WO2004010933A2 - Insulin-producing cell compositions and related methods - Google Patents

Abstract

The disclosure relates, in part, to insulin-producing cell compositions, methods for generating insulin-producing cell compositions by using a cell proliferation inhibitor, and therapeutic and non-therapeutic methods for using the insulin-producing cell compositions.

Description

INSULIN-PRODUCING CELL COMPOSITIONS AND RELATED METHODS

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 60/398,939, filed July 25, 2002, entitled "Insulin-Producing Cell Compositions", naming inventors S. K. Kim, Y. Hori and I. Rulifson and U.S. Provisional Application No. 60/426,632, filed November 14, 2002, entitled "Insulin-Producing Cell Compositions", naming inventors S. K. Kim, Y. Hori and I. Rulifson. The entire teachings of the referenced applications are incorporated by reference herein in their entirety. BACKGROUND OF THE INVENTION

Diabetes mellitus (DM) is a major cause of morbidity and mortality worldwide, and incidence rates of type I and type II DM are increasing. In type I DM, destruction of insulin- producing pancreatic islets leads to a prolonged illness often culminating in devastating multisystem organ failure and early mortality. Clinical trials demonstrate that tight glucose regulation can prevent the development of diabetic complications, but attempts to achieve this regulation by exogenous insulin administration are only partially successful. Recent evidence suggests that islet cell transplantation with improved systemic immunosuppression may provide a short-term durable remission in insulin requirements in type I diabetics (Shapiro et al, 2000, N Engl J Med. 343: 230-238 ; Ryan et al, 2001, Diabetes 50: 710-719). However, in DM and the vast majority of other human diseases amenable to treatment by tissue replacement, there is an extreme shortage of engraftable donor tissues. An expandable source of tissues like human stem cells may provide the best promise for tissue replacement strategies for human diseases.

Stem cells, including embryonic stem (ES) cells and various adult stem cells provide a promising potential means for cell-replacement therapy in human diseases. Stem cells may provide serve as an inexhaustible source for the production of replacement islets for transplantation in diabetic humans. However, conditions to produce stably-differentiated functional insulin-producing cell compositions (ICCs) with stem cells generally, and particularly ES stem cells, have not been developed to a clinically satisfactory level.

Methods to provide a renewable source of replacement islets from stem cells could transform therapeutics in DM.

SUMMARY In certain embodiments, the invention relates to methods for making insulin-producing cell compositions by a method that comprises culturing a suitable cell composition in the presence of a cell proliferation inhibitor. In certain embodiments, the invention relates to methods for making insulin-producing cell compositions by a method that comprises culturing a suitable cell composition in the presence of a phosphatidylinositol 3' -kinase (PI3K) pathway inhibitor. Optionally, the suitable cell composition is a stem-cell derived cell composition, such as a composition derived from a mammalian embryonic stem cell line or adult stem cell line. In certain embodiments, the stem cell-derived cell composition comprises a detectable amount of a marker that is indicative of neural, hepatic or pancreatic cells. In preferred embodiments, the suitable cell composition is cultured in the presence of both a PI3K pathway inhibitor and a poly adenosine diphosphate ribosyl transferase (ADPRT) inhibitor, such as a nicotinamide agent or a benzamidine.

In certain embodiments, a method of making an insulin-producing cell composition comprises culturing a cell composition comprising stem cells so as to produce a cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, and then further culturing the cell composition in the presence of a cell proliferation inhibitor to obtain an insulin-producing cell composition. In certain embodiments, the stem cells are from a stem cell line. Optionally the one or more markers are selected from the group consisting of: nestin, neurogenin, islet- 1, glucagon and insulin. Optionally the cell line is initially cultured in the presence of leukemia inhibitory factor, or a functional variant thereof. Optionally, the cell composition is cultured in the presence of insulin, transferrin, selenium and fibronectin, or functional analogs thereof, followed by culturing in the presence of basic fibroblast growth factor, or a functional analog thereof. In preferred embodiments, the cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, is cultured in the presence of both a cell proliferation inhibitor and a poly adenosine diphosphate ribosyl transferase (ADPRT) inhibitor, such as a nicotinamide agent or a benzamidine.

In certain embodiments, a method of making an insulin-producing cell composition comprises culturing a cell composition comprising stem cells so as to produce a cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, and then further culturing the cell composition in the presence of a PI3K pathway inhibitor to obtain an insulin-producing cell composition, h certain embodiments, the stem cells are from a stem cell line. Optionally the one or more markers are selected from the group consisting of: nestin, neurogenin, islet- 1, glucagon and insulin. Optionally the cell line is initially cultured in the presence of leukemia inhibitory factor, or a functional variant thereof. Optionally, the cell composition is cultured in the presence of insulin, transferrin, selenium and fibronectin, or functional analogs thereof, followed by culturing in the presence of basic fibroblast growth factor, or a functional analog thereof. Optionally, the PI3K pathway inhibitor is a PI3K inhibitor, such as an agent that inhibits PI3K activity or an agent that decreases the level of PI3K protein subunit(s) in the cell.

In certain embodiments, the invention provides insulin-producing cell compositions generated according to any of the methods disclosed herein. In certain embodiments, an insulin-producing cell composition comprises at least about 1000 nano grams of insulin per milligram of total protein, and preferably at least about 5000 or 10000 nano grams of insulin per milligram of total protein. Optionally, an insulin-producing cell composition of the invention is such that at least about 80%, 90%, 95% or 99% of the cells of the insulin-producing cell composition produce insulin.

In additional embodiments, the invention relates to therapeutic cell compositions comprising an insulin-producing cell composition disclosed herein and a therapeutically acceptable excipient. The insulin-producing cell compositions may also, in certain embodiments, b e used for therapeutic purposes without an excipient. C ertain aspects o f the invention also relate to methods of making a therapeutic cell compositions, comprising mixing an insulin-producing cell composition with a therapeutically acceptable excipient. In further embodiments, the invention provides for the use of an msulin-producing cell composition d isclosed h erein for t he m anufacture o f a m edicament for t he amelioration o f a condition related to insufficient pancreatic function, such as a form of diabetes. Optionally, the insulin-producing cell composition is mixed or otherwise combined with a therapeutically acceptable excipient. The insulin-producing cell compositions may also, in certain embodiments, be used for therapeutic purposes without an excipient. An insulin-producing cell composition for in the manufacture of a medicament may be an insulin-producing cell composition generated according to any of the methods disclosed herein. In certain embodiments, the insulin-producing cell composition comprises at least about 1000 nanograms of insulin per milligram of total protein, and preferably at least about 5000 or 10000 nanograms of insulin per milligram of total protein. Optionally, the insulin-producing cell composition is such that at least about 80%, 90%, 95% or 99% of the cells of the insulin-producing cell composition produce insulin. In yet further embodiments, the invention relates to methods of ameliorating, in a subject, a condition related to insufficient pancreatic function, such as a form of diabetes. In certain embodiments, a method comprises administering to a subject an effective amount of an insulin producing cell composition disclosed herein or made according to a method disclosed herein. In certain preferred embodiments, the administration of an insulin-producing cell composition causes an increase in blood insulin levels in the subject. In further preferred embodiments, the administration of an insulin-producing cell composition improves blood glucose homeostasis. In yet additional preferred embodiments, the insulin producing cell composition c auses a neoplasm in fewer than 30% or 1 5% o f subjects and, in a p articularly preferred embodiment, fewer than 5% or fewer than 1% of subjects. In certain embodiments, the invention relates to methods for testing the developmental potential of a cell of interest. An embodiment of such a method comprises subjecting a stem cell line to an in vitro differentiation process so as to cause a cell of the stem cell-derived cell composition to give rise to a cell that produces a pancreatic hormone, mixing a cell of interest with the stem cell-derived cell composition at a point in the differentiation process and determining the identity of cells derived from the cell of interest, thereby testing the developmental potential of the cell of interest. Optionally, the cell of interest is obtained from a pancreatic tissue, and in preferred embodiments the method is operated repeatedly (in series, in parallel, orb oth) t o a ssess the d evelopmental p otential o f a v ariety o f cells o btained from a pancreatic tissue, h certain embodiments, the in vitro differentiation process comprises contacting the stem cell-derived cell composition with a cell proliferation inhibitor. In a preferred embodiment, the in vitro differentiation process comprises contacting the stem cell- derived cell composition with a PI3K pathway inhibitor. In further embodiments, methods for testing developmental potential comprise forming a mixed cell composition comprising the cell of interest and a cell composition suitable for culturing in the presence of a PI3K inhibitor to create a mixed cell composition, culturing the mixed stem cell-derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining a mixed insulin-producing cell composition; and determining the identity of cells derived from the cell of interest, thereby testing the developmental potential of the cell of interest. In other embodiments, the cell of interest is mixed with the starting cell culture (e.g. cells of a stem cell line) or at another stage of the differentiation process . hi additional embodiments, the invention relates to methods for identifying a candidate gene related to pancreatic development. An embodiment of such a method comprises measuring a first level of a gene product in a stem cell-derived cell composition, culturing the stem-cell derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining an insulin-producing cell composition, and measuring a second level of the gene product in the msulin-producing cell composition, wherein a gene that encodes the gene product that has a significantly different first and second levels is a candidate gene related to pancreatic development. Gene products include transcripts, proteins, post-translational modifications etc., and may be measured, for example, by nucleic acid or protein array systems (e.g. microarrays) or other methods such as two-dimensional gel electrophoresis, mass spectroscopy, etc.

In yet further embodiments, insulin-producing cell compositions may be contacted with a test compound to screen for compounds that have desired effects, such as proliferative or insulin-production promoting effects, on insulin-producing cells. It is further contemplated that insulin-producing cells may be genetically modified. For example, insulin-producing cells may be genetically modified to become hyperproliferative, and such hyperproliferative cells may be used to test for anti-neoplastic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Similarities of gene expression and mitotic status in pancreatic β cells and insulin- producing cells comprising stage 5NL ICCs. Images were obtained by confocal microscopy from at least 3 samples for each immunohistochemical probe. Simultaneous immunofiuorescent detection of insulin and (A-C) glucagon, (D-F) α-fetoprotein (α-FP), (G-I) MAP2, or (J-L) β- tubulinlll (β tublll). Insulin staining appears as a pale gray in each box, while glucagon, α-FP, MAP2, or β tublll appear as darker gray. In mature islets there is localized expression of insulin and glucagon (A), with insulin appearing primarily in the center of the field and glucagon most apparent around the periphery. There is no detectable expression of α-FP (D) or the neuronal markers MAP2 (G) and β tublll (J). hi each of these fields, the only visible immunofluorescence is for insulin. Stage 5NL ICCs cellular homogeneity (B, E, H, K) contrasts with cellular heterogeneity o f stage 5NB ICCs (C, F, I, L). In B , E , H and K, the immunofluorescence is almost entirely or entirely insulin. In C, F, I and L, there is substantial immunofluorescence from the other marker. In C, there is roughly equal signal from insulin and glucagon. In F, there is somewhat more α-FP signal than insulin signal. In I, there is roughly two to five times more MAP2 signal than insulin signal, i L, there is roughly equal to double β tublll signal relative to insulin signal. (M-O) Immunofiuorescent detection of Ki67, a nuclear marker of proliferating cells, shown in gray-scale. Stage 5NL ICCs (N) are predominantly non- proliferating, similar to mature pancreatic islets (M), whereas stage 5NB culture conditions result in significant numbers of proliferating cells (O). (P) Teratoma (white arrows) formation 3 weeks after transplantation of 300 stage 5NB ICCs under the left kidney capsule (pink tissue to left of rumor). (Q) Lack of tumor growth following renal transplantation of 300 stage 5NL ICCs (graft indicated by dashed lines and white arrow). (R) hnmunohistochemical detection of insulin (pale gray) and glucagon (dark gray) expression in stage 5NL ICCs three weeks after engraftment; the signal is almost exclusively insulin immunofluorescence. Magnification in panels A-L and R is equal. Panel R inset shows a merge (lighter gray) of simultaneous immunohistochemical detection of insulin (pale gray) and C-peptide expression (dark gray) in these engrafted ICCs; most of the signal indicates overlap in insulin and C-peptide expression. Figure 2. Stage 5NL ICCs express several characteristic pancreatic β cell markers. Immunofiuorescent staining of adult pancreatic islets and stage 5NL ICCs for insulin and C- peptide (A and B) (showing substantial overlap in expression of these markers), insulin and proinsulin (C and D) (showing interspersed insulin positive cells and proinsulin positive cells in the islet, with a somewhat greater extent of overlap in the 5 NL ICCs), Pdxl (E and F) (dark gray, abundant in both fields), Glut-2 (G and H) (dark gray, abundant in each field), and glucokinase (I and J) (dark gray, abundant in both fields). K, RT-PCR analysis of ICC gene expression during stages 1-4, and in hand-isolated 5NB and 5NL ICCs, and wildtype pancreatic islets. Islet- 1 and neurogenin-3 are transcription factors required for islet formation in mice (25, 26) and Nestin is an intermediary filament protein (2). In stage 5 ICCs applicants detected carboxypeptidase A, but not pancreatic amylase, both products of pancreatic exocrine (acinar) cells. Amylase transcripts are routinely detected by RT-PCR in pancreatic islet preparations because of adherent acinar cells (13). Other gene products are described in the examples, below.

Figure 3. Transplanted stage 5NL ICCs function in vivo to rescue and ameliorate diabetic phenotypes. (A) Kaplan-Meier survival distribution following stage 5NL ICC transplantation (n= 7) or sham transplant (n=17). ICC graft removal 20 days after engraftment resulted in increased mortality, in contrast to unilateral nephrectomy in surviving control sham-transplanted mice. (B) Random-fed blood glucose levels in STZ treated mice with transplantation of stage 5NL ICCs (n=7) or in surviving sham-transplanted mice (n=8). Two asterisks indicate P <0.01, three asterisks indicate P <0.005. (C) Intraperitoneal glucose challenge in fasted mice 21 days after transplant with ICCs (n=8; black squares) or sham-transplant (n=14; gray circles). Two asterisks indicate P <0.01. Asterisk indicates P <0.05. Figure 4. In v itro i nsulin r elease from s tage 5 NL and 5 NB ICCs w as m easured a s follows. Plates were washed with no glucose RPMI 1 640 (GIBCO) three times. 10 ICCs w ere h and- picked and incubated with 25 mM glucose in RPMI 1640 for each time at 37oC and 5% CO₂. The supernatant was collected and insulin levels were determined using mouse insulin ELISA kit (ALPCO, Widham, NH). Figure 5. PI3K inhibition followed by PI3K activation induces ICC growth. Immunofluorescence staining of ICCs treated with only stage 5NL conditions (A) or stage 5NL and stage 6NI conditions (B) express insulin (pale gray). Greyscale conversion of A and B, C and D respectively, demonstrates not only an overall increase in insulin expression (black) but also an overall increase in cell growth with the addition of stage 6 conditions. Figure 6. Integration, survival, and β-galactosidase expression of ROSA-lacZ mouse pancreatic cells (dark, arrows) in ICCs (spheres) after 14 days co-culture. 400X magnification

DETAILED DESCRIPTION

1. Definitions For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "adult stem cell" is used herein to refer to a stem cell obtained from any non- embryonic tissue. For example, cells derived from fetal tissue and amniotic or placental tissue are included in the term adult stem cell. Cells of these types tend to have properties more similar to cells derived from adult animals than to cells derived from embryonic tissue, and accordingly, for the purposes of this application stem cells may be sorted into two categories: "embryonic" and "adult" (or, equivalently, "non-embryonic").

A "patient" or "subject" to be treated by the method of the invention can mean either a human or non-human animal, preferably a mammal. A "cell composition" is any composition of matter g enerated by human manipulation that comprises viable cells as a substantial component. A cell composition may comprise more than one type of viable cell. An "enriched cell composition" is a cell composition comprising a substantially greater purity (i.e. at least twice as pure) of a recognizable cell type than is found in any natural tissue. A "pure cell composition" is a cell composition that comprises at least about 75%, and optionally at least about 85%, 90% or 95% of a recognizable cell type. A recognizable cell type is generally one that has a reasonably uniform morphology, a characteristic set of two or more molecular markers and a functional characteristic. It is understood that there is likely to be some variation in certain characteristics even within a recognizable cell type. A cell composition may comprise, in addition to cells, essentially any component(s) that are compatible with the intended use for the cell composition. For example, a cell composition may include media, growth factors, pharmaceutically acceptable excipients, preservatives, a solid or semi-solid substrate, a porous matrix or scaffold, nonviable cells or a therapeutic agent.

A "cyclic AMP stimulating agent" or "cAMP stimulating agent" is any agent that causes an increase in cAMP mediated cell signaling. Exemplary cyclic AMP stimulating agents include forskolin and membrane diffusible cAMP analogues and phosphodiesterase inhibitors including 3-isobutly-l -methyl xanthine (IBMX).

A later cell is "derived" from an earlier cell if the later cell is descended from the earlier cell through one or more cell divisions. Where a cell culture is initiated with one or more initial cells, it may be inferred that cells growing up in the culture, even after one or more changes in culture condition, are derived from the initial cells. A later cell may still be considered derived from an earlier cell even if there has been an intervening genetic manipulation.

The term "culturing" includes exposing cells to any condition. While "culturing" cells is often intended to promote growth of one or more cells, "culturing" cells need not promote or result in any cell growth, and the condition may even be lethal to a substantial portion of the cells.

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

An "insulinogenic agent", as the term is used herein, is an agent that promotes proliferation, maturation, survival or differentiation of insulin producing cells, or that promotes insulin production, maturation, storage or secretion. Exemplary insulinogenic agents include: IGF-1, HGF, a cyclic AMP stimulating agent, exendin, GLP1, PPARγ ligand, sonic hedgehog, PACAP and growth hormone.

The term "LY294002" or "L", as used herein, refers to the phosphatidylinositol 3-kinase inhibitor, 2-(4-Mθ holinyl)-8-phenyl-4 H-l-benzopyran-4-one; as described by Vlahos, et al. (1994) J. Biol., Chem., 269(7) 5241-5248, and is available from Calbiochem Corp., La Jolla Calif.

The term "marker" as used herein refers to a detectable aspect of a cell. For example, an insulin marker may include an insulin transcript or an insulin polypeptide, such as proinsulin, the alpha chain, the beta chain or the C peptide. A cell is "positive" for a marker if that marker is convincingly detected in the cell. The term "nicotinamide agent" includes nicotinamide and analogs thereof that are biocompatible. Optionally, a nicotinamide agent has ADPRT inhibitory activity.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.

The term "percent identical" refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.

Expression a s a p ercentage o f i dentity refers t o a function o f t he n umb er o f i dentical a mino acids or nucleic acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin,

Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the

National Center for Biotechnology Information, National Library of Medicine, National

Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division o f Harcourt Brace & C o., S an Diego, C alifornia, USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

The term "PI3K" refers to a phosphatidylinositol (PI) 3'-kinase, a family of proteins that phosphorylate the inositol ring of PI in the D-3 position. The canonical mammalian PI3K is a heterodimeric complex that contains p85 and a 110-Kd protein (pi 10) (Carpenter et al. (1990) J.

Biol. Chem. 265, 19704). The purified p85 subunit has a regulatory role while the 110-Kd subunit harbors the catalytic activity.

The "PI3K pathway" refers to the collection of molecular actors that are influenced by the primary effects of PI3K activity, as well as the collection of molecular actors that have primary effects on PI3K activity. It is understood that a cellular signaling event, such as an increase in the activation of a PI3K, will generally produce effects that occur independent of any changes in gene expression, referred to herein as "primary" effects, such as certain changes in protein-protein interactions, certain changes in protein activities and certain changes in post- translational modifications. The PI3K pathway is not intended to encompass the vast array of actors that become involved only as a result of changes in gene expression.

A "PI3K pathway inhibitor" is any substance or mixture of substances that decreases a cellular activity that is increased in response to PI3K activity. A PI3K pathway inhibitor may be a " PI3K i nhibitor", m eaning t hat t he i nhibitor a cts b y d ecreasing P I3K a ctivity. A P I3K inhibitor may, for example, inhibit an activity of a PI3K enzyme or decrease the level of protein of one or more PI3K subunits.. Alternatively, a PI3K pathway inhibitor may act by affecting a part of the PI3K pathway. Exemplary PI3K pathway inhibitors include wortmannin, LY294002, a PI3K-targeted RNAi, etc.

A "poly-adenosine diphosphate ribosyl transferase inhibitor" or "ADPRT inl ibitor" includes any compound or treatment that inhibits the ADPRT enzyme. Exemplary ADPRT inhibitors include nicotinamide and N-substituted benzamidines.

The term "stem cell" as used herein refers to an undifferentiated cell which is capable of proliferation and giving rise t o at 1 east o ne m ore d ifferentiated c ell t ype. "Totipotent s tem cells" are stem cells that are capable of giving rise to any cell type of the organism from which the stem cells were obtained. "Pluripotent stem cells" are stem cells that are capable of giving rise to cells of the three major embryonic lineages, the endoderm, mesoderm and ectoderm. "Multipotent stem cells" are stem cells that are capable of giving rise to more than one type of more differentiated cell. The term "stem cell" is also intended to include cells of varying developemental potential that may be obtained by somatic cell nuclear transfer or by causing a differentiated cell to undergo de-differentiation. For the purposes of this disclosure, a stem cell is named by the tissue from which it was obtained. For example, an "embryonic stem cell" is a stem cell obtained from an embryo and a "pancreatic stem cell" is a stem cell obtained from a pancreas, understanding that many different stem cell types may usually be obtained from a single tissue source.

A "stem cell line" is an enriched or pure cell composition comprising a recognizably distinct stem cell type that, when cultured in appropriate conditions, self-propagates. The term "stem cell line" is explicitly intended to include cell compositions that have not been genetically manipulated or extensively passaged, just so long as there has been enrichment for stem cells.

A "stem cell line-derived cell composition" or (in shorter but interchangeable form) a "stem cell-derived cell composition" is a cell composition that is obtained by culturing a stem cell line to obtain the desired cell composition. A stem cell line may be cultured through a series of different culture conditions or selections (such as fluorescence activated cell sorting or other methods for separating different cell types) to obtain the stem cell-derived cell composition.

2. Methods for Generating Insulin-Producing Cell Compositions In certain embodiments, the invention relates to methods of generating insulin-producing cell compositions by exposing a suitable cell composition to an inhibitor of cell proliferation. Preferably the suitable cell composition is exposed to an inhibitor of the PI3K pathway. A suitable cell composition is one that, when exposed to an effective amount of an inhibitor of cell proliferation in an appropriate medium develops increased insulin production. In some preferred embodiments, the suitable cell composition is a stem cell-derived cell composition. While not wishing to be bound to any particular mechanism, it is expected that cell proliferation inhibitors and PI3K pathway inhibitors have a stabilizing effect on the cell identity of stem cell derived cells, thereby decreasing the likelihood that such cells will become hyperproliferative (e.g., tumorigenic) upon transplantation into a subject. In certain embodiments, a stem cell-derived cell composition for use in the methods disclosed herein may be essentially any composition of cells that results from culturing cells of a stem cell line. Suitable stem cell lines include embryonic stem cell lines and adult stem cell lines, whether totipotent, pluripotent, multipotent or of lesser developmental capacity. Stem cell lines are preferably derived from mammals, such as rodents (e.g. mouse or rat), primates (e.g. monkeys, chimpanzees or humans), pigs, and ruminants (e.g. cows, sheep and goats). Examples of mouse embryonic stem cells include: the JM1 ES cell line described in M. Qiu et al., Genes Dev 9, 2523 (1995), and the ROSA line described in G Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cells described in US Patent No. 6,190,910. Many other mouse ES lines are available from Jackson Laboratories (Bar Harbor, Maine). Examples of human embryonic stem cells include those available through the following suppliers: Arcos Bioscience, Inc., Foster City, California, CyThera, Inc., San Diego, California, BresaGen, Inc., Athens, Georgia, ES Cell International, Melbourne, Australia, Geron Corporation, Menlo Park, California, Gδteborg University, Gδteborg, Sweden, Karolinska Institute, Stockholm, Sweden, Maria Biotech Co. Ltd. - Maria Infertility Hospital Medical Institute, Seoul, Korea, MizMedi Hospital - Seoul National University, Seoul, Korea, National Centre for Biological Sciences/ Tata Institute of Fundamental Research, Bangalore, India, Pochon CHA University, Seoul, Korea, Reliance Life Sciences, Mumbai, India, Technion University, Haifa, Israel, University of California, San Francisco, California, and Wisconsin Alumni Research Foundation, Madison, Wisconsin. In addition, examples of embryonic stem cells are described in the following U.S. patents and published patent applications: 6,245,566; 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780; 20020045259; 20020068045. hi preferred embodiments, the human ES cells are selected from the list of approved cell lines provided by the National Institutes of Health and accessible at http://escr.nih.gov. Examples of human adult stem cells include those described in the following U.S. patents and patent applications: 5,486,359; 5,766,948; 5,789,246; 5,914,108; 5,928,947; 5,958,767; 5,968,829; 6,129,911; 6,184,035; 6,242,252; 6,265,175; 6,387,367; 20020016002; 20020076400; 20020098584; and, for example, in the PCT application WO 0111011. In certain preferred embodiments, a stem cell line is selected from the group consisting of: the WA09 line obtained from Dr. J. Thomson (Univ. of Wisconsin) and the UC01 and UC06 lines, both on the current NTH registry. In other preferred embodiments, an adult stem cell line is a neurosphere-type neural stem cell line or a multipotential adult stem cell as described by Verfaillie et al. in WO 0111011. In certain embodiments, a suitable cell composition may be derived from a cell fusion or dedifferentiation process, such as described in the following US patent application: 20020090722, and in the following PCT applications: WO200238741, WO0151611, WO9963061, WO9607732. A stem cell line, as the term is used herein, may include cells cultured directly from a tissue sample in such a way as to enrich for one or more types of stem cells. A passaged stem cell line is one that has been propagated through at least two media changes or growth substrate changes since being obtained from a tissue sample.

In some preferred embodiments, a stem cell line should be compliant with good tissue practice guidelines set for the by the U.S. Food and Drag Administration (FDA) or equivalent regulatory agency in another country. Methods to develop such a cell line may include donor testing, and avoidance of exposure to non-human cells and products during derivation of the stem cell lines. Preferably the stem cell line can be prepared and used without the use of a feeder layer or any type of virus or viral vector.

In certain preferred embodiments, the cells of the insulin-producing cell composition have a wild-type genotype, meaning that the genotype of the cells is a genotype that may be found in a subject organism naturally. For example, cells having chromosomal rearragements as a result of culture treatments are not cells having a wild-type genotype. As a further example, cells that have been transfected with an integrating nucleic acid construct will not (except in cases of perfect excision) have a wild-type genotype. The term "genotype" does not refer to peripheral modifications to the genomic nucleic acids, such as methylation, and therefore, cells having a naturally occurring genetic makeup may have unnatural phenotypes as an effect of changes in methylation or other modifications.

In certain embodiments the suitable cell composition, such as a stem cell-derived cell composition, is positive for markers that are traditionally associated with neural cell types (referred to herein as "markers that are indicative of neural cell types"), such as: nestin, neurogenin, MAP2 and beta-tubulin III. Optionally, at least about 5%, at least about 20%>, at least about 50%, at least about 75%, or at least about 90% of the cells of the stem cell-derived cell composition are positive for a marker that is indicative of a neural cell type. In certain embodiments, the stem cell-derived cell composition is positive for markers that are traditionally associated with pancreatic cell types (referred to herein as "markers that are indicative of pancreatic cell types") such as: insulin (including proinsulin, alpha chain, beta chain or C peptide), glucagon, GLUT2, islet amyloid polypeptide (IAPP), glucokinase, HNF3β , HNFlα, HNF4α, HNF3α, or PDX1 (also known as IPF1, STF1 and IDX1). In certain embodiments, the stem cell-derived cell composition is positive for markers that are traditionally associated with hepatic cell types or "markers that are indicative of hepatic cell types", such as:, GLUT2, HNF3β , HNFlα, HNF4α, HNF3α, alpha-fetoprotein and glucokinase. As will be appreciated by one of skill in the art, certain markers are associated with both pancreatic and hepatic lineages. In certain preferred embodiments, insulin-producing cell compositions may be derived from hematopoietic or mesenchymal stem cells, such as stem cell populations dervied from adult human bone marrow. Recent studies suggest that marrow-derived hematopoietic (HSCs) and mesenchymal stem cells (MSCs), which are readily isolated, have a broader differentiation potential than previously recognized. Purified HSCs not only give rise to all cells in blood, but can also develop into cells normally derived from endoderm, like hepatocytes (Krause et al., 2001, Cell 105: 369-77; Lagasse et al., 2000 Nat Med 6: 1229-34). In at least one report (Lagasse et al, 2000 Nat Med 6: 1229-34), the possibility of somatic cell fusion was ruled out. MSCs appear to be similarly multipotent, producing progeny that can, for example, express neural cell markers (Pittenger et al., 1999 Science 284: 143-7; Zhao et al., 2002 Exp Neurol 174: 11-20).

In certain embodiments, insulin-producing cell compositions are derived from an autologous source or an HLA-type matched source. For example, HSCs may be obtained from the bone marrow of a subject in need of msulin-producing cell compositions and cultured by a method described herein to generate an autologous insulin-producing cell compositions. Other sources of stem cells are easily obtained from a subject, such as stem cells from muscle tissue, stem cells from skin (dermis or epidermis) and stem cells from fat. Insulin producing cell compositions may also be derived from banked stem cell sources, such as banked amniotic epithelial stem cells or banked umbilical cord blood cells.

Certain embodiments of the methods disclosed herein are advantageous in part because they permit the generation of insulin-producing cell compositions from starting materials, such as many of the passaged stem cell lines identified herein, that are available, as a practical matter, in sufficient quantities for formation o f a therapeutically e ffective insulin-producing implant. By contrast, for example, fetal pancreatic tissue, and particularly human fetal pancreatic tissue, is only available in small quantities, making it difficult or impossible to assemble sufficient material to form a therapeutically effective implant. h certain embodiments, a stem cell-derived cell composition suitable for treatment with a PI3K pathway inhibitor may be generated according to the following steps. First, cells of the stem cell line are cultured in the presence of leukemia inhibitory factor (LIF), or a functional analog thereof. Optionally the cells are cultured on a feeder layer, and exemplary feeder layers include irradiated fibroblasts, preferably species-matched (e.g. mouse fibroblasts for mouse stem cells, human fibroblasts for human stem cells) and Matrigel (BD Biosciences, Lexington, Connecticut). Then the cells are cultured in the absence of the LLF (or LIF functional analog). Optionally, the cells are also cultured in the absence of the feeder layer on an adhesive or non- adhesive surface. Subsequently, the cells are cultured in the presence of ITSFn (a mixture of insulin, transferrin, selenium and fibronectin, or functional analogs thereof). An example of an ITSFn medium is described in Lee et al. Nature Biotechnol. 18: 675 (2000). The resulting cells are cultured in the presence of basic fibroblast growth factor and neurotrophic factors (e.g. B27 supplement), or functional analogs thereof, thereby generating a stem cell-derived cell composition for use in making an insulin-producing cell composition.

Once a suitable stem cell-derived cell composition or other suitable cell composition has been obtained, it may be cultured in the presence of an amount of a cell proliferation inhibitor sufficient to decrease the rate of cell proliferation. A PI3K pathway inhibitor may be used to achieve an inhibition of cell proliferation. A PI3K inhibitor may also be selected for effects that are independent of effects on cell proliferation. A cell proliferation inhibitor may be selected as agent that increases the expression and/or activity of one or more cell cycle inhibitors, such as cyclin-dependent kinases (cdks), e.g., pl5, pl7, pl8, p21, p27, or p57. Members of the TGF- beta family, and particularly GDFl l, have been demonstrated to have such activity (Wu et al. 2003 Neuron Jan 23 ;37(2): 197-207), and accordingly, GDFll is a preferred cell proliferation inhibitor. Optionally, the cell composition is also contacted with an inhibitor of ADPRT, such as a nicotinamide agent or a benzamidine agent. In certain preferred embodiments, the cell composition is contacted with the PI3K pathway inhibitor in the absence of B27 supplement, optionally in the absence of neurotrophic factors. A variety of PI3K pathway inhibitors may be employed in the methods disclosed herein, including direct inhibitors that act directly on a PI3K subunit (or decrease the amount thereof) and pathway inhibitors that affect one or more other molecular actors in the PI3K pathway. PI3K pathway inhibitors may have both direct and indirect actions.

Phosphatidylinositol 3-kinase, has been found to phosphorylate the 3-position of the inositol ring of phosphatidylinositol (PI) to form phosphatidylinositol 3-phosphate (PI-3P) (Whitman et al.(1988) Nature, 322: 664-646). In addition to PI, this enzyme also can phosphorylate phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PIP3), respectively (Auger et al. (1989) Cell, 57: 167-175). Direct PI3K inhibitors include materials that reduce or eliminate either or both of these activities of P3 K. Such inhibitors include direct PI3K inhibitors such as Ly294002 (Calbiochem Corp., La Jolla, Calif.) and wortmannin (Sigma Chemical Co., St. Louis Mo. The chemical properties of Ly294002 are described in detail in J. Biol, Chem., (1994) 269: 5241-5248. Ly294002 is stable in tissue culture medium and is membrane permeable. Wortmannin tends to degrade in cell cultures with a half-life of about 4-5 hours.

Other inhibitors of PI3K include viridin, viridiol, demethoxyviridin, and demethoxyviridiol (see, U.S. Pat. No. 5,276,167). Once viridin, viridiol, demethoxyviridin, and demethoxyviridiol, or other PI3K inhibitors are isolated and purified, analogs of each may be prepared via well known methods to provide generally known compounds such as those illustrated by formula I of U.S. Pat. No. 5,276,167 (see, also, Grove et al. (1965) J. Chem. Soc, June: 3803-3811, Hanson et al. (1985) J. Chem. Soc. Perkin Trans. I: 1311-1314. Aldridge et al. (1975) J. Chem. Soc. Perkin Trans. I: 943-945 (1975), and Blight et al. J. Chem. Soc. Perkin Trans I: 1317-1322).

Suitable derivatives and analogues include, but are not limited to alpha/beta- viridin, 1- acetylviridin, 1-methylether of viridin, demethoxyviridin, demethoxyviridin mono-acetate, dehydroxyviridin, demethoxyviridin mono-methanesulfonate, diacetyldemethoxyviridol OAc, viridiol, 1-O-acetylviridiol, 1-O-methyl-methylether of viridiol, demethoxyviridiol, 1- acetyldemethoxyviridiol, 1-O-methylether dimethoxyviridiol (see, U.S. Pat. No. 5,276,167). Other derivatives include, but are not limited to Wortmannin stereochemical alcohol and ester derivatives, such as 11 -substituted, 17-substituted and 11, 17 disubstituted derivatives of wortmannin (see, U.S. Pat. No. 5,480,906), and the like.

Direct PI3K inhibitors may also include nucleic acid or antibody reagents (or other targeted polypeptide or peptide analogs) that are directed against a PI3K subunit (e.g. p85 or pi 10) or a nucleic acid encoding the subunit. Nucleic acid inhibitors may include RNAi oligonucleotides and antisense nucleic acids. An antisense construct can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a PI3K pathway protein. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a PI3K pathway gene.

Oligonucleotides that are complementary to the 5' end of the mRNA, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well.

(Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of a gene could be used in an antisense approach to inhibit translation of that mRNA. Whether designed to hybridize to the 5', 3 ' or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably less that about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length. Inhibitor RNA (RNAi, also referred to as siRNA) may also be used to inhibit a component of the P I3K p athway, and in certain embodiments, RNAi approaches may be d esirable b ecause such approaches are thought to be temporary and to cause no permanent changes in the genome of the target cell. RNAi may be achieved as described in any of the following patent applications: WO9932619, WO0044914, WO0129058, WO0175164, and WO0136646. Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate olgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. Antibody reagents may include single chain antibodies or other antibody forms that can be introduced into a cell, either as a protein or as a nucleic acid competent to express the protein, to act directly on PI3K.

Targets for indirect inhibitors include PI3K pathway proteins, such as insulin receptor,

IGF receptors, protein kinase C (PKC), IRS proteins, protein kinase B (PKB or Akt), NFAT

(which may be modulated, for example, with lithium or cyclosporine A), various mitogen- activated protein kinases (MAPKs), MTOR, GSK3 and calcineurin (which may be modulated, for example, with FK506 and cyclosporine A).

Stem cell lines may be engineered to permit conditional expression of PI3K or actors in the pathway. For example, a cell line may be engineered with the relevant gene placed under control of an inducible promoter, such as a tet promoter. As another example, a cell line may be engineered with the relevant gene placed between recombinase sites (such as lox sites for the Cre recombinase) that permit excision upon expression of Cre, and Cre expression may be controlled by an inducible promoter.

The functionality of a PI3K inhibitor may be assessed according to any of a variety of art-recognized assays, hi general, the assays for direct PI3K inhibitors involve comparing the activity of PI3K in the presence and absence of the putative inhibitor. Examples of such assays are described in detail in U.S. Pat. No. 5,480,906. PI3K activity may be measured as described by Matter et al. (1992) Biochem. Biophys. Res. Comm., 186: 624-631. Briefly, inhibitor candidates are initially dissolved, e.g., in DMSO and then diluted e.g., 10-fold with 50 mM of HEPES buffer, pH 7.5, containing 15 mM of MgC12 and 1 mM of EGTA. Ten microliters of this solution are incubated with purified PI3K and phosphatidylinositol in 50 mM of HEPES buffer, pH 7.5, containing 1 mM of EGTA. Reactants are preincubated 10 minutes at ambient temperature and then the enzyme reaction is started upon addition of 32P-ATP (2 mCi/mL, 500 mu M of stock solution; 0.08 mCi/mL, 20 mu M of final concentration; Dupont New England Nuclear, Boston, Mass.). The reaction is allowed to proceed for 10 minutes at ambient temperature with frequent mixing, after which time the reaction is quenched by addition of 40 mu L of IN HC1. Lipids are extracted with addition of 80 μl L CHC13 :MeOH (1:1, v:v). The samples are mixed and centrifuged, and the lower organic phase is applied to a silica gel TLC plate (EM Science, Gibbstown, N.J.), which is developed in CHC13 :MeOH:H2O:NH4OH (45:35:8.5:1.5, v:v). Plates are dried, and the kinase reaction visualized by autoradiography. The phosphatidylinositol 3-monophosphate region is scraped from the plate and quantitated using liquid scintillation specfroscopy with ReadyProtein (Beckman Instruments, Inc., Fullerton, Calif.) used as the scintillation cocktail. The level of inhibition for the putative inhibitor is determined as the percentage of 32P-counts per minute compared to controls.

The activity of a candidate PI3K inhibitor may also be assessed by testing its effect on expression of a PI3K-responsive gene in a cell, particularly where the promoter of the PI3K- responsive gene is linked to a reporter gene.

Any of the various inhibitors and reagents described herein may be replaced with functional analogs. A functional analog is a structurally similar molecule having at least 10%, and preferably at least 50%>, of the activity of the inhibitor or reagent. In the case of polypeptide factors, a functional analog may be simply a version using one or more modified amino acids but retaining the same sequence, or a functional analog may be a polypeptide having at least

80%) amino acid sequence identity to the polypeptide factor, and preferably at least 90% or 95% sequence identity. Functional analogs may be identified from combinatorial libraries by the use of high-throughput screens. A combinatorial chemical library is a collection of diverse chemical compounds. Such libraries may be generated by chemical synthesis or biological synthesis by combining a number of simpler chemical subunits. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of amino acids in as many ways as possible for a given polypeptide length. The functionality of a candidate functional analog may be evaluated by using a published assay for the activity of the agent to be replaced. Millions of chemical compounds can be synthesized through such combinatorial mixing of subunits. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26, 1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993), random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidyl phosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See, generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 274: 1520- 1522, and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepines U.S. Pat. No. 5,288,514, and the like).

In certain embodiments, an insulin-producing cell composition is exposed to an additional culture condition. An additional culture condition may be simply the removal of the PI3K pathway inhibitor or cell proliferation inhibitor. In certain preferred embodiments, the removal of the inhibitor stimulates growth of cells in the insulin-producing cell composition and increased insulin production, h addition or independent of removal of the inhibitor, the insulin- producing cell composition may be treated with any of the various agents, and functional analogs t hereof, t hat a re k nown t o s timulate i nsulin p roduction or b eta-cell p roliferation. In certain embodiments, such agents are only effective in increasing insulin production if used subsequent to or simultaneously with the PI3K pathway inhibitor of cell proliferation inhibitor. Examples of such agents include Igf-1 (e.g. at a concentration of lOng/ml), GLP-1, exendin-4, HGF, activin, sonic hedgehog (and other suitable hedgehog family members, as well as hedgehog agonists) and reagents that increase cAMP levels, such as membrane permeable forms of cAMP and forskolin.

3. Insulin-Producing Cell Compositions In certain embodiments, the invention relates to insulin-producing cell compositions produced according to any of the methods disclosed herein. Insulin-producing cell compositions may be in any form, including, preferably, insulin-producing cell clusters, but optionally in dispersed cell suspensions, confluent cell cultures, seeded on a matrix or other cell support, mixtures of the foregoing, etc. In further embodiments, the invention relates to insulin-producing cell compositions in which at least about 50% of the cells are positive for insulin production, optionally at least 75% of the cells are positive for insulin production and preferably at least 85%, 90% or 95% of the cells are positive for insulin production, hi certain embodiments, most of the cells, and preferably greater than 80%>, 90% or 95% of the cells, that produce insulin are negative for other pancreatic hormones that are not naturally produced by native pancreatic insulin producing cells, such as glucagon. hi certain embodiments, an insulin-producing cell composition comprises at least about 1000 nanograms (ng) of insulin per milligram of total protein, optionally at least about 5000 nanograms of insulin per milligram of total protein and preferably at least about 10000 nanograms of insulin per milligram of total protein, h embodiments where the insulin- producing cell composition comprises islet-like cell clusters of roughly 300-400 μm in diameter, the clusters produce greater than 0.2 ng of insulin per hour, and preferably greater than 0.5 ng of insulin per hour. In preferred embodiments, insulin production by the insulin-producing cell composition is stimulated by exposure to glucose. hi certain embodiments, insulin-producing cell compositions comprise cells that are positive for one or more of the following markers: insulin (any of the various chains), islet- 1, nestin, neurogenin 3, PDX1, GLUT2 and glucokinase. In certain embodiments, at least about 50% of the cells in an insulin-producing cell composition are not proliferative. Proliferating cells may be detected by a variety of ways known in the art, including staining with Ki67, a nuclear marker of proliferating cells, or incorporation of labeled nucleotide (e.g. tritiated thymidine or bromodeoxyuridine). In preferred embodiments, insulin-producing cell compositions do not form neoplastic growths when implanted in a subject. It is understood that biological systems are tremendously variable and, depending on host and implant characteristics, even a very safe insulin-producing cell composition is likely to form, or appear to form, a neoplastic growth at some low frequency. In certain embodiments the insulin- producing cell compositions of the invention produce a neoplastic growth in a fewer than 30% of implanted subject, optionally in fewer than 1% of implanted subjects and preferably in fewer than 0.1% of implanted subjects.

4. Administration of Insulin-Producing Cell Compositions

In additional embodiments, the invention relates to methods for ameliorating, in a subject, a condition related to insufficient pancreatic function by administering to the subject an effective amount of an insulin producing cell composition. Preferrably, a sufficient amount of insulin-producing cell composition is administered to a subject to cause an increase in blood insulin levels or an improvement in glucose homeostasis. Glucose homeostasis may be tested by administering a dose of glucose and monitoring the kinetics with which blood glucose levels decline. Conditions related to insufficient pancreatic function include the various forms of diabetes mellitus (e.g. type I and type II), NOD mice (a type I diabetes model), the streptozotocin-induced diabetes rodent model, surgically-induced diabetes models and diseases resulting from dysfunctional islet growth (e.g. insulinomas). Administration of an insulin- producing cell composition may not produce a permanent ameliorating effect, and periodic dosing, such as on a weekly, monthly or yearly basis may be beneficial.

In preferred embodiments, an effective dose of insulin-producing cell composition comprises administering at least about one islet-like cell cluster of the invention (or an equivalent number of cells) per islet that is naturally present in the subject organism. For example, mice have about 300-500 islets, rats have about 3000-5000 islets and humans have about 1,000,000 islets, and accordingly, a preferred dosage is about 300-500 islet-like cell clusters for a mouse, about 3000-5000 islet-like cell clusters for a rat and about 1,000,000 isletlike cell clusters for a human. The number of islets per organism is proportional to average body mass (20-30 grams, mouse, 200-300 grams, rat, 60-70 kilograms, human) and it may be desirable to administer a dosage that is proportional to body mass of the subject, hi instances when an islet-like cell cluster is less efficient at producing insulin than a native islet, or where an insulin-producing cell composition is subject to cell mortality (e.g. in the case of host immune system-mediated rejection), the dosage may be increased proportionally. In certain embodiments, the invention relates to therapeutic compositions comprising insulin-producing cell compositions and methods for making such therapeutic compositions, as well as the use of insulin-producing cell compositions in the manufacture of a medicament for the treatment of subjects having insufficient pancreatic function. Therapeutic compositions may include an insulin-producing cell compostions disclosed herein and/or made by the methods disclosed herein, as well as mixtures comprising such insulin-producing cell compositions and a therapeutic excipient. Examples of therapeutic excipients include matrices, scaffolds or other substrates to which cells may attach (optionally formed as solid or hollow beads, tubes, or membranes), as well as reagents that are useful in facilitating administration (e.g. buffers and salts), preserving the cells (e.g. chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics), or promoting engraftment.

Cells may be encapsulated in a membrane to avoid immune rejection. By manipulation of the membrane permeability, so as to allow free diffusion of glucose and insulin back and forth through the membrane, yet block passage of antibodies and lymphocytes, normoglycemia may be maintained (Sullivan et al. (1991) Science 252:718). In a second approach, hollow fibers containing cells may be immobilized in a polysaccharide alginate. (Lacey et al. (1991) Science 254:1782). Cells may be placed in microcapsules composed of alginate or polyacrylates. (Lim et al. (1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Ada. 840:133; Sugamori et al. (1989) Trans. Am. Soc. Artif Intern. Organs 35:791; Levesque et al. (1992) Endocrinology) 130:644; and Lim et al. (1992) Transplantation 53:1180). Additional methods for encapsulating cells are known in the art. (Aebischer et al. U.S.

Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Hoffman et al. (1990) Expt.

Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al.

(1991) J. Biomech. Eng. 113:178-183, U.S. Patent No. 4,391,909; U.S. Patent No. 4,353,888;

Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55).

The site of implantation of insulin-producing cell compositions may be selected by one of skill in the art. hi general, such as site preferably has adequate blood perfusion to allow the cells to sense blood conditions and secrete hormones and other factors into the general circulation. Exemplary implantation sites include the liver (via portal vein injection), the peritoneal cavity, the kidney capsule and the pancreas.

5. Certain Additional Uses for Insulin-Producing Cell Compositions hi certain embodiments, the invention relates to methods employing insulin-producing cell compositions disclosed herein.

In certain embodiments, methods of the invention relate to the identification of pancreatic developmental markers. For example, expression patterns of established markers of endoderm and islet development may be monitored at one or more stages of differentiation into insulin-producing cell compositions. Markers may be assessed using standard methods, including Northern blotting, RT-PCR, in situ hybridization (ISH), immunohistochemistry (LHC) as well as nucleic acid or protein array or microarray-based methods. In certain embodiments, monitoring production of one or more gene products will be useful to identify candidate cell- surface proteins for FACS-based purification strategies for insulin-producing cell precursors. Yet another aspect of the present invention provides methods for screening various compounds for their ability to modulate insulin-producing cells, such as, for example, by affecting growth, proliferation, maturation or differentiation, or by affecting insulin production, secretion or storage, as well as compounds that may improve graft performance (e.g. result in a longer-lasting graft, improved insulin production, or changes in proteins that interact with the host immune system). In an illustrative embodiment, the subject cells can be used to screen various compounds or natural products, such as small molecules or growth factors. Such compounds may be tested for essentially any effect, with exemplary effects being cell proliferation or differentiation, insulin production, or cell death. In further embodiments, insulin-producing cells may used to test the activity of compounds/factors to promote survival and maturation, and further, since certain cells produced according to methods disclosed herein have one or more properties of islet cells, specifically β-cells, such cells may be used to identify factors ( or g enes) that regulate production, processing, storage, secretion, and degradation of insulin or other relevant proteins (like LAPP, glucagon, including pro-glucagon, GLPs, etc) produced in pancreatic islets. In further embodiments, an msulin-producing cell may be modified, such as by genetic modification, to become hyperproliferative. Such hyperproliferative cells may be contacted with compounds to identify, for example, anti- proliferative and anti-neoplastic agents (e.g. agents that inhibit cell growth or promote cell death). The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the compound. A control assay can also be performed to provide a baseline for comparison. Identification of the progenitor cell population(s) amplified in response to a given test agent can be carried out according to such phenotyping as described above. Assays such as those described above may be carried out in vitro (e.g. with cells in culture) or in vivo (e.g. with cell implanted in a subject).

In further embodiments, the invention provides non-human animals that comprise an insulin-producing cell composition as disclosed herein. Such animals may be useful, for example, for screening compounds that may affect graft performance in vivo. 6. Methods for Identifying Stem Cells

In certain embodiments, the invention relates to methods for identifying a cell that has the potential to develop into a pancreatic cell, and particularly an insulin-producing cell. In one aspect, the method comprises providing a stem cell line, or other multipotent cell line, and differentiating the cell line so as to obtain an insulin-producing cell composition. At the beginning of the differentiation process, or at some stage within the differentiation process, the differentiating cells are mixed with a cell of interest. The differentiation of the cell of interest may then be assessed. A cell of interest that is able to differentiate into an insulin-producing cell is a cell that has the potential to develop into an insulin-producing cell. In further embodiments, the cell may be assessed for the production of other pancreatic products, such as glucagons, to identify cells that have the potential to develop into other types of pancreatic cells. In certain embodiments, a pancreatic tissue (e.g. ductal tissue, adult pancreatic tissue, fetal pancreatic tissue, etc.) may be dissociated into a cell suspension, and clumps of cells or single cells are used as the cell of interest in the above method embodiments, thereby permitting a rapid screen of pancreatic cells for candidate pancreatic progenitors. In one embodiment, insulin-producing cell compositions and methods for generating such compositions may be used to assess the developmental potential of a cell of interest. In some embodiments, the developmental potential of a cell of interest may be determined by mixing t he c ell o f i nterest w ith c ells d uring t he process o f m aking a n i nsulin-producing cell composition. The cell of interest is then tracked (for example by a transgenic marker) to determine the types of cells that arise from it. In an exemplary embodiment, the cell of interest is mixed with differentiating ES cells at one of the culturing steps preceding treatment with a PI3K inhibitor. h certain embodiments, culture systems for making insulin-producing cell compositions may be used as part of an assay to identify candidate pancreatic endocrine precursor cells. Current evidence suggest that such precursors exist as single cells or small cell clusters within or closely associated with pancreatic epithelium. If so, an assay to identify pancreatic progenitor cells could comprise detecting isolated single cells, h certain embodiments, cell compositions in the process of differentiating into insulin-producing cell compositions provide the appropriate cellular microenvironment to permit pancreas-derived endoderm to integrate and differentiate. In certain embodiments, cells of a pancreatic tissue are fractionated and mixed, either as populations of cells or as single cells, into cells being differentiated into insulin-producing cell compositions. Cells of the pancreatic tissue that develop into msulin-producing cells are candidate pancreatic stem cells.

Upon review of this specification and the appended claims, additional uses for the insulin-producing cell compositions will be apparent to one of skill in the art, and such uses are intended to be encompassed as part of the present invention.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Example 1 : Rescue of diabetes phenotypes with insulin-producing grafts derived from ES cells

This example describes experimental strategies for developing insulin-producing cell clusters (ICCs) from ES cells that increase circulating insulin levels, improve glucose homeostasis, and rescue survival in an experimental model of diabetes mellitus.

Materials and Methods: The stem cells were the JM1 mouse ES cell line described in M. Qiu et al., Genes Dev 9,

2523 (1995), and the ROSA line described in G. Friedrich, P. Soriano, Genes Dev 5, 1513 (1991). These distinct lines gave comparable r esults, b ut the JM 1 line consistently produced greater levels of insulin than the ROSA line.

The undifferentiated mouse ES cells (stage 1) were cultured on a feeder layer of irradiated mouse embryonic fibroblasts with media containing Knockout-DMEM, penicillin-streptomycin, 0.0007%) β-mercaptoethanol, 2 mM L -glutamine, 100 mM non-essential amino acids (Gibco, Rockville, MD), 15% FCS (Hyclone, Logan, UT), and 1000 U/ml Leukemia Inhibitory Factor (LIF; Chemicon, Temecula, CA). Media was changed daily for 4 days, and then cells were harvested and placed in fresh culture plates (Fisher, Santa Clara, CA). After 2 days cells were placed in low-adhesion plates (Corning International, Corning, NY) and cultured in media without LIF (stage 2). Resultant embryoid bodies were transferred to culture dishes and allowed to adhere, then cultured for 6 days (stage 3) in ITSFn serum-free media (S. H. Lee et al., Nature Biotechnol. 18, 675, 2000). Cells were transferred to plates coated with poly-L-ornithine (Sigma, Saint Louis, MO) and fibronectin (Gibco) and cultured for 6 days (stage 4) in N₂ media (S. H. Lee et al., Nature Biotechnol. 18, 675, 2000) supplemented with 10 ng/ml bFGF (R&D Systems, Minneapolis, MN) and B27 supplement (Gibco). During stage 5NB, ICCs were cultured i n N ₂ m edia s upplemented w ith B27 a nd 1 0 m M n icotinamide ( Sigma). S tage 5 NL ICCs were cultured in N₂ media supplemented with 10 mM nicotinamide and 10 μM LY294002 (Calbiochem, La Jolla, CA). Media was changed during stage 5 every other day for at least 6 days. Islets were isolated by intra-ductal collagenase perfusion. Total cellular insulin content from islets and ICCs, and serum insulin levels from ICC engrafted mice, was measured using a mouse insulin ELISA kit (ALPCO, Windham, NH). Pancreatic glucagon and insulin, and serum glucagon and insulin were measured by radio-immunoassay (Linco, St. Louis, MO). Applicants measured in vitro insulin release from isolated islets and ICCs by static batch incubation. In vitro insulin release from stage 5NL and 5NB ICCs was measured as follows. Plates were washed with no glucose RPMI 1640 (GIBCO) three times. 10 ICCs were hand-picked and incubated with 25 mM glucose in RPMI 1640 for each time at 37oC and 5% CO₂. The supernatant was collected and insulin levels were detennined using mouse insulin ELISA kit (ALPCO, Widham, NH). For immunohistochemistry, cells were fixed in 4% paraformaldehyde, embedded in

Matrigel (Becton Dickinson, Bedford, MA), and then embedded in paraffin. Applicants performed immunohistochemistry on 6 μm tissue sections prepared by microtomy (Leica, San Jose, CA) using standard protocols. Applicants used primary antibodies at the following dilutions: guinea pig anti-insulin 1:200 (Linco Research Inc., St. Charles, MO), mouse anti- glucagon 1:500 (Sigma, St. Louis, MO), mouse anti-α-fetoprotein 1:500 (Sigma), mouse anti- MAP2 1:500 (Sigma), mouse anti-β tubulin III 1:500 (Sigma), mouse anti-Glut-2 1:200 (ADI, San Antonio, TX), mouse anti-glucokinase 1:200 (C. Newgard, U.T. Southwestern). Rabbit anti-C-peptide 1 :500 and mouse anti-proinsulin 1:500 (O. M adsen, H agedorn, D enmark) and rabbit anti-Pdxl 1:500 (C. Wright, Nashville, TN). Confocal immunofluorescence microscopy with an optical slice thickness of 0.5 μm was performed on aBioRad MRC1000.

Total RNA was prepared using an RNeasy Kit (Qiagen) and RQ1 RNase-free DNase

(Promega). For cDNA synthesis, oligo dT primers (Invitrogen) were used to prime reverse transcription reactions and synthesis was carried out by Thermoscript RT (Invitrogen). PCR was performed using Taq polymerase (Applied Biosystems), and an Opti-Prime Optimization kit (Stratagene). In addition to β-actin, GAPDH expression (not shown) was used to normalize input template cDNA to analyze relative gene expression. Primer sequences for insulin, glucagon and β-actin were as described in N. Lumelsky et al., Science 292, 1389, 2001. All animal studies were performed in accordance with Stanford University Animal Care and Use guidelines. Experimental diabetes was induced 7 days prior to transplant in 9- to 10- week-old male NOD scid mice (Jackson Laboratory, Bar Harbor, ME) by intraperitoneal injection of 175 mg/kg streptozotocin (STZ; Sigma, St Louis, MO). Blood glucose was measured with a glucometer (Bayer Corp., Elkhart, IN). Statistical significance was calculated as described in S. K. Kim et al, Nat Genet 30, 430 (2002). Blood glucose level was 135 ± 12 mg/dL in untreated control mice (n=6) during random-feeding, and 85 + 10 mg/dL (n^ό) after 15 hour overnight fast. Under general anesthesia, mice were engrafted with 300 handpicked ICCs or received a sham transplant of saline solution in the left subcapsular renal space. Stage 5NL ICC diameter was approximately 300-400 μm, 2-3 times larger than pancreatic islets, and transplantation of more ICCs in a single renal graft site was not feasible. Grafts were removed after 3 weeks by unilateral nephrectomy for analysis. Survival distributions were determined using the standard product limit method described in E. L. Kaplan, P. Meier, J. Am. Stat. Assoc. 53, 457 (1958). By 3 weeks, applicants detected tumors in 4 of 4 mice transplanted with handpicked s tage 5 NB ICCs. Tumors following transplantation o f s tage 5NL ICCs were not observed 8 weeks after engraftment, the maximum period of observation (n=3). hi sham-transplanted mice that did not survive, average blood glucose on the day preceding death was 532 ± 33 mg/dL (n=4). ICC graft removal 3 weeks after transplantation resulted in comparable hyperglycemia (674 ± 117 mg/dL , n=4).

3 weeks after ICC transplantation, glucose tolerance testing was performed with 1 g glucose/kg body weight as described by S. K. Kim et al, Nat Genet 30, 430 (2002).

Results:

Undifferentiated ES cells were exposed to a sequence of conditions described above that culminated in treatment with nicotinamide and LY294002 (stage 5NL). Other cells were treated with nicotinamide and B27 supplement (stage 5NB). Direct comparison revealed that intracellular insulin content (3.7 ± 1.3 ng/ICC) in stage 5NL ICCs was 7% of levels in isolated pancreatic islets (52 ± 6.3 ng/ islet). Normalized to protein content, the insulin level in stage 5NL ICCs was 4900 ± 820 ng/mg protein, a 3000% increase compared to the insulin level i ICCs produced by others after treatment of ES cells with nicotinamide and B27. Consistent with these results, stage 5NL, but not stage 5NB, ICCs were stained by ditbizone, a chromogen (R. M. Jindal, Pancreas 11, 316, 1995) used to detect stored intracellular insulin in pancreatic β- cells (data not shown). Applicants obtained similar results with a combination of wortmannin, a different PI 3K inhibitor and nicotinamide (See Example 2).

To elucidate cellular differentiation in ICCs, applicants examined ICC morphology and expression of gene products that govern or define pancreatic cell fates. Stage 5NL ICCs were smaller and had more contacts between insulin cells than stage 5NB ICCs (Fig.l). hi stage 5NL insulin^4" cells and pancreatic β cells, co-expression of glucagon, and other markers like α- fetoprotein, a marker of primitive endoderm and liver, or microtubule-associated protein 2 (MAP2) and β-tubulinlll, neuronal-specific markers, was rare or not detected (Fig. 1A-B, D-E, G-H, J-K). Insulin^"1" cells in stage 5NL ICCs expressed numerous pancreatic β cell markers, including C-peptide, proinsulin, the transcription factor PDX1, type 2 glucose transporter (GLUT2), and glucokinase (Fig. 2A-J). Reverse transcriptase-polymerase chain reaction (RT- PCR) analysis showed that isolated ICCs expressed Islet- 1, nestin, and neurogenin-3, markers also expressed in isolated pancreatic islets (Fig. 2K). Immunohistochemical detection of Ki67, a nuclear marker of proliferating cells, demonstrated that the majority of cells in stage 5NL ICCs were not proliferating, similar to cells in pancreatic islets (Fig. IM, N). Our analyses (Fig. 2K and data not shown) also revealed the presence of stage 5NL cells lacking insulin that produced carboxypeptidase A (carbA), a pancreatic exocrine gene product not detected in ICCs described in p revious s tudies. In c ontrast to t hese results with s tage 5 NL ICCs, many i nsulin^"1" c ells i n stage 5NB ICCs co-expressed glucagon or α- fetoprotein (Fig. 1C, F), and stage 5NB ICCs were comprised mainly of MAP2 or β-tubulin III expressing cells (Fig. II, L). Ki67 staining revealed that approximately 25%> of cells in stage 5NB ICCs were proliferating (Fig. 1O). Thus, insulin*^" cells produced from our methods displayed hallmark molecular features of pancreatic β cells and comprised ICCs with remarkable similarities to pancreatic islets. 5NL ICCs released substantially more insulin than the 5NB ICCs (Fig. 4)

To determine the feasibility of transplanting ES cell-derived ICCs to treat diabetes mellitus, applicants performed a series of ICC grafting experiments in immunocompromised recipient mice. Undifferentiated or partially-differentiated ES cell grafts often form tumors, particularly teratomas. In contrast, transplantation of pancreatic islets does not produce these lethal tumors.. Stage 5NB and stage 5NL ICC fates were monitored following engraftment in the left subcapsular renal space of NOD scid mice. Within 3 weeks after transplantation of stage 5NB ICCs, tumors formed (Fig. IP) with features characteristic of teratomas (data not shown). In contrast, stage 5NL grafts maintained high levels of insulin and C-peptide expression in clustered cells that did not co-express glucagon, and did not form tumors (Fig. 1Q, R). hιsulin⁺ cells from renal grafts appeared larger than those in stage 5NL ICCs prior to engraftment (Fig IB, R). Exposure to PI 3K inhibitors can reduce cell size, suggesting that engraftment of stage 5NL ICCs in an environment lacking LY294002 permitted cell enlargement. Thus, engrafted insulin c ells from s tage 5 NL s urvived, grew, p roduced i nsulin, and r emained d ifferentiated, leading to superior performance in vivo.

To investigate stage 5NL ICC function in vivo, ICCs were transplanted into NOD scid mice with streptozotocin (STZ)-induced diabetes mellitus. 7 days after STZ injection, NOD scid mice were engrafted with ICCs or received a sham transplant. In mice with STZ-induced diabetes, the most striking benefit of engraftment with 300 stage 5NL ICCs was complete rescue of survival (Fig. 3A). Endogenous pancreatic insulin content in ICC-transplanted mice remained severely reduced at the completion of these transplantation experiments (Table 1), indicating that recovery o f host pancreatic insulin secretion did not account for the observed phenotypic rescue. In support of this conclusion, applicants observed that removal of the kidney containing the ICC graft 21 days after transplantation resulted in rapid onset of severe hyperglycemia and death, whereas removal of the sham-transplanted kidney in surviving control mice had no detectable effects (Fig. 3A,B). Together, these results show that improved outcomes following ICC transplantation in mice with fatally severe diabetes mellitus resulted from the ICC graft.

21 d ays a fter s ham t ransplantation, s urviving c ontrol m ice h ad r educed s erum i nsulin levels (0.05 ± 0.01 ng/ml; Table 1), hyperglycemia during random feeding (468 ± 37 mg/dL) and a 17%> average reduction in body mass. This level of hyperglycemia (Fig. 3B) in surviving mice under-estimated the effects of STZ, since most STZ-treated mice had expired by this time. In STZ-treated NOD scid mice engrafted with stage 5NL ICCs (n=7), hyperglycemia (305 ± 35 mg/dL) and weight loss (6%; P < 0.05) were attenuated, and serum insulin levels were increased approximately 6-fold (0.28 ± 0.06 ng/ml; Table 1) compared to sham-transplanted controls. Thus, ICC engraftment resulted in circulating insulin levels during random-feeding which were 17%) of those in untreated control mice, similar to in vitro levels of glucose-dependent insulin release by ICCs and consistent with the moderate hyperglycemia observed in random-fed mice after ICC transplantation. ICC-engrafted mice also achieved improved glucose regulation compared to sham-transplanted control mice in glucose tolerance tests after overnight fasting (Fig. 3C). Our data support the conclusion that transplantation of stage 5NL ICCs in this diabetes model increased levels of circulating insulin, leading to better glucose regulation and maintenance of body mass. Rapid restoration of normoglycemia in mice with STZ-induced diabetes by pancreatic islet transplantation requires engraftment of 500-1000 islets per mouse; thus, further improvements in glycemic control by ICC transplantation are feasible, either by increasing ICC graft mass, or insulin expression.

The results presented in this Example show that simple manipulations of ES cell culture conditions produce relatively homogeneous clusters of insulin-secreting cells with important similarities to islets. These advances allowed ES cell-derived ICCs to rescue and ameliorate disease phenotypes in an experimental model of diabetes. Endocrine and exocrine cells are likely derived from a common precursor in the embryonic pancreas and detection of both cell types associated with ICCs is consistent with the possibility that a similar precursor cell may be produced during ICC development in vitro. Expression of liver and pancreatic acinar cell markers during ICC differentiation from ES cells suggests that our methods may be adapted to model the differentiation of pancreatic exocrine cells, hepatocytes, and other endodermal derivatives, in addition to insulin-secreting cells. This study demonstrates for the first time, to our knowledge, that insulin-producing cell grafts derived from ES cells can significantly improve outcomes in diabetic animals.

Table 1. ICC grafts increase circulating insulin levels in mice with STZ-induced diabetes mellitus. Insulin and glucagon in blood and pancreas from untreated control mice (n=6), and in STZ-treated diabetic mice 2 1 days a fter r eceiving a sham transplant (n^δ) or ICC transplant (n=7).

Blood Pancreas insulin glucagon insulin (ng/g glucagon (ng/g

(ng/ml) (pg/ml) tissue) tissue)

Untreated 1.65 ± 0.3 75 ± 9 97,800 ± 595 ± 71 10,400

Sham 0.05 ± 0.01 107 ± 14 600 ± 145 700 ± 80 transplant

ICC 0.28 ± 0.06 151 ± 26 466 ± 66 520 ± 37 transplant Example 2: Detailed Protocol for the Production of ICCs from Mouse ES Cells

Cells:

Mouse Embryonic Feeders (MEF): C57B1/6 (Jackson Laboratories, Bar Harbor, ME)

Mouse ES cells: JM1 (129/SvJ)

Cell Culture Medium

Cell culture medium should be prepared using aseptic technique.

Solutions should be pre-warmed in the 37°C water bath unless otherwise noted.

Solutions should be combined in a Nalgene Filter Unit with a 0.2 μm filter and need not be mixed before applying a vacuum. Once prepared the solutions should be stored in the refrigerator at least 4°C on the top shelf for up to a week.

hicubators, Refrigerators, and Freezers

The tissue culture are kept at 37°C and 5% CO .

Reagents

Apo-Transferrin (Bovine) [Sigma #T-1428]

B-27 Serum-Free Supplement 50X [Gibco-BRL #17504-044] basic Fibroblast Growth Factor (bFGF) lOOx (1 μg/ml) [R&D Systems #133-F-025]

D(+)-Glucose Hybri-Max [Sigma #G-5146]

Defined Fetal Calf Serum (Hyclone #SH30070.01)

Dissociation Buffer A (DisA)

DMEM (+Glutamax, +Na Pyruvate) [Gibco-BRL #10569-010]

DMEM/F-12 [Gibco-BRL #10565-018]

EDTA (4% (Ethylenediaminetatraacetic Acid) [Sigma E6635, lOOg]

Fibronectin [preferred supplier Gibco-BRL #33010-018, alternate Sigma #F-4759]

Gelatin (0.2%) [Sigma #G-9391)]

Hydrochloric acid (HC1 (IN)

ITSFn Insulin 5 mg/ml [Sigma #1-6634]

KNOCKOUT D-MEM [Gibco-BRL #10829-018]

Leukemia Inhibitory Factor (LIF) lOOx [Chemicon #ESG1106] L-Glutamine lOOx [Gibco-BRL #25039-149]

L-GLutamine [Sigma #G-8540]

2-Mercaptoethanol (0.07%) (2ME) [Sigma #M-7522]

N² Insulin 5 mg/ml [Sigma #1-6634]

Nicotinamide 100X [Sigma #N0636]

Non-Essential Amino Acids (NEAA) lOOx [Gibco-BRL #11140-050]

Penicillin-Streptomycin (Pen/Strep) lOOx [Gibco-BRL #15140-122]

Phosphate-Buffered Saline (PBS) [Gibco-BRL #14190-250]

Poly-ornithine [Sigma #P-4957]

Progesterone 2mM stock (PG) [Sigma #P-8783]

Putrescine 1 M stock (Ptr) [Sigma #P-5780]

Sodium Bicarbonate [Sigma #S-5761]

Sodium Selenite (300 μM [Sigma #S-5261]

Ly294002 (lOμM [Calbiochem #440202]

60 mm Tissue Culture Dishes [Fisher #08-772F, Falcon #35-3004]

100 mm Tissue Culture Dishes [Fisher #08-772-22]

Transferrin 2 mg/ml [Sigma #T-3309]

Trypsin-EDTA (DisB) [GIbco-BRL#25300-054]

12 Well Plates [Fisher #12-565-321, Nalge Nunc International #150628]

Ultra Low Attachment Plates six well [Corning, Costar #3471]

Media

Stage 0: MEF media (Mouse Embryonic Feeder)

88 ml DMEM (±Glutamax and Na Pyruvate) 10 ml FCS l ml lOOx NEAA l ml lOOx Pen/Strep

Stage 1 and 2: KO media, LIF (+ or -) 80 ml KNOCKOUT D-MEM

15 ml FCS l ml lOOx EAA l ml lOOx Pen/Strep 1 ml 2 ME

l ml LΓF

Stage 3: ITSFn media (Insulin, Transferrin, sodium Selenite, Fibronectin)

50 ml DMEM/F12

1.25 ml Transferrin, 2 mg/ml

50 ml ITSFn insulin, 5 mg/ml

5 ml Sodium selenite, 200 mM 250 ml Fibronectin, 1 mg/ml

500 ml lOOx Pen/Strep

Stage 4 and 5: N2 media

250 ml DMEM/F12 media 50 mg Apotransferrin

775 mg Glucose 36.5 mg Glutamine 845 mg Sodium bicarbonate

Adjust the pH to 7.1 to 7.2 with cell culture grade IN HCl. Adjust the volume to 500 ml with pure ddH2O. Filter through a 0.22 mm filter.

Stage 4 X ml N2 media

5 Ox B27 supplement lOOx bPGF

Stage 5 X ml N2 media lOμM Ly294002 lOOx Nicotinamide Procedure

Stage 0: Day 1. (1)

• Prepare and pre-warm MEF media.

• Add 5 ml of gelatin to 2x 100 mm tissue culture dishes. Incubate for 20 minutes at room temperature.

• Retrieve one vial of MEF cells from the cryostorage unit and immediately place in the 37°C water bath for two minutes.

• Once the cells are thawed and warmed, gently add the cell suspension to MEF media and centrifuge for 5 minutes at 1000 m to pellet the cells. While the cells are spinning aspirate off the gelatin off the tissue culture plates and add 8 ml of MEF media to each dish.

• Once the cells are done spinning, aspirate off the supernatant and add 4 mil of MEF media and completely resuspend the MEF cells breaking up the clumps as well as possible.

• Add 2 ml of cell suspension to each dish and place in the incubator.

• Incubate for at least 5-6 hours or overnight.

Stage 1: Day 1(2)

1. Prepare and pre-warm KO media LLF(+).

2. Thaw ES cells in the 37 °C water bath for two minutes.

3. Once the cells are thawed and warmed, gently add the cell suspension to the KO LLF(+), centrifuge for five minutes. While the cells are spinning, aspirate the MEP media from feeder cells plates and add 8 ml of KO LIF(+) media.

4. Aspirate the supernatant from the ES cells and very gently add 4 ml of KO L1F(+) media. Gently resuspend the ES cells and add 2 ml to each dish and place the cells in the incubator.

Stage 1: Day 2 (3)

1. Pre-warm KO LIF(+) media.

2. Carefully remove tissue culture dishes from incubator and aspirate off the supernatant.

3. Gently add fresh KO LLF(+) media by touching the end of the pipette to the side of the tissue culture dish near the bottom of the dish and dispense the solution at less than 0.2 ml/sec. Return the cells to the incubator.

Stage 1: Day 3 (4) 1. Change media of ES cells.

Stage 1: Day 4 (5)

1. Add 5 ml of gelatin to 6 x 100 mm tissue culture plates.

2. Warm KO LIF(+) media. Warm DisA to room temperature. 3. Wash the E5 cell plates with DisA buffer.

4. Add 1 ml of DisB buffer for 5 minutes at room temperature. Complete dissociation of cells is desired.

5. Add 2 ml KO LIF(+) media to each dish and dissociate the cell clusters completely by passing the cell suspension though a plastic pipette. Transfer the cells to the Falcon tube. Wash the plate one more time with KO LIF(+) and check for >98% transfer.

6. Pellet the cells at 1000 m for 5 minutes.

7. Aspirate the gelatin solution from the new plates and add 8 ml of KO LTF(+) solution.

8. Resuspend the cells in 12 mi of KO LIF(+). The cell pellet tends to stick together to pipette up and down until a homogeneous solution is obtained, about 20-30 times. (Although this will not completely dissociate the cell clusters.)

9. Add 2 ml cell suspension to each plate. Check for the size of cell clusters formed. It is likely that a number of large clusters will still be present. The break up these clusters use a PI 000 set to 800 μl to forcefully titrate 20-30 times without creating any bubbles. This can best be done by using the 200 to 600 μl range of the pipetman. It is important to get close to a single cell suspension to keep the cells from differentiating and also to separate any sick or dead cells from the healthy cells.

10. Check the amount of dissociation and return the cells to the incubator.

Stage 2: Day 1 (6) 1. Pre-warm DisA to room temperature.

2. Prepare KO LIF(-) media.

3. Add 2 ml KO LIF(-) to 6-well low adhesion tissue culture plates.

4. Wash plates with cells with DisA buffer.

5. Add 1 ml of DisB to plates for 5 minutes at room temperature. 6. Add 3 ml KO LJF(-) media to each the dish. Transfer the cells to a 50ml tube. Wash plates serially with a 3 ml of KO LIF(-) and check for >98% transfer. Pellet the cells far 5 minutes at lOOOφrn. 7. Aspirate the supernatant, resuspend in KO LIF(-), Pipette up and down until a homogeneous solution is obtained. 8. Add 1 ml cell suspension to each well of a 6-well low adhesion tissue culture dish. Be very careful to make sure the cell density is the same. Incubate at 37°C.

Stage 2: Day 3 (8) (early miming)

1. Pre-warm KO LTF(-) media and transfer all the cells from their individual wells into a 50 ml tube using a 10ml pipet. Let the cells settle for 5-10 minutes. Aspirate off media.

2. Add 3 ml/well fresh media (54 ml for 18 wells) and gently titrate the solution up and down in the 10 ml pipette to break up the embryoid body clumps, but so as to not break up the embryoid bodies.

3. 3 ml of solution was added to each well trying to keep the same cell density in each well.

Stage 2: Day 3 (8) (early evening) 1. Collect embryoid bodies and wash each well with 3 ml of media.

2. Let the embryoid bodies settle for 10 minutes.

3. Aspirate off the solution and add KO LIF(-) media.

4. Plate on 6 cm tissue culture dishes (3 ml total). Incubate at 37°C for about 26 to 36 hours to ensure embryoid body adhesion to the dish surface.

Stage 3: Day 1 (10)

1. Make ITSFn media.

2. Pre-warm DMEM/F-12 media.

3 Very gently aspirate off the solution from the attached embryoid bodies. Very gently add 3 ml of DMEM/F-12 and aspirate to wash the cells and eliminate any FCS.

4. Add 3 ml of ITSFn media to each dish and incubate at 37°C.

Stage 3: Day 3 (12) 1. Warm ITSFn media. 2. Aspirate off the old ITSFn media

3. Gently add 3 ml fresh media.

Stage 3: Day 5 (14) 1. Warm ITSFn media.

2. Aspirate off the old ITSFn media

3. Gently add 3 ml fresh media.

4. Coat plates with poly-ornithine.

5. Add .5 ml of this solution to each well of 12-well. 6. Add 1 ml of this solution to each 6 cm tissue culture dish.

7. Place in 37°C incubator overnight.

Stage 3: Day 6 (15)

1. Aspirate off poly-ornithine solution.

2 Add PBS (0.5 ml/well or 1 ml/dish). 3. Incubate at 37°C for at least 1 hour. 4 Prepare 1 μg/ml fibronectin solution and filter. 5. Aspirate off PBS from cells. 6 Wash once more with PBS (do not incubate). 7 Add 1 μg/ml filtered fibronectin solution. 8 Incubate 1 hour to overnight up to 2 days maximum.

Stage 4: Day 1 (16)

I . Make N₂ media (-B27, bFGF, and nicotinamide). 2. Prepare an aliquot of N₂ media with 50X B27 and 100X bFGF.

3. Prepare DMEM/F-12 + 10% FCS.

4. Aspirate off ITSFn media from plates and wash with 3 ml of DisA, aspirate off.

5. Add l ml of DisB for 5 minutes and check for complete cell cluster dissociation from the plate. 6. Add 1 ml of DMEM F-12 +10% FCS to each plate and transfer to a 50 ml Falcon tube. Wash plates serially with 3 ml of DMEM F-12 + 10% FCS and add to the 50 ml tube.

7. Centrifuge for 5 minutes at lOOOφrn, remove the supernatant and resuspend the cell clusters in 10 ml of N₂ + B27 + bFGF.

8. Count cells. 9. If fibronectin solution has not been aspirated off yet and the plates and wells washed with lx with PBS, perform this step now. 10. Aspirate off PBS and immediately add cells once PBS is removed.

I I . Plate cells at a density of 2.3-3 x 10⁵ cells/cm². Stage 4: Day 3 (18) 1. Stage 4 media change

Stage 4: Day 5 (20) 1. Stage 4 media change

Stage 5: Day 1(22)

1. Change to Stage 5 media conditions.

Stage 5 Day 3 (24)

1. Stage 5 media change

Stage 5: Day 5 (26) 1. Stage 5 media change.

Stage 5: Day 6 (27)

1. Collect cells for analysis or experiment.

Example 3: Withdrawal of PI3K Inhibitor Induces ICC Growth mES cells were processed through the 5NL stage as described above. A portion of the

ICCs were then transferred to a 6NI medium, lacking the PI3K inhibitor. The effects of removal of the inhibitor are shown in Fig 5, as well as Fig. IR.

PI3K inhibition followed by PI3K activation induces ICC growth. Immunofluorescence staining of ICCs treated with only stage 5NL conditions (A) or stage 5NL and stage 6NI conditions (B) express insulin (green). Greyscale conversion of A and B, C and D respectively, demonstrates not only an overall increase in insulin expression (black) but also an overall increase in cell growth with the addition of stage 6 conditions. These insulin expressing cells at stage 6NI also expressed C-peptide, but as in stage 5NL, there was no significant expression of MAP2, β-tubulinm, glucagon, or α-fetoprotein in stage 6 NI ICCs.

Example 4: Influence of different manipulations on ICC growth and insulin production The effects of different combinations of PI3K inhibitors and their withdrawal on insulin production by ICCs was tested.

Comparison of different stage 5 and stage 6 media conditions on insulin and protein content per ICC and the ratio of insulin per protein per ICC. Both stage 5 and 6 conditions last 6 days with media changes every other day. Wortmannin was added every day. N= nicotinamide (lOμM), B= B27 (50x), L = LY294002 (lOμM), W= Wortmannin (100nM), I= Igf-1 (lOng/ml).

Table 2: Comparison of insulin and protein content following treatment of ES cell-derived ICCs with different combinations of growth factors.

The results demonstrate that both wortmannin and LY294002 cause an increase in the insulin production per unit of total protein in ICCs. Wortmannin had a smaller effect than LY294002, but this difference may be explained by the instability of wortmannin. A comparison of NB, NW and NL indicates that N alone does not have a substantial effect on insulin production in these cells. A comparison of NBL to NL indicates that removal of the B27 components is useful for increasing insulin production per unit of total protein in ICCs. In subsequent experiments, ng insulin/mg protein ratios for the NL group reached as high as 12,000. Example 5: The Effects of Mixing Cells of Interest With Differentiating ES Cells

Cells from the pancreatic anlage of E10.5 and El 1.5 ROSA mouse embryos were isolated and mixed with differentiating ES cell-derived ICCs. ROSA mice express lacZ from the cell-type independent ROSA promoter, which therefore allows subsequent rapid, efficient detection of input mouse cells and their progeny. ROSA-derived cells were mixed with suspended stage 3 or stage 4 ICC cells and co-cultured until stage 5 when cell clusters were visible, β -galactosidase+ c ells i n ICCs w ere d etected w ith X gal ( Fig 6 ). T o d eteπnine if β- galactosidase+ cells express islet markers, and are therefore endoderm-derived, applicants have performed IHC double labeling with antibodies to detect co-expression of β-galactosidase and insulin or other islet cell hormones. A subset of β-galactosidase+ cells express insulin. Detection of insulin+ β-galactosidase+ cells suggests that ICCs provide the appropriate environment to support islet cell development and/or survival. These data indicate that ICC cultures may be used to fractionate candidate islet precursor cell populations derived from the pancreas.

Example 6: Additional Detailed Protocol for the Production of ICCs from Mouse ES Cells

Cells:

Mouse Embryonic Feeders (MEF): C57B1/6 (Jackson Laboratories, Bar Harbor, ME)

Mouse ES cells: JMl (129/SvJ, UCSF, R.Pedersen & J. Meneses)

Cell Culture Medium

Cell culture medium should be prepared using aseptic technique.

Solutions should be pre-warmed in the 37 °C water bath unless otherwise noted. Solutions should be combined in a Nalgene Filter Unit with a 0.2 μm filter and need not be mixed before applying a vacuum.

Once prepared the solutions should be stored in the refrigerator at 4 °C on the top shelf for up to a week.

Incubators, Refrigerators, and Freezers The tissue culture are kept at 37 °C and 5% CO .

Reagents

Apo-Transferrin (Bovine) [Sigma #T- 1428]

B-27 Serum-Free Supplement 50X [Gibco-BRL #17504-044] basic Fibroblast Growth Factor (bFGF) lOOx (1 μg/ml) [R&D Systems #133-FB-025]

D(+)-Glucose Hybri-Max [Sigma #G-5146]

Defined Fetal Calf Serum (Hyclone #SH30070.01)

Dissociation Buffer A (DisA)

DMEM (+Glutamax, +Na Pyruvate) [Gibco-BRL #10569-010]

DMEM/F-12 [Gibco-BRL #10565-018] EDTA (4%) (Ethylenediaminetetraacetic Acid) [Sigma E6635, lOOg]

Fibronectin [preferred supplier Gibco-BRL #33010-018, alternate Sigma #F-4759]

Gelatin (0.2%) [Sigma #G-9391]

Hydrochloric acid (HCl) (IN)

ITSFn Insulin 5 mg/ml [Sigma #1-6634]

KNOCKOUT D-MEM [Gibco-BRL #10829-018]

Leukemia Inhibitory Factor (LIF) lOOx [Chemicon #ESG1106]

L-Glutamine lOOx [Gibco-BRL #25030-149]

L-Glutamine [Sigma #G-8540]

2-Mercaptoethanol (0.07%) (2ME) [Sigma #M-7522]

N? Insulin 5 mg/ml [Sigma #1-6634]

Nicotinamide 100X [Sigma #Ν0636]

Non-Essential Ammo Acids (NEAA) lOOx [Gibco-BRL #11140-050]

Penicillin-Streptomycin (Pen/Strep) lOOx [Gibco-BRL #15140-122]

Phosphate-Buffered Saline (PBS) [Gibco-BRL #14190-250]

Poly-ornithine [Sigma #P-4957]

Progesterone 2 mM stock (PG) [Sigma #P-8783]

Putrescine 1 M stock (Ptr) [Sigma #P-5780]

Sodium Bicarbonate [Sigma #S-5761]

Sodium Selenite (300 μM) [Sigma #S-5261]

Ly294002 (10DM) [Calbiochem #440202]

60 mm Tissue Culture Dishes [Fisher # 08-772F, Falcon #35-3004]

100 mm Tissue Culture Dishes [Fisher # 08-772-22]

Transferrin 2 mg/ml [Sigma #T-3309]

Trypsin-EDTA (DisB) [Gibco-BRL #25300-054]

12 Well Plates [Fisher # 12-565-321, Nalge Nunc International #150628]

Ultra Low Attachment Plates six well [Corning, Costar #3471]

Media

Stage 0: MEF media (Mouse Embryonic Feeder) 88 ml DMEM (+ Glutamax and Na Pyruvate)

10 ml FCS l ml lOOx NEAA l ml lOOx Pen/Strep

Stage 1 and 2 : KO media, LIF (+ or -)

80 ml KNOCKOUT D-MEM

15 ml FCS l ml lOO NEAA l ml lOOx Pen/Strep l ml lOO L-glutamine l ml 2ME

l ml LΓF

Stage 3: ITSFn media (Insulin, Transferrin, sodium Selenite, Fibronectin)

50 ml DMEM/F12

1.25 ml Transferrin, 2 mg/ml

50 μl ITSFn insulin, 5 mg/ml

5 μl Sodium selenite, 200 μM 250 μl Fibronectin, 1 mg/ml

500 μl lOOx Pen/Strep

Stage 4 and 5: N2 media 250 ml DMEM/F12 media 50 mg Apotransferrin

775 mg Glucose

36.5 mg Glutamine

845 mg Sodium bicarbonate

Adjust the pH to 7.1 to 7.2 with cell culture grade IN HCl. Adjust the volume to 500 ml with pure ddH₂O. Filter through a 0.22 μm filter. For 500ml, now add the following:

2.5ml N₂ insulin

100 μl lOOμM Progesterone

50μl Putrescine (IM)

50μl Selenium (300μM)

5ml Pen/Strep

Stage 4

X ml N₂ media

50x B27 supplement lOOx bFGF

Stage 5

X ml N₂ media lOμM Ly294002 lOOx Nicotinamide

DisA Buffer

5 ml of 4% EDTA into 495ml PBS .

Procedure

Stage 0: Day 1 (1)

• Prepare and pre-warm MEF media.

• Add 5 ml of gelatin to 4 x 10cm tissue culture dishes. Incubate for 20 minutes at room temperature. • Retrieve one vial of MEF cells ( 3 x 10⁶ cells/vial) from cryostorage and immediately thaw in the 37 °C water bath for two minutes (gently rotate the vial to thaw).

• Once the cells are thawed, gently add the cell suspension to MEF media and centrifuge for 5 minutes at 1000 φm to pellet the cells. While the cells are spinning aspirate off the gelatin off the tissue culture plates and add 8 ml of MEF media to each dish. • Once the cells are done spinning, aspirate off the supernatant and add 4 ml of MEF media and completely resuspend the MEF cells breaking up the clumps as well as possible.

• Add 2 ml of cell suspension to each dish and place in the incubator.

• Incubate for at least 5-6 hours or overnight.

Stage 1: Day 1 (3)

1. Prepare and pre-warm KO media LIF(+).

2. Thaw 3 vials of ES cells (5 x 10^δ cells/vial) in the 37 °C water bath for two minutes.

3. Once the cells are thawed, gently add the cell suspension to the KO LLF(+), centrifuge for five minutes. While the cells are spuming, aspirate the MEF media from feeder cells plates and add 8 ml of KO LIF(+) media.

4. Aspirate the supernatant from the ES cells and very gently add 4 ml of KO LLF(+) media. Gently resuspend the ES cells and add 2 ml to each dish and place the cells in the incubator.

Stage 1: Day 2 (4)

1. Pre-warm KO LIF(+) media.

2. Carefully remove tissue culture dishes from incubator and aspirate off the supernatant.

3. Gently add fresh KO LIF(+) media by touching the end of the pipette to the side of the tissue culture dish neat the bottom of the dish and dispense the solution at less than 0.2 ml/sec. Return the cells to the incubator.

Stage 1: Day 3 (5)

1. Add 5 ml of gelatin to 6 x 10cm tissue culture plates.

2. Warm KO LIF(+) media. Warm DisA to room temperature.

3. Wash the ES cell plates with DisA buffer. 4. Add 1 ml of DisB buffer for 5 minutes at room temperature. Complete dissociation of cells is desired. 5. Add 2 ml KO LIF(+) media to each dish and dissociate the cell clusters completely by passing the cell suspension though a plastic pipette. Transfer the cells to the Falcon tube. Wash the plate one more time with KO LLF(+) and check for >98% transfer. 6. Pellet the cells at 1000 φm for 5 minutes.

7. Aspirate the gelatin solution from the new plates and add 8 ml of KO LIF(+) solution.

8. Resuspend the cells in 12 ml of KO LIF(+). The cell pellet tends to stick together to pipette up and down until a homogeneous solution is obtained, about 20-30 times. (Although this will not completely dissociate the cell clusters.) 9. Add 2 ml cell suspension to each plate. Check for the size of cell clusters formed. It is likely that a number of large clusters will still be present. The break up these clusters use a PI 000 set to 800 μl to forcefully titrate 20-30 times without creating any bubbles. This can best be done by using the 200 to 600 μl range of the pipetman. It is important to get close to a single cell suspension to keep the cells from differentiating and also to separate any sick or dead cells from the healthy cells.

10. Check the amount of dissociation and return the cells to the incubator.

Stage 2: Day 1 (6)

1. Pre-warm DisA to room temperature.

2. Prepare KO LLF(-) media.

3. Add 2 ml KO LLF(-) to 3 x 6-well low adhesion tissue culture plates.

4. Wash plates with cells with DisA buffer.

5. Add 1 ml of DisB to plates for 5 minutes at room temperature. 6. Add 3 ml KO LIF(-) media to each the dish. Transfer the cells to a 50ml tube. Wash plates serially with a 3 ml of KO LLF(-) and check for >98% transfer. Pellet the cells for 5 minutes at lOOOφrn. 7. Aspirate the supernatant, resuspend in KO LIF(-). Pipette up and down until a homogeneous solution is obtained. 8. Add 1 ml cell suspension to each well of a 6-well low adhesion tissue culture dish. Be very careful to make sure the cell density is the same. Incubate at 37 °C.

Stage 2: Day 2 (7) 1. Pre-warm KO LIF(-) media and transfer all the cells from their individual wells into a 50 ml tube using a 10ml pipet. Let the cells settle for 5-10 minutes. Aspirate off media.

2. Add 3 ml/well fresh media (54 ml for 18 wells) and gently titrate the solution up and down in the 10 ml pipette to break up the embryoid body clumps, but so as to not break up the embryoid bodies.

3. 3 ml of solution was added to each well trying to keep the same cell density in each well.

Stage 2: Day 3 (8)

1. Collect embryoid bodies and wash each well with 3 ml of media.

2. Let the embryoid bodies settle for 10 minutes.

3. Aspirate offthe solution and add KO LIF(-) media.

4. Plate on 20-30 x 6cm tissue culture dishes (3 ml total). Incubate at 37 °C for about 26 to 36 hours to ensure embryoid body adhesion to the dish surface.

Stage 3: Day 1 (10)

1. Make ITSFn media.

2. Pre-warm DMEM/F-12 media. 3. Very gently aspirate offthe solution from the attached embryoid bodies. Very gently add 3 ml of DMEM F-12 and aspirate to wash the cells and eliminate any FCS. 4. Add 3 ml of ITSFn media to each dish and incubate at 37 °C.

Stage 3: Day 3 (12)

1. Warm ITSFn media.

2. Aspirate offthe old ITSFn media

3. Gently add 3 ml fresh media.

Stage 3: Day 5 (14)

1. Warm ITSFn media.

2. Aspirate offthe old ITSFn media 3. Gently add 3 ml fresh media.

4. Coat plates with poly-ornithine.

5. Add .5 ml of this solution to each well of 12-well or, add 1 ml of this solution to each 6 cm tissue culture dish or each well of a 6-well plate, (usually there are enough cells for 4 6- well plates). 6. Place in 37 °C incubator overnight.

Stage 3: Day 6 (15)

1 Aspirate off poly-ornithine solution.

2 Add PBS (0.5 ml/well or 1 ml/dish). 3 Incubate at 37 °C for at least 1 hour. 4 Prepare 1 μg/ml fibronectin solution and filter. 5 Aspirate off PBS from cells. 6 Wash once more with PBS (do not incubate). 7 Add 1 μg/ml filtered fibronectin solution. 8 Incubate 1 hour to overnight up to 2 days maximum.

Stage 4: Day 1 (16)

1. Make N₂ media (- B27, bFGF, and nicotinamide).

2. Prepare an aliquot of N₂ media with 50X B27 and 100X bFGF.

3. Prepare DMEM/F-12 + 10% FCS.

4. Aspirate off ITSFn media from plates and wash with 3 ml of DisA, aspirate off.

5. Add 1 ml of DisB for 5 minutes and check for complete cell cluster dissociation from the plate.

6. Add 1 ml of DMEM/F-12 +10% FCS to each plate and transfer to a 50 ml Falcon tube. Wash plates serially with 3 ml of DMEM/F-12 + 10% FCS and add to the 50 ml tube.

7. Centrifuge for 5 minutes at lOOOφm, remove the supernatant and resuspend the cell clusters in 10 ml of N₂ + B27 + bFGF. 8. Count cells.

9. If fibronectin solution has not been aspirated off yet and the plates and wells washed with lx with PBS, perform this step now.

10. Aspirate off PBS and immediately add cells once PBS is removed. 11. Plate cells at a density of 2.3-3 x 10 ⁵ cells/cm².

Stage 4: Dav 3 (18)

1. Stage 4 media change

Stage 4: Day 5 (20)

1. Stage 4 media change

Stage 5: Day 1 (22)

1. Change to Stage 5 media conditions.

Stage 5: Day 3 (24)

1. Stage 5 media change

Stage 5: Day 5 (26)

1. Stage 5 media change.

Stage 5: Day 6 (27)

1. Collect cells for analysis or experiment.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incoφorated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incoφorated by reference. In case of conflict, the present application, including any definitions herein, will control.

References to be incoφorated herein include the following:

A. M. Shapiro et al, N Engl J Med 343, 230 (2000).

N. Lumelsky et al, Science 292, 1389 (2001).

B. Soria et al., Diabetes 49, 157 (2000).

S. Assady et al, Diabetes 50, 1691 (2001). N. M. Le Douarin, Cell 53, 169 (1988).

S. K. Kim, M. Hebrok, Genes Dev. 15, 111 (2001).

S. Okabe, K. Forsberg-Nilsson, A. C. Spiro, M. Segal, R. D. McKay, Meek Dev. 59, 89 (1996).

S. H. Lee, N. Lumelsky, L. Studer, J. M. Auerbach, R. D. McKay, Nature Biotechnol. 18, 675 (2000).

A. Ptasznik et al., J Cell Biol 137, 1127 (1997).

R. M. Jindal, Pancreas 11, 316 (1995).

J. A. Thomson et al, Science 282, 1145 (1998).

D. L. Eizirik et al, Proc Natl Acad Sci US A. 91, 9253 (1994).

Y. Guz, I. Nasir, G. Teitelman, Endocrinology 142, 4956 (2001).

D. B. Kaufman et al., J. Exp. Med. 172, 291 (1990).

P. L. Herrera, Development 121, 2317 (2000). G. Gu, J. Dubauskaite, D. A. Melton, Development 129, 2447 (2002).

U. Ahlgren, S. L. Pfaff, T. M. Jessell, T. Edlund, H. Edlund, Nature 385, 257 (1997).

G. Gradwohl, A. Dierich, M. LeMeur, F. Guillemot, Proc Natl Acad Sci USA 97, 1607 (2000).

K. Ueki, et al., Proc Natl Acad Sci U S A 99, 419-24. (2002)

Y. Terauchi, et al., Nat Genet 21, 230-5. (1999) K. Eto, et al., Diabetes 51, 87-97. (2002)

B. Soria, Differentiation 68, 205-19. (2001)

S. Okabe, Differentiation of Embryonic Stem Cells., Current Protocols in Neuroscience (1997), vol. 3

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

WE CLAIM:

1. A method for making an insulin-producing cell composition, the method comprising culturing a stem-cell derived cell composition in the presence of a cell proliferation inhibitor in an amount sufficient to enrich for insulin-producing cells.

2. The method of claim 1 , wherein the cell proliferation inhibitor is a PI3K pathway inhibitor.

3. The method of claim 1, wherein the insulin-producing cell composition comprises at least about 1000 nanograms of insulin per milligram of total protein.

4. The method of claim 1, wherein at least about 80% of the cells of the insulin-producing cell composition produce insulin. 5. The method of claim 1 , wherein the insulin-producing cell composition does not undergo neoplastic growth after transplantation into a mammalian host. 6. The method of claim 1 , wherein the stem cell-derived cell composition is derived from a cell line selected from the group consisting of: an embryonic stem cell line, a multipotent adult stem cell line and a pluripotent adult stem cell line. 7. The method of claim 1, wherein the stem cell-derived cell composition comprises a detectable amount of nestin protein.

8. The method of claim 1, wherein the stem cell-derived cell composition is not derived from a pancreatic stem cell line.

9. The method of claim 1, wherein the majority of the cells of the stem cell-derived cell composition have a wild-type genotype.

10. The method of claim 2, wherein the PI3K pathway inhibitor is a PI3K inhibitor.

11. The method of claim 2, wherein the PI3K inhibitor is LY294002 or wortmannin.

12. The method of claim 2, wherein the method comprises culturing the stem cell-derived cell composition in the presence of a PI3K pathway inhibitor and an ADPRT inhibitor. 13. The method of claim 2, wherein the method comprises culturing the stem cell-derived cell composition in the presence of a PI3K pathway inhibitor and a nicotinamide agent.

14. The method of claim 1, further comprising a step of removing the cell proliferation inhibitor.

15. The method of claim 14, further comprising, after removing the cell proliferation inhibitor, culturing the insulin producing cell composition in the presence of an insulinogenic agent and a nicotinamide agent.

16. The method of claim 15, wherein the insulinogenic agent is selected from the group consisting of: IGF-1, HGF, a cyclic AMP stimulating agent, exendin, GLP1, PPARγ ligand, sonic hedgehog, PACAP and growth hormone. 17. The method of claim 16, wherein the cyclic AMP stimulating agent is forskolin or

ΓBMX. 18. A method of making an insulin-producing cell composition, the method comprising: a) culturing a stem cell line to produce a stem cell-derived cell composition comprising cells expressing one or more proteins that are indicative of neural stem cells or pancreatic stem cells; b) culturing the stem cell-derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining an insulin-producing cell composition.

19. The method of claim 18, wherein the stem cell-derived cell composition is positive for the following markers: nestin, neurogenin, islet-1, glucagon and insulin.

20. The method of claim 18, wherein (a) comprises: i) culturing the stem cell line on a feeder layer in the presence of leukemia inhibitory factor (LIF), or a functional analog thereof to generate a first cell composition; ii) culturing the first cell composition in the absence of LIF, or a functional analog thereof to generate a second cell composition; iii) culturing the second cell composition in the presence of insulin, transferrin, selenium and fibronectin, or functional analogs thereof to generate a third cell composition; and iv) culturing the third cell composition in the presence of basic fibroblast growth factor and B27 supplement, or functional analogs thereof, to generate the stem cell-derived cell composition for use in (b). 21. An insulin-producing cell composition made according the method of any of claims 1 -

20.

22. An insulin-producing cell composition derived from a stem cell line and comprising at least about 1000 nanograms of insulin per milligram of total protein.

23. The insulin-producing cell composition of claim 22 that is derived from an embryonic stem cell line.

24. The insulin-producing cell composition of claim 22 that is derived from a human embryonic stem cell line.

25. The insulin-producing cell composition of claim 22, wherein at least about 80% of the cells of the insulin-producing cell composition produce insulin.

26. The insulin-producing cell composition of claim 22, wherein the cells are tumorigenic upon implantation in fewer than 1%> of subjects.

27. A therapeutic cell composition comprising an insulin-producing cell composition of claim 21 and a therapeutically acceptable excipient.

28. A therapeutic cell composition comprising an insulin-producing cell composition of any of claims 22-26 and a therapeutically acceptable excipient.

29. A method of ameliorating, in a subject, a condition related to insufficient pancreatic function, the method comprising administering to the subject an effective amount of an insulin producing cell composition produced by a method comprising culturing a stem- cell derived cell composition in the presence of a cell proliferation inhibitor in an amount sufficient to enrich for insulin-producing cells.

30. The method of claim 29, wherein the effective amount of insulin-producing cell composition causes an increase in blood insulin levels in the subject. 31. The method of claim 29, wherein the effective amount of insulin-producing cell composition causes an increased rate of glucose-induced insulin production in the subject. 32. The method of claim 29, wherein the subject has a diabetes caused by beta-cell insufficiency. 33. The method of claim 29, wherein the effective amount of insulin producing cell composition causes a neoplasm in fewer than 1%> of subjects. 34. The method of claim 29, wherein the effective amount of insulin producing cell composition is at least one islet-like cluster per islet that is, on average, naturally present in the subject. 35. A method for testing the developmental potential of a cell of interest, the method comprising: a) subjecting a stem cell line to an in vitro differentiation process so as to cause a cell of the stem cell-derived cell composition to give rise to a cell that produces a pancreatic hormone; b) mixing a cell of interest with the stem cell-derived cell composition at a point in the differentiation process; and c) determining the identity of cells derived from the cell of interest, thereby testing the developmental potential of the cell of interest.

36. The method of claim 35, wherein the cell of interest is obtained from a pancreatic tissue.

37. The method of claim 35, wherein the in vitro differentiation process comprises contacting the stem cell-derived cell composition with a PI3K pathway inhibitor.

38. A method for identifying a candidate gene related to pancreatic development, the method comprising: a) Measuring a first level of a gene product in a stem cell-derived cell composition; b) Culturing the stem-cell derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining an insulin-producing cell composition; and c) Measuring a second level of the gene product in the insulin-producing cell composition, wherem a gene that encodes the gene product that has a significantly different first and second levels is a candidate gene related to pancreatic development.

39. The method of claim 38, wherein measuring a first and second level of a gene product comprises performing a nucleic acid microarray assay.

40. The method of claim 38, wherein measuring a first and second level of a gene product comprises performing a protein measurement assay selected from the group consisting of: a protein microarray assay, a two-dimensional gel electrophoresis and a mass spectroscopy analysis.

41. A non-human animal comprising an insulin-producing cell composition of claim 21.

42. A non-human animal comprising an insulin-producing cell composition of any of claims 22-26.

43. Use of an insulin-producing cell composition of claim 21 for preparing a medicament for the treatment of a condition related to insufficient pancreatic function.

44. Use of an insulin-producing cell composition of any of claims 22-26 for preparing a medicament for the treatment of a condition related to insufficient pancreatic function.