acid compatible with the intended use for the cell composition. (RA) for 7 days, followed by exposure for 7 days to (10) 100 For example, a cell composition may include media, growth nM RA, (11) 10 mM Nicotinamide, (12) 10 micromolar factors, pharmaceutically acceptable excipients, preserva LY294.002, (13) Both 10 nM Nicotinamide and 10 micro tives, a solid or semi-solid Substrate, a porous matrix or molar LY294.002, (14) 10 ng/mL GDF8, (15) 10 nM Nico scaffold, nonviable cells or a therapeutic agent. tinamide and 10 ng/mL GDF8, (16) 10 mM Nicotinamide 0029. The term “culturing includes exposing cells to any and 10 micromolar LY294.002 and 10 ng/mL GDF8, (17) 2 condition. While “culturing cells is often intended to pro nM activin A, (18) 10 mM Nicotinamide and 2 nM activin mote growth of one or more cells, “culturing cells need not A. Results are average of triplicate samples. promote or result in any cell growth, and the condition may DETAILED DESCRIPTION even be lethal to a substantial portion of the cells. 0030) A later cell is "derived” from an earlier cell if the 1. Definitions later cell is descended from the earlier cell through one or 0023 For convenience, certain terms employed in the more cell divisions. Where a cell culture is initiated with one specification, examples, and appended claims are collected or more initial cells, it may be inferred that cells growing up here. Unless defined otherwise, all technical and scientific in the culture, even after one or more changes in culture terms used herein have the same meaning as commonly condition, are derived from the initial cells. A later cell may understood by one of ordinary skill in the art to which this still be considered derived from an earlier cell even if there invention belongs. has been an intervening genetic manipulation. 0024. The articles “a” and “an are used herein to refer to 0.031) A member of the “GDF8/GDF11 subfamily” is a one or to more than one (i.e., to at least one) of the polypeptide (or an encoding nucleic acid) comprising an grammatical object of the article, unless context clearly amino acid sequence that is at least 80% identical to an indicates otherwise. By way of example, “an element' amino acid sequence of a mature, naturally occurring GDF8 means one element or more than one element. or GDF11 polypeptide, such as the mature human or mouse 0025. An "ActRIIA or ActRIIB signaling pathway” refers GDF8 and GDF11 sequences, a fragment thereof. A member to polypeptides and polypeptide interactions that participate of the GDF8/GDF11 subfamily should also retain the ability in transducing or otherwise effectuating changes in the to stimulate a receptor-mediated signaling pathway. properties of a cell upon stimulation of an ActRIIA and/or 0032. The term “including is used herein to mean, and ActRIIB receptor (e.g. by contacting the receptor with a is used interchangeably with, the phrase “including but not natural ligand such as a GDF8 or GDF11), including the limited to. receptors themselves. An example of an ActRIIA or ActRIIB signaling pathway is the ActRII-ActRI-Smad2/3-Smada 0033. The term “islet precursor cell” refers to any cell transcriptional regulation pathway. that has differentiated so as to be recognizably of pancreatic lineage and that differentiates under appropriate conditions 0026. The term “adult stem cell” is used herein to refer to to give rise to beta cell precursor cells. a stem cell obtained from any non-embryonic tissue. For example, cells derived from fetal tissue and amniotic or 0034. The term 'or' is used herein to mean, and is used placental tissue are included in the term adult stem cell. interchangeably with, the term “and/or, unless context Cells of these types tend to have properties more similar to clearly indicates otherwise. cells derived from adult animals than to cells derived from 0035. The term “percent identical refers to sequence embryonic tissue, and accordingly, for the purposes of this identity between two amino acid sequences or between two application stem cells may be sorted into two categories: nucleotide sequences. Percent identity can be determined by "embryonic' and “adult' (or, equivalently, “non-embry comparing a position in each sequence which may be onic'). aligned for purposes of comparison. Expression as a per 0027) “Beta cell precursor cells' are cells having gener centage of identity refers to a function of the number of ally mesenchymal, non-cell-cell adherent qualities that form identical amino acids or nucleic acids at positions shared by insulin producing cells under appropriate conditions. the compared sequences. Various alignment algorithms and/ or programs may be used, including FASTA, BLAST, or 0028. A "cell composition' is any composition of matter ENTREZ. FASTA and BLAST are available as a part of the generated by human manipulation that comprises viable GCG sequence analysis package (University of Wisconsin, cells as a Substantial component. A cell composition may Madison, Wis.), and can be used with, e.g., default settings. comprise more than one type of viable cell. An "enriched ENTREZ is available through the National Center for Bio cell composition' is a cell composition comprising a Sub technology Information, National Library of Medicine, stantially greater purity (i.e. at least twice as pure) of a National Institutes of Health, Bethesda, Md. In one embodi recognizable cell type than is found in any natural tissue. A ment, the percent identity of two sequences can be deter "pure cell composition' is a cell composition that comprises mined by the GCG program with a gap weight of 1, e.g., at least about 75%, and optionally at least about 85%, 90% or 95% of a recognizable cell type. A recognizable cell type each amino acid gap is weighted as if it were a single amino is generally one that has a reasonably uniform morphology, acid or nucleotide mismatch between the two sequences. a characteristic set of two or more molecular markers and a 0036). Other techniques for alignment are described in functional characteristic. It is understood that there is likely Methods in Enzymology, Vol. 266: Computer Methods for US 2007/O 155661 A1 Jul. 5, 2007

Macromolecular Sequence Analysis (1996), ed. Doolittle, chymal cell type shift, but has not undergone a reorganiza Academic Press, Inc., a division of Harcourt Brace & Co., tion back to the epithelial cell traits that are characteristic of San Diego, Cali., USA. Preferably, an alignment program mature pancreatic beta cells. that permits gaps in the sequence is utilized to align the 0040. While not wishing to be bound to theory, it is sequences. The Smith-Waterman is one type of algorithm contemplated that the development of pancreatic islets that permits gaps in sequence alignments. See Meth. Mol. involves a shifting of cell characteristics from epithelial to Biol. 70: 173-187 (1997). Also, the GAP program using the mesenchymal and then back to epithelial again. At an early Needleman and Wunsch alignment method can be utilized to stage of pancreatic development, the cells are organized in align sequences. An alternative search strategy uses a pancreatic duct. The cells of the pancreatic duct have MPSRCH software, which runs on a MASPAR computer. epithelial characteristics, meaning that the cells adhere MPSRCH uses a Smith-Waterman algorithm to score closely, possibly through tight junctions, to form sheets. To sequences on a massively parallel computer. This approach form islets, cells of the duct become mesenchymal in improves ability to pick up distantly related matches, and is character, meaning that the adhesions to neighboring cells especially tolerant of Small gaps and nucleotide sequence dissolve and the cells move away from the two dimensional errors. Nucleic acid-encoded amino acid sequences can be sheet. Then the cells develop adhesive, epithelial qualities used to search both protein and DNA databases. again and form into islets, separated from the original ductal 0037. The term “stem cell as used herein refers to an cell sheet. In addition, the final steps of islet cell maturation undifferentiated cell which is capable of proliferation and are marked by a cessation of proliferation. Members of the giving rise to at least one more differentiated cell type. BMP family may encourage the formation of epithelial “Totipotent stem cells' are stem cells that are capable of structures, thereby directing the dispersed mesenchymal giving rise to any cell type of the organism from which the cells (e.g. beta cell precursors) to form multicellular islet stem cells were obtained. “Pluripotent stem cells' are stem structures. Members of the BMP family also inhibit prolif cells that are capable of giving rise to cells of the three major eration, in part by causing expression of inhibitors of cyclin embryonic lineages, the endoderm, mesoderm and ecto dependent kinases (cdks). Such as p15, p17, p 18, p21, p27. derm. “Multipotent stem cells' are stem cells that are or p57. Inhibitors of the BMP signaling pathway may capable of giving rise to more than one type of more stimulate the formation of beta cell precursor cells in two differentiated cell. The term “stem cell' is also intended to ways. First, such inhibitors may stimulate epithelial cells of include cells of varying developmental potential that may be the duct to adopt mesenchymal characteristics, and second, obtained by somatic cell nuclear transfer or by causing a such inhibitors may prevent formation of the islet epithelial differentiated cell to undergo de-differentiation. For the Structures. purposes of this disclosure, a stem cell is named by the tissue 0041. Without regard to theories of pancreatic develop from which it was obtained. For example, a “neural stem ment, the present application demonstrates that the devel cell' is a stem cell obtained from a neural tissue (or a fluid, opment of islet cells and various precursors can be manipu such as cerebrospinal fluid that is in contact with neural lating by altering BMP signaling. tissue), a “neuroendocrine stem cell is a stem cell derived from a neuroendocrine tissue. Such as the adrenal gland or 0.042 A“BMP family member” may be selected from the the pituitary gland, but specifically excluding the pancreas. naturally occurring members of the TGF-beta/BMP family, An "embryonic stem cell is a stem cell obtained from an and may also be a functional analog thereof. Naturally embryo. Many “tissues are complex and actually contain occurring members of this family are characterized by several different stem cell types. For example, the skin may common sequence homology and as being proteins that are be considered a tissue, but skin contains neural stem cells of translated as preproprotein precursors. A preprotein precur the peripheral nervous system, skin stem cells from the Sor contains an N-terminal signal peptide, a prodomains and dermis, and stem cells from the blood circulating through the a mature portion. The mature portion contains six to nine skin. Accordingly, in determining the classification of a stem conserved cysteine residues that participate in forming inter cell, the true origin, including sub-tissue structures, should molecular disulfide bonds, although a few members (e.g. be carefully considered. GDF-9, BMP-15, GDF-3, lefty-1 and lefty-2) contain a serine substitution for a cysteine involved in disulfide bond 0038 A “stem cell line” is an enriched or pure cell formation. A dendrogram presented in Chang et al. (2002) composition comprising a recognizably distinct stem cell Endocrine Rev. 23(6):787-823 lists a number of vertebrate type that, when cultured in appropriate conditions, self BMP family members, including the following: TGF-beta2. propagates. TGF-beta3, TF-betal, GDF-15, GDF-9, BMP-15, BMP-16, 2. Methods for Promoting Formation of Beta Cell Precursors BMP-3, GDF-10, BMP-9, BMP-10, GDF-6, GDF-5, GDF and Increasing Insulin Production 7, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, BMP-4, GDF 3, GDF-1, GDF 11, GDF8, Activins betaC, betaE, betaA and 0039. In certain aspects the application provides methods betaB, BMP-14, GDF-14, MIS, Inhibin alpha, Lefty 1, for increasing insulin-production in a Suitable cell compo Lefty2, GDNF, Neurteurin, Persephin and Artemin. The sition by culturing the cell composition in the presence of a term “BMP family member is intended to include variant BMP family member or other activator of a BMP signaling forms of any of the naturally occurring polypeptides, includ pathway. Suitable cell populations will generally include ing fusion proteins, truncated proteins, and mutant forms, any cell populations containing stem cells that retain the although only if such variants retain the ability to stimulate ability to develop into insulin-producing cells. As used a receptor-mediated signaling pathway and further have herein, the term “stem cell' includes cells that are islet cell Sufficient sequence similarity to a naturally occurring family or beta cell precursors. Preferably a suitable cell population member as to be recognizable using a sequence comparison comprises a cell that has undergone an epithelial to mesen algorithm such as BLAST or PILEUP. The term “BMP US 2007/O 155661 A1 Jul. 5, 2007

family member is also intended to refer both to single 0046. In certain embodiments, the application provides polypeptides and homo- and hetero-dimers or other multi methods for increasing the number of beta cell precursor meric forms, as well as nucleic acids encoding polypeptides cells in a cell composition by culturing cells in the presence that are BMP family members. Several subgroupings are of an inhibitor of a BMP pathway. Inhibitors of a BMP also identifiable, including the “GDF8/GDF11 subfamily' pathway include noggins, chordins, follistatins, twisted gas (e.g. GDF8, GDF11), the “Activin C subfamily” (e.g. trulation (TSG), Dan, Cerberus and Xenopus nodal related 3 Activins betaC, betaE, and BMP-14), the “Activin A sub (Xnr3), to name only a few. In addition, antibodies that bind family’ (e.g. Activin betaA, betab), the “TGF-beta subfam to BMP family members and prevent receptor activation ily” (e.g. TGF-beta2. TGF-beta3, TF-beta1), the “BMP-5 may also be used as BMP pathway inhibitors. Public data subfamily” (e.g. BMP-5, BMP-6, BMP-7), the “BMP-8 base references for preferred BMP pathway inhibitors are set subfamily” (e.g. BMP-8), the “GDF-6 subfamily” (e.g. forth in Table 2, and Such references provide guidance as to GDF-5, GDF-6, GDF-7), the “BMP-9 subfamily” (e.g. the mature and pro-polypeptide sequences. The database BMP-9, BMP-10), the “BMP-3 subfamily” (e.g. BMP-3, entries listed in Table 2 are incorporated herein by reference, GDF-10), the “GDF-9 subfamily” (e.g. GDF-9, BMP-15) in their entirety. and the “GDF-15 subfamily” (e.g. GDF-15). TABLE 2 0.043 ABMP family member may be replaced by, or used in conjunction with, a BMP signaling pathway activator. A Selected Inhibitors of BMP Signaling BMP signaling pathway activator is a compound that stimu lates one or more of the polypeptides or interactions that Polypeptide Nucleic Acid participate in transducing or otherwise effectuating changes Protein Name Organism Database Ref. No. Ref. No. in the properties of a cell in response to a BMP family Noggin Human Q13253 BCO34O27 member. A BMP signaling pathway includes BMP family Noggin Mouse P97466 NM 008711 Chordin Human Q9H2XO XM 209529 members themselves. An example of a BMP signaling Chordin Mouse NP 034023 NM OO9893 pathway is the GDF11-ActRII-ActRI-Smad3/3-Smada tran Follistatin Human P19883 M1948O scriptional regulation pathway. The BMP family member M19481 binds to the extracellular ligand binding domain portion of Follistatin Mouse S4S321 X83377 the ActRII receptor and then forms a complex with ActRI, leading to the inhibition of the Smad7 negative regulator and phosphorylation of the Smad2/Smad3 complex. The Smad3/ 0047. In certain embodiments, a cell composition com Smad3 complex associates with Smad4 to regulate expres prising stem cells may be cultured sequentially with an sion of certain genes. In certain embodiments, activation of inhibitor of BMP signaling and then with an activator of a BMP signaling pathway leads to activation of small BMP signaling, thereby obtaining insulin-producing cells. inhibitors of cyclin-dependent kinases (cdks). Such as p15, p17, p.18, p21, p27 and p57. A BMP signaling pathway 0048 Stem cells for use in the methods disclosed herein activator is also a compound that antagonizes an inhibitor of may be essentially any stem cell that has not lost the a BMP signaling pathway, Such as an anti-noggin or anti potential to become a pancreatic hormone-producing cell. chordin antibody. The term “stem cell as used herein refers to an undiffer 0044 Accordingly, certain method embodiments of the entiated cell which is capable of proliferation and giving rise application comprise culturing stem cells, preferably embry to at least one more differentiated cell type. Stem cells may onic stem cells, to a desired stage and then contacting the be totipotent, pluripotent stem cells or multipotent. Stem cells with a BMP family member, preferably a GDF8/ cells may also be obtained by somatic cell nuclear transfer GDF11 subfamily member. Public database references for or by causing a differentiated cell to undergo de-differen preferred GDF8/GDF11 are set forth in Table 1, and such tiation. In certain embodiments, stem cells for use with the references provide guidance as to the mature and pro disclosed methods may be impure, Such as stem cells nested polypeptide sequences. The database entries listed in Table in a tissue or in a suspension obtained from a tissue sample. 1 are incorporated herein by reference, in their entirety. It is now widely believed that most adult tissues include Small populations of stem cells, as that term is used herein. 0045. In an exemplary embodiments, human embryonic Stem cells may also be enriched from tissue samples, and stem cells are cultured in the presence of a retinoid com may optionally be purified stem cells. Stem cells may also pound. Such as all-trans retinoic acid and then cultured in the be used from stem cell lines, and preferably from well presence of a GDF8/GDF11 subfamily member, thereby characterized and established stem cell lines. Tissue may be generating a cell composition comprising insulin-producing embryonic or “adult as the term is used herein, including cells. fetal, infant, child and mature animal tissue. Cells need not be obtained from a tissue, and other cell-containing sources TABLE 1. that are not generally considered “tissues may be employed (e.g. cerebrospinal fluid and mucus or secreted fluids of the Selected GDF3 GDF11 Subfamily Members lung or gut). Polypeptide Nucleic Acid Protein Name Organism Database Ref. No. Database Ref. No. 0049. In certain embodiments a stem cell for use in a disclosed method is an embryonic stem cell. Examples of GDF11 Human O95390 NM O05811 mouse embryonic stem cells include: the JM1 ES cell line GDF11 Mouse Q971W4 XM 125935 GDF8 Human O14793 AFO19627 described in M. Qiu et al., Genes Dev 9, 2523 (1995), and GDF8 Mouse OO8689 U84OOS the ROSA line described in G. Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cells described in U.S. Pat. No. 6,190,910. Many other mouse ES lines are available US 2007/O 155661 A1 Jul. 5, 2007

from Jackson Laboratories (Bar Harbor, Maine). Examples 225; 5,972,703, those described in PCT publication nos. WO of human embryonic stem cells include those available 00/53795; WO 00/02654; WO 98/20907, and those through the following Suppliers: Arcos BioScience, Inc., described in Pittenger et al. and Zhao et al., Supra. Foster City, Cali. CyThera, Inc., San Diego, Cali. BresaGen, Inc., Athens, Ga., ES Cell International, Mel 0052 Stem cell lines are preferably derived from mam bourne, Australia, Geron Corporation, Menlo Park, Calif., mals, such as rodents (e.g. mouse or rat), primates (e.g. Göteborg University, Göteborg, Sweden, Karolinska Insti monkeys, chimpanzees or humans), pigs, and ruminants tute, Stockholm, Sweden, Maria Biotech Co. Ltd.—Maria (e.g. cows, sheep and goats), and particularly from humans. Infertility Hospital Medical Institute, Seoul, Korea, Miz In certain embodiments, stem cells are derived from an Medi Hospital Seoul National University, Seoul, Korea, autologous source or an HLA-type matched source. For National Centre for Biological Sciences/Tata Institute of example, stem cells may be obtained from a Subject in need Fundamental Research, Bangalore, India, Pochon CHA Uni of pancreatic hormone-producing cells (e.g. diabetic patients versity, Seoul, Korea, Reliance Life Sciences, Mumbai, in need of insulin-producing cells) and cultured by a method India, Technion University, Haifa, Israel, University of Cali described herein to generate autologous insulin-producing fornia, San Francisco, Calif., and Wisconsin Alumni cells. Other sources of stem cells are easily obtained from a Research Foundation, Madison, Wis. In addition, examples Subject, Such as stem cells from muscle tissue, stem cells of embryonic stem cells are described in the following U.S. from skin (dermis or epidermis) and stem cells from fat. patents and published patent applications Nos.: 6.245,566: Insulin-producing cells may also be derived from banked 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780; stem cell sources, such as banked amniotic epithelial stem 20020045259; 20020068045. In preferred embodiments, the cells or banked umbilical cord blood cells. human ES cells are selected from the list of approved cell 0053. In certain embodiments, a stem cell may be derived lines provided by the National Institutes of Health and from a cell fusion or dedifferentiation process, such as accessible at http://escr.nih.gov. In certain preferred embodi described in the following US patent application: ments, an embryonic stem cell line is selected from the 20020090722, and in the following PCT applications: group consisting of the WA09 line obtained from Dr. J. WO200238741, WOO151611, WO9963.061, WO9607732. Thomson (Univ. of Wisconsin) and the UC01 and UC06 lines, both on the current NIH registry. 0054. In some preferred embodiments, a stem cell line 0050. In certain embodiments, a stem cell for use in should be compliant with good tissue practice guidelines set disclosed methods is a stem cell of neural or neuroendocrine for the by the U.S. Food and Drug Administration (FDA) or origin, such as a stem cell from the central nervous system equivalent regulatory agency in another country. Methods to (see, for example U.S. Pat. Nos. 6,468,794; 6,040, 180; develop Such a cell line may include donor testing, and 5,753,506; 5,766,948), neural crest (see, for example, U.S. avoidance of exposure to non-human cells and products Pat. Nos. 5,589,376; 5,824,489), the olfactory bulb or during derivation of the stem cell lines. Preferably the stem peripheral neural tissues (see, for example, Published US cell line can be prepared and used without the use of a feeder Patent Applications 20030003574; 20020123143: layer or any type of virus or viral vector. 20020016002 and Gritti et al. 2002 J Neurosci 22(2):437 0055. In certain preferred embodiments, both the stem 45), the spinal cord (see, for example, U.S. Pat. Nos. cells and differentiated cells of the methods and composi 6,361.996, 5,851,832) or a neuroendocrine lineage, such as tions disclosed herein have a wild-type genotype, meaning the adrenal gland, pituitary gland or certain portions of the that the genotype of the cells is a genotype that may be found gut (see, for example, U.S. Pat. Nos. 6,171,610 and PC12 in a subject organism naturally. For example, cells having cells as described in Kimura et al. 1994. J. Biol. Chem. 269: chromosomal rearragements as a result of culture treatments 18961-67). In preferred embodiments, a neural stem cell is are not cells having a wild-type genotype. As a further obtained from a peripheral tissue or an easily healed tissue example, cells that have been transfected with an integrating from a patient in need of cells that produce a pancreatic nucleic acid construct will not (except in cases of perfect hormone, thereby providing an autologous population of excision) have a wild-type genotype. The term 'genotype cells for transplant. does not refer to peripheral modifications to the genomic 0051. In certain embodiments, hematopoietic or mesen nucleic acids, such as methylation, and therefore, cells chymal stem cells may be employed in a disclosed method. having a naturally occurring genetic makeup may have Recent studies Suggest that marrow-derived hematopoietic unnatural phenotypes as an effect of changes in methylation (HSCs) and mesenchymal stem cells (MSCs), which are or other modifications. readily isolated, have a broader differentiation potential than 0056 Certain embodiments of the methods disclosed previously recognized. Purified HSCs not only give rise to herein are advantageous in part because they permit the all cells in blood, but can also develop into cells normally generation of insulin-producing cell compositions from derived from endoderm, like hepatocytes (Krause et al., starting materials, such as certain stem cell lines, that are 2001, Cell 105: 369-77: Lagasse et al., 2000 Nat Med 6: available, as a practical matter, in Sufficient quantities for 1229-34). MSCs appear to be similarly multipotent, produc formation of a therapeutically effective insulin-producing ing progeny that can, for example, express neural cell implant. By contrast, for example, fetal pancreatic tissue, markers (Pittenger et al., 1999 Science 284: 143-7; Zhao et and particularly human fetal pancreatic tissue, is only avail al., 2002 Exp Neurol 174: 11-20). Examples of hematopoi able in Small quantities, making it difficult or impossible to etic stem cells include those described in U.S. Pat. Nos. assemble sufficient material to form a therapeutically effec 4,714,680; 5,061,620; 5,437,994; 5,914,108; 5,925,567; tive implant. 5,763, 197; 5,750,397; 5,716,827; 5,643,741; 5,061,620. Examples of mesenchymal stem cells include those 0057. In certain embodiments, an insulin-producing cell described in U.S. Pat. Nos. 5,486,359; 5,827,735; 5,942, is exposed to an additional culture condition. For example, US 2007/O 155661 A1 Jul. 5, 2007 insulin-producing cells may be treated with any of the 0062. In additional embodiments, the application pro various agents, and functional analogs thereof, that are vides methods for ameliorating, in a subject, a condition known to stimulate insulin production or beta-cell prolifera related to insufficient pancreatic function by administering tion. Examples of Such agents include IGF-1 (e.g. at a to the Subject an effective amount of an insulin producing concentration of 10 ng/ml), glucagon-like peptides (e.g. cell composition. In certain embodiments, administration of GLP-1), exendin-4, HGF, and reagents that increase cAMP a BMP signaling activator may be used to ameliorate a levels, such as membrane permeable forms of cAMP and condition related to insufficient pancreatic function. In yet forskolin. another embodiments, administration of a BMP signaling 3. Insulin-Producing Cell Compositions inhibitor may be used to ameliorate a condition related to 0.058. In certain embodiments, the application provides insufficient pancreatic function. The benefit from an inhibi insulin-producing cell compositions produced according to tor versus an activator of BMP signaling may depend on any of the methods disclosed herein. Insulin-producing cell whether regeneration of insulin-producing cells is limited by compositions may be in any form, including, preferably, in a failure in maturation of beta cell precursor cells, or a insulin-producing cell clusters, but optionally in dispersed failure in the formation of beta cell precursor cells. Option ally, a subject may be administered a BMP signaling inhibi cell Suspensions, confluent cell cultures, seeded on a matrix tor followed by an activator. BMP signaling inhibitors or or other cell Support, etc. activators may be coadministered with insulin-producing 0059. In further embodiments, the invention relates to cells. Preferrably, a sufficient amount of one or more of the insulin-producing cell compositions in which at least about above therapeutic compositions is administered to a subject 50% of the cells are positive for insulin production, option to cause an increase in blood insulin levels or an improve ally at least 75% of the cells are positive for insulin ment in glucose homeostasis. Glucose homeostasis may be production and preferably at least 85%, 90% or 95% of the tested by administering a dose of glucose and monitoring the cells are positive for insulin production. In certain embodi kinetics with which blood glucose levels decline. Conditions ments, most of the cells, and preferably greater than 80%, related to insufficient pancreatic function include the various 90% or 95% of the cells, that produce insulin are negative forms of diabetes mellitus (e.g. type I and type II), NOD for other pancreatic hormones that are not naturally pro mice (a type I diabetes model), the Streptozotocin-induced duced by native pancreatic insulin producing cells, such as diabetes rodent model, Surgically-induced diabetes models glucagon. and diseases resulting from dysfunctional islet growth (e.g. 0060. In certain embodiments, an insulin-producing cell insulinomas). Administration of an insulin-producing cell composition comprises at least about 1000 nanograms (ng) composition may not produce a permanent ameliorating of insulin per milligram of total protein, optionally at least effect, and periodic dosing, such as on a weekly, monthly or about 5000 nanograms of insulin per milligram of total yearly basis may be beneficial. protein and preferably at least about 10000 nanograms of insulin per milligram of total protein. In embodiments where 0063. In preferred embodiments, an effective dose of the insulin-producing cell composition comprises islet-like insulin-producing cell composition comprises administering cell clusters of roughly 300-400 um in diameter, the clusters at least about one islet-like cell cluster of the invention (or produce greater than 0.2 ng of insulin per hour, and prefer an equivalent number of cells) per islet that is naturally ably greater than 0.5 ng of insulin per hour. In preferred present in the Subject organism. For example, mice have embodiments, insulin production by the insulin-producing about 300-500 islets, rats have about 3000-5000 islets and cell composition is stimulated by exposure to glucose. humans have about 1,000,000 islets, and accordingly, a 0061. In certain embodiments, insulin-producing cell preferred dosage is about 300-500 islet-like cell clusters for compositions comprise cells that are positive for one or amouse, about 3000-5000 islet-like cell clusters for a rat and more of the following markers: insulin (any of the various about 1,000,000 islet-like cell clusters for a human. The chains), islet-1, PDX1, GLUT2, glucokinase, a cdk inhibi number of islets per organism is proportional to average tor, and a tight junction protein, such as a connexin. In body mass (20-30 grams, mouse, 200-300 grams, rat, 60-70 certain embodiments, at least about 50% of the cells in an kilograms, human) and it may be desirable to administer a insulin-producing cell composition are not proliferative. dosage that is proportional to body mass of the Subject. In Proliferating cells may be detected by a variety of ways instances when an islet-like cell cluster is less efficient at known in the art, including staining with Ki67, a nuclear producing insulin than a native islet, or where an insulin marker of proliferating cells, or incorporation of labeled producing cell composition is Subject to cell mortality (e.g. nucleotide (e.g. tritiated thymidine or bromodeoxyuridine). in the case of host immune system-mediated rejection), the In preferred embodiments, insulin-producing cell composi dosage may be increased proportionally. tions do not form neoplastic growths when implanted in a 0064. In certain embodiments, the invention relates to Subject. It is understood that biological systems are tremen therapeutic compositions comprising insulin-producing cell dously variable and, depending on host and implant char compositions and methods for making Such therapeutic acteristics, even a very safe insulin-producing cell compo compositions. Therapeutic compositions include an insulin sition is likely to form, or appear to form, a neoplastic producing cell compositions disclosed herein and/or made growth at Some low frequency. In certain embodiments the by the methods disclosed herein, as well as mixtures com insulin-producing cell compositions of the invention pro prising Such insulin-producing cell compositions and a duce a neoplastic growth in a fewer than 30% of implanted therapeutic excipient. Examples of therapeutic excipients subject, optionally in fewer than 1% of implanted subjects include matrices, scaffolds or other substrates to which cells and preferably in fewer than 0.1% of implanted subjects. may attach (optionally formed as Solid or hollow beads, 4. Administration of Insulin-Producing Cell Compositions tubes, or membranes), as well as reagents that are useful in and/or Factors facilitating administration (e.g. buffers and salts), preserving US 2007/O 155661 A1 Jul. 5, 2007 the cells (e.g. chelators such as sorbates, EDTA, EGTA, or 0070. In a further method embodiment, a cell is cultured quaternary amines or other antibiotics), or promoting with the test agent; and an activity of a BMP signaling engraftment. pathway is detected, wherein a test agent that increases the activity of a BMP signaling pathway has an increased 0065 Cells may be encapsulated in a membrane to avoid likelihood of being effective for increasing insulin produc immune rejection. By manipulation of the membrane per tion in a cell. Test agents for promoting formation of beta meability, so as to allow free diffusion of glucose and insulin cell precursor cells may similarly be tested by determining back and forth through the membrane, yet block passage of whether they cause a decreases in the activity of a BMP antibodies and lymphocytes, normoglycemia may be main signaling pathway. Cells for use in Such assays preferably tained (Sullivan et al. (1991) Science 252:718). In a second express an ActRIIA and/or ActRIIB receptor. Optionally, approach, hollow fibers containing cells may be immobi assay cells comprise a reporter gene, wherein expression of lized in a polysaccharide alginate. (Lacey et al. (1991) the reporter gene is positively or negatively regulated by a Science 254:1782). Cells may be placed in microcapsules BMP signaling pathway. A reporter gene may be designed to composed of alginate or polyacrylates. (Lim et al. (1980) be activated by Smada, which is positively regulated by Science 210:908; O'Shea et al. (1984) Biochim. Biochys. BMP signaling. Examples of Smad4 regulated genes include Acta. 840: 133: Sugamori et al. (1989) Trans. Aim. Soc. Artif. cdk inhibitors, such as p15, p17, p 18, p21, p27 and p57. The Intern. Organs 35:791; Levesque et al. (1992) Endocrinol activity of a BMP signaling pathway may also be assessed ogy 130:644; and Lim et al. (1992) Transplantation by measuring a change in one or more components of the 53:1180). BMP signaling pathway. For example, phosphorylation of 0.066 Additional methods for encapsulating cells are one or more pathway proteins, such as ActRIIA, ActRIIB, known in the art. (Aebischer et al. U.S. Pat. No. 4,892,538; Smad2 and Smad3, may be evaluated. Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) 7. Methods for Assessing Candidate Islet Cell Differentia Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. tion Factors and Other Test Compounds Biomech. Eng. 113:178-183, U.S. Pat. No. 4,391,909; U.S. 0071. In certain embodiments, the application provides Pat. No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artif. methods for obtaining beta cell precursor cell populations as Intern. Organs 35:791-799: Sefton et al. (1987) Biotehnol. well as insulin-producing cells, and Such cells may be used Bioeng. 29:1135-1143; and Aebischer et al. (1991) Bioma for a variety of purposes, such as the identification of terials 12:50-55). markers for these cell types. 0067. The site of implantation of insulin-producing cell 0072. In certain aspects the application provides methods compositions may be selected by one of skill in the art. In for assessing whether a test agent has beta cell precursor cell general. Such as site preferably has adequate blood perfusion differentiation activity. An exemplary embodiment of such a to allow the cells to sense blood conditions and secrete method may comprise contacting beta cell precursor cells hormones and other factors into the general circulation. with a test agent and detecting a beta cell marker. Generally, Exemplary implantation sites include the liver (via portal a test agent that stimulates the formation of cells expressing vein injection), the peritoneal cavity, the kidney capsule and islet cell markers has beta cell precursor cell differentiation the pancreas. activity activity. The term “beta cell marker” is intended to include any phenotype that is distinctive of one or more islet 6. Methods for Identifying Agents for Modulation of Pan cell types, including various protein, nucleic acid, morpho creatic Cell Development logical, biochemical (e.g. metabolic or transport) or other 0068. In certain aspects, the application provides meth phenotypes. Examples of beta cell markers include the ods for assessing the effectiveness of a test agent for following polypeptides or the corresponding RNA tran modulating the development of insulin producing cells. Script: insulin (any of the various chains, including, for Such methods may be used to screen libraries of test agents. example, C-peptide, mature insulin or proinsulin), GLUT2, In general, a method of this type involves assessing the glucokinase, PDX-1, IAPP. SUR1, PC1/3, PC2, KIR6.2, ability of the test agent to interfere with or promote BMP pancreatic polypeptide, Somatostatin, glucagon, glucokinase pathway signaling. Two non-limiting categories of assays and C-peptide. In an illustrative embodiment, the subject include biochemical assays and cell-based assays. cells can be used to screen various compounds or natural products, such as Small molecules or growth factors. The 0069. In certain embodiments, a test agent may be mixed efficacy of the test agent can be assessed by generating dose with a BMP pathway polypeptide; and a test agent that binds response curves. A control assay can also be performed to the BMP pathway polypeptide specifically and with an affinity of at least 10 M, and preferably 10, 10, 107 or provide a baseline for comparison. less, has an increased likelihood of being effective for 0073. In certain embodiments, methods of the application modulating the development of insulin producing cells. An relate to the identification of pancreatic developmental agent that binds may inhibit signaling, as in the case of an markers. For example, expression patterns of established agent that binds and titrates a way a BMP family member. markers may be monitored at one or more stages of differ Alternatively, an agent that binds may activate, as in the case entiation of stem cells into beta cell precursors and insulin of an agent that binds to an ActRIIA or ActRIIB receptor and producing cells. Markers may be assessed using standard mimics activation by a BMP family member. Test agents methods, including Northern blotting, RT-PCR, in situ may be assessed in an assay system that measures binding hybridization (ISH), immunohistochemistry (IHC) as well between a BMP family member, such as a GDF8/GDF11 as nucleic acid or protein array or microarray-based meth family member, and a receptor, such as an ActRIIA or ods. In certain embodiments, monitoring production of one ActrB. or more gene products will be useful to identify candidate US 2007/O 155661 A1 Jul. 5, 2007 cell-surface proteins for FACS-based purification strategies by genetic modification, to become hyperproliferative. Such for insulin-producing cell precursors. hyperproliferative cells may be contacted with compounds 0074. In certain embodiments, the application provides to identify, for example, anti-proliferative and anti-neoplas methods for identifying affinity reagent that bind to cells at tic agents (e.g. agents that inhibit cell growth or promote cell various stages of pancreatic development. Affinity reagents death). The efficacy of the compound can be assessed by include antibodies, and preferably monoclonal antibodies, generating dose response curves from data obtained using targeting peptides (e.g. peptides selected from a high diver various concentrations of the compound. A control assay can sity phage display library), RNA or DNAaptamers. The term also be performed to provide a baseline for comparison. “antibody' as used herein is intended to include whole Identification of the progenitor cell population(s) amplified antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), in response to a given test agent can be carried out according and includes fragments thereof which are also specifically to Such phenotyping as described above. Assays such as reactive with a vertebrate, e.g., mammalian, protein. Anti those described above may be carried out in vitro (e.g. with bodies can be fragmented using conventional techniques and cells in culture) or in vivo (e.g. with cell implanted in a the fragments screened for utility and/or interaction with a Subject). specific epitope of interest. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared por 8. Methods for Identifying Stem Cells tions of an antibody molecule that are capable of selectively 0076. In certain embodiments, the application relates to reacting with a certain protein. Non-limiting examples of methods for identifying a cell that has the potential to Such proteolytic expressing and/or recombinant fragments develop into a pancreatic cell, and particularly an insulin include Fab, F(ab')2, Fab'. Fv, and single chain antibodies producing cell. In one aspect, the method comprises pro (scFv) containing a VL and/or VH domain joined by a viding a stem cell line, or other multipotent cell line, and peptide linker. The sclv’s may be covalently or non-co differentiating the cell line so as to obtain an insulin valently linked to form antibodies having two or more producing cell composition. At the beginning of the differ binding sites. The term antibody includes polyclonal, mono entiation process, or at Some stage within the differentiation clonal, or other purified preparations of antibodies and process, the differentiating cells are mixed with a cell of recombinant antibodies. In certain embodiments, beta cell interest. The differentiation of the cell of interest may then precursors or insulin producing cells may be used to Screen be assessed. A cell of interest that is able to differentiate into a plurality of affinity reagents. The cells themselves may be an insulin-producing cell is a cell that has the potential to used for the screening, or membrane or protein extracts may develop into an insulin-producing cell. In further embodi be used. Likewise, cell surface proteins may be selectively ments, the cell may be assessed for the production of other labeled and used to Screen a plurality of affinity reagents. In pancreatic products, such as glucagons, to identify cells that a preferred embodiment, the plurality of affinity reagents to have the potential to develop into other types of pancreatic be screened is a library of monoclonal antibodies. An affinity cells. In certain embodiments, a pancreatic tissue (e.g. ductal reagent detected as binding to a cell Such as an beta cell tissue, adult pancreatic tissue, fetal pancreatic tissue, etc.) precursor cell may be tested on tissue samples for capability may be dissociated into a cell Suspension, and clumps of to detect particular subpopulations of pancreatic or pre cells or single cells are used as the cell of interest in the pancreatic cells, and it is of particular interest to identify above method embodiments, thereby permitting a rapid affinity reagents that are useful in the identification of natural screen of pancreatic cells for candidate pancreatic progeni populations of cells that are precursors of beta cells or other tors. By using inhibitors of BMP signaling, it will be islet cells. possible to obtain large numbers of beta cell precursor cells, 0075 Yet another aspect of the present application pro which enables identification of markers for this population vides methods for screening various compounds for their of cells. ability to modulate insulin-producing cells, such as, for 0077. In one embodiment, insulin-producing cell compo example, by affecting growth, proliferation, maturation or sitions and methods for generating such compositions may differentiation, or by affecting insulin production, Secretion be used to assess the developmental potential of a cell of or storage, as well as compounds that may improve graft interest. In some embodiments, the developmental potential performance (e.g. result in a longer-lasting graft, improved of a cell of interest may be determined by mixing the cell of insulin production, or changes in proteins that interact with interest with cells during the process of making beta pre the host immune system). In an illustrative embodiment, the cursor or insulin-producing cells (i.e. co-culturing). The cell Subject cells can be used to screen various compounds or of interest is then tracked (for example by a transgenic natural products, such as Small molecules or growth factors. marker) to determine the types of cells that arise from it. In Such compounds may be tested for essentially any effect, an exemplary embodiment, the cell of interest is mixed with with exemplary effects being cell proliferation or differen differentiating neural or neuroendocrine stem cells. tiation, insulin production, or cell death. In further embodi ments, insulin-producing cells may used to test the activity 0078. In certain embodiments, culture systems for mak of compounds/factors to promote Survival and maturation, ing insulin-producing cell compositions may be used as part and further, since certain cells produced according to meth of an assay to identify candidate pancreatic endocrine pre ods disclosed herein have one or more properties of islet cursor cells. Current evidence Suggests that such precursors cells, specifically B-cells, such cells may be used to identify exist as single cells or Small cell clusters within or closely factors (or genes) that regulate production, processing, Stor associated with pancreatic epithelium. In certain embodi age, Secretion, and degradation of insulin or other relevant ments, cell compositions in the process of differentiating proteins (like IAPP, glucagon, including pro-glucagon, into beta cell precursor cells or insulin-producing cells GLPs, etc) produced in pancreatic islets. In further embodi provide the appropriate cellular microenvironment to permit ments, an insulin-producing cell may be modified. Such as pancreas-derived endoderm to integrate and differentiate. In US 2007/O 155661 A1 Jul. 5, 2007

certain embodiments, cells of a pancreatic tissue are frac 0082 Mice heterozygous for Smad2 mutation are viable, tionated and mixed, either as populations of cells or as single and similar to ActRIIB-f- and ActRIA+/-IIB+/- mutant cells, into cells being differentiated into insulin-producing mice, have (1) increased production of cells in the embry cell compositions. Cells of the pancreatic tissue that develop onic pancreas expressing neurogenin3, a marker of pancre into insulin-producing cells are candidate pancreatic stem atic islet precursors (2) increased numbers of immature cells. In certain embodiments, instead of a co-culture, a pancreatic B-cells late in gestation (e.g., cells expressing the fraction of cells that are in the process of differentiating into transcription factor Nkx6.1 but not insulin) (3) evidence of insulin-producing cell compositions may be used in the impaired islet maturation after birth, with islet hypoplasia culture medium of the cells of interest. Fractions that may be and reduced B-cell mass, (4) normal islet architecture, used include conditioned media or other preparations of without evidence of other organ malformations (5) impaired secreted material, extracellular matrix, membrane prepara glucose tolerance, and (6) inadequate blood insulin levels. tions, total soluble protein, soluble cellular protein and other These results are encompassed in a manuscript in prepara portions of cells that are in the process of differentiating into tion (Harmon et al). Thus, our studies of pancreas develop beta cell precursors or insulin-producing cells. ment and glucose physiology in Smad3 mutant provide unexpected evidence for a molecular connection between EXAMPLES TGF-B signaling and (I) regulation of islet precursor cell 0079 The invention now being generally described, it progenitor development and (II) B-cell maturation. These will be more readily understood by reference to the follow studies corroborate the studies of GDF-11 described below. ing examples, which are included merely for purposes of Data are shown in FIG. 5. illustration of certain aspects and embodiments of the present invention, and are not intended to limit the inven Example 3 tion. In Vitro Studies of Noggin, Chordin. GDF11 and Example 1 GDF8 Activity on Mouse ES Cells During Formation of Insulin-producing Cell Clusters GDF-11 and Pancreatic Development (IPCCs). 0080 Growth and differentiation factor 11 (GDF-11; also 0083) GDF8 (which is ~80% identical to GDF11 in the known as GDF11) is a recently identified member of the mature region and has similar in vitro activities in a neural TGF-Bligand superfamily (Nakashima et al., 1999; Gameret cell development assay as shown by published studies from al., 1999). Homozygous null GDF-11 mutants manifest the laboratory of Thomas Jessell, Columbia University) several defects including skeletal transformations, and promotes insulin production in mouse ES cell-derived abnormal Hox gene expression that resemble defects IPCCs. In duplicate experiments, we detect approximately observed in ActRIIB mutants (McPherron and Lee, 1999). 6000 ng insulin/mg protein produced by mouse embryoid GDF-11 expression in pancreatic epithelium begins by E9.5- bodies treated with a sequence of growth factors including 10 in pancreatic epithelium, continues there throughout GDF8. Data are shown in FIG. 7. gestation, and is later abundantly expressed in isolated adult islets. We have characterized foregut development in GDF INCORPORATION BY REFERENCE 11 mutant embryos, and found (1) hyposplenism with 0084 All publications and patents mentioned herein are splenic malformations, (2) defects in axial patterning of hereby incorporated by reference in their entirety as if each stomach epithelium and mesenchyme, and (3) severe pan individual publication or patent was specifically and indi creatic defects including islet hypoplasia, defects in islet cell vidually indicated to be incorporated by reference. In case of differentiation, increased accumulation of neurogenin3-ex conflict, the present application, including any definitions pressing cells (presumptive islet cell precursors) during herein, will control. embryonic pancreas formation, and reduced B-cell mass. Thus, morphogenetic defects in pancreas development, are Equivalents strikingly similar in GDF-11 and ActRIIB mutants (Kim et 0085 While specific embodiments of the subject inven al., 2000). Our analysis identifies GDF-11 as a candidate tions have been discussed, the above specification is illus TGF-B ligand that regulates development of pancreatic islet trative and not restrictive. Many variations of the inventions precursor cells and Subsequent maturation of pancreatic will become apparent to those skilled in the art upon review B-cells to functioning insulin-producing and secreting cells. of this specification and the claims below. The full scope of Data are shown in FIGS. 1-4 and 6. the inventions should be determined by reference to the claims, along with their full scope of equivalents, and the Example 2 specification, along with Such variations. Smad2 and Pancreatic Development 1. A method for increasing insulin production in a cell 0081) Numerous in vitro and in vivo studies demonstrate composition comprising stem cells, the method comprising that Smad2 activity is regulated both by ActRIIA and contacting the cell composition with a BMP family member. ActRIIB. Smad2 encodes a transcription factor which is 2. The method of claim 1, wherein the BMP family phosphorylated by type I activin or TGF-B receptors after member is a member of a GDF8/GDF11 subfamily. ligand binding to type II and type I receptors. In mice, 3. (canceled) Smad3, ActRIIA, ActRIIB and other TGF-B signaling com 4. The method of claim 1, wherein the BMP family ponents are expressed in embryonic pancreas, and later in member binds to an ActRIIA and/or ActRIIB receptor. adult islets. We investigated roles of Smad2 in pancreas 5. The method of claim 4, wherein the BMP family development and function. member increases one or more of the following in a stem US 2007/O 155661 A1 Jul. 5, 2007 cell: (a) Smad3 phosphorylation (b) Smad3 phosphorylation 23. A method for culturing a cell composition comprising and (c) expression of a gene that is positively regulated by stem cells to obtain an insulin producing cell composition, Smada. the method comprising: 6. (canceled) a) culturing the cell composition in the presence of a 7. The method of claim 1, wherein the cell composition substance that inhibits a BMP signaling pathway; and comprises beta cell precursor cells. 8. A method for promoting the maturation of beta cell b) culturing cells from (a) in the presence of a Substance precursor cells, the method comprising contacting beta cell that stimulates a BMP signaling pathway. precursor cells with a BMP family member. 24. The method of claim 23, wherein the substance that 9. The method of claim 8, wherein the beta cell precursor inhibits a BMP signaling pathway is a secreted polypeptide cells express neuroD, nkx6.1 and/or nk33 2.2. that binds directly to a BMP family member. 10. A method for increasing insulin production in a cell 25. The method of claim 23, wherein the substance that composition comprising stem cells, the method comprising inhibits BMP signaling is selected from the group consisting contacting the cell composition with a Substance that stimu of a noggin and a chordin. lates an ActRIIA and/or ActRIIB signaling pathway. 26. The method of claim 23, wherein the substance that 11. The method of claim 10, wherein the substance causes stimulates a BMP signaling pathway is a BMP family an increase in one or more of the following (a) Smad3 member. function (b) Smad3 function and (c) expression of a gene 27. The method of claim 26, wherein the BMP family that is positively regulated by Smada. member is a member of a GDF8/GDF11 subfamily. 12. (canceled) 28. (canceled) 13. (canceled) 29. The method of claim 23, wherein the stem cells are 14. The method of claim 10, wherein the substance is a islet precursor cells. member of the GDF8/GDF11 subfamily. 30. (canceled) 15. The method of claim 10, wherein the substance is a 31. A method for increasing insulin production in a nucleic acid comprising a nucleic acid sequence encoding a Subject, the method comprising administering to the Subject positive regulator of the ActRIIA and/or ActRIIB signaling a BMP family member in an amount sufficient to increase pathway. insulin production. 16. The method of claim 10, wherein the positive regu 32. The method of claim 31, wherein the subject has been lator is selected from the group consisting of Smad2. diagnosed with type I or type II diabetes. Smad3, Smad4 and a GDF8/GDF11 subfamily member. 33. The method of claim 31, wherein the BMP family 17. The method of claim 10, wherein the substance causes member is a member of a GDF8/GDF11 subfamily. the inhibition of an inhibitor of the ActRIIA and/or ActRIIB 34. A method for increasing the production of islet pre signaling pathway. cursor cells in a Subject, the method comprising adminis tering to the subject a substance that inhibits a BMP signal 18. (canceled) ing pathway. 19. A method for promoting the formation of beta cell 35. The method of claim 34, wherein the substance that precursor cells in a cell composition comprising stem cells, inhibits BMP signaling is selected from the group consisting the method comprising contacting the cell composition with of a noggin, a chordin and a follistatin. a substance that inhibits a BMP signaling pathway. 36. A method for increasing insulin production in a 20. The method of claim 19, wherein the substance that Subject comprising administering to the Subject a composi inhibits a BMP signaling pathway is a secreted polypeptide tion comprising insulin producing cells produced according that binds directly to a BMP family member. to the method of any of claims 1, 8, 10 or 23. 21. The method of claim 19, wherein the substance that 37. A method for increasing the number of beta cell inhibits a BMP signaling pathway is selected from the group precursor cells in a Subject comprising administering to the consisting of a noggin, a chordin and a follistatin. Subject a composition comprising beta cell precursor cells 22. The method of claim 19, wherein the substance that antagonizes a BMP signaling pathway is a nucleic acid produced according to the method of claim 19. comprising a coding sequence for a polypeptide antagonist 38.-46. (canceled) of a BMP signaling pathway.