Type I diabetes is an autoimmune disease of humans caused by destruction of pancreatic islet β cells. Transplantations of whole pancreas or isolated islet cells are effective treatments for Type I diabetes to restore insulin independence, when combined with immunosuppressive therapy. Successful transplantation of isolated islets from human cadaver donors is a proof-in-principle that a cell-based therapy for human diabetes can be successful. However, the lack of available organs and islet cells has restricted this therapy to very few patients. The amount of islet cells which can be harvested from human cadavers is extremely limited. Therefore, technologies capable of producing significant quantities of cells of the pancreatic lineage are highly desirable.
Stem cells are cells that are capable of differentiating into many cell types. Embryonic stem cells are derived from embryos and are potentially capable of differentiation into all of the differentiated cell types of a mature body. Certain types of stem cells are “pluripotent,” which refers to their capability of differentiating into many cell types. One type of pluripotent stem cell is the human embryonic stem cell (hESC), which is derived from a human embryonic source. Human embryonic stem cells are capable of indefinite proliferation in culture, and therefore, are an invaluable resource for supplying cells and tissues to repair failing or defective human tissues in vivo.
Similarly, induced pluripotent stem (iPS) cells, which may be derived from non-embryonic sources, can proliferate without limit and differentiate into each of the three embryonic germ layers. It is understood that iPS cells behave in culture essentially the same as ESCs. Human iPS cells and ES cells express one or more pluripotent cell-specific markers, such as Oct-4, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and Nanog (Yu et al. Science, Vol. 318. No. 5858, pp. 1917-1920 (2007)). Also, recent findings of Chan, suggest that expression of Tra 1-60, DNMT3B, and REX1 can be used to positively identify fully reprogrammed human iPS cells, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT, and NANOG are insufficient as markers of fully reprogrammed human iPS cells. (Chan et al., Nat. Biotech. 27:1033-1037 (2009)). Subsequent references herein to hESCs and the like are intended to apply with equal force to iPS cells.
One of most significant features of hESCs is their ability to self-renew: hESCs can proliferate into multiple progeny hESCs, each having the full potential of its immediate ancestor. In other words, the progeny are pluripotent and have all the developmental and proliferative capacity of the parental cell. Self-renewal appears mutually exclusive with differentiation, as only undifferentiated hESCs are capable of indefinite self-renewal. Upon commitment toward any cell lineage, the attribute of perpetual self-renewal is lost. Therefore, until culture conditions are discovered that provide the ability to direct the commitment and subsequent differentiation of hESCs to a desired cell lineage, care must be taken to maintain the cells in an undifferentiated state.
Under nonselective culture conditions, it has been previously demonstrated that a wide variety of stem cells, including mouse embryonic stem cells and hESCs, differentiate spontaneously into cells of many lineages including the pancreatic lineage. Such differentiated cells can express the pancreatic duodenal homeobox 1 (PDX1) gene, a transcription factor specifying the pancreatic lineage, and can also express insulin. However, without selective conditions, stem cells will spontaneously and simultaneously differentiate in the same culture dish into a wide variety of different lineages with only a small proportion of the cells being differentiated towards any particular lineage.
Culture systems that allow the spontaneous differentiation of hESCs into insulin-staining cells have been reported (Assady, S. et al., Insulin production by human embryonic stem cells. Diabetes 50, 1691-1697 (2001); and Segev, H. et al., Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22, 265-274 (2004)). However, these studies neither investigated endoderm marker expression nor demonstrated development of cells possessing stereotypical characteristics of β cells: simultaneous expression of C-peptide and PDX1, which is required for pancreas formation and co-activates the insulin promoter (Jonsson, J. et al., Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371, 606-609 (1994)). Because non-β cells such as neuronal cells, may express insulin (Sipione, S. et al., Insulin expressing cells from differentiated embryonic stem cells are not β cells. Diabetologia 47, 499-508 (2004)), and insulin present in the culture media may be taken up into other cell types under certain conditions in vitro (Rajagopal, J. et al., Insulin staining of ES cell progeny from insulin uptake. Science 299, 363 (2003)), it is important that the endoderm and pancreatic origin of insulin-staining cells derived from hESCs be ascertained.
Spontaneous differentiation of hESCs has produced PDX1+/FOXA2+ cells and co-transplantation of these differentiated cells with mouse dorsal pancreas (E13.5) produced PDX1+/insulin+ cells, and co-staining of insulin and C-peptide was observed (Brolen, G. K. et al., Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing β-cell-like cells. Diabetes 54, 2867-2874 (2005)). Thus, pancreatic lineage cells can be induced from spontaneously differentiating hESCs by signals emanating from the embryonic pancreas. However, the experimental methods used to reach such observations would be impractical to adopt into a high-throughput culture protocol. Moreover, the nature of the molecular signals was not revealed by the study. In addition, unselected stem cell populations are tumorigenic, meaning that they will generate non-malignant tumors, known as teratomas, in immunodeficient animals like undifferentiated ES cells do.
Several studies have evaluated the effects of growth factors on hESC differentiation to endoderm (Schuldiner, M. et al., Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97, 11307-11312 (2000) and D'Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534-1541 (2005)). However, highly efficient differentiation to pancreatic precursors and islet cells has not been routinely achievable. Furthermore, insulin producing cells generated using previously reported methods are less responsive to glucose, in that, they appear less functionally mature than adult human β cells and are believed to possess a phenotype more like immature β cells. Taken together, these studies indicate that additional signals may be necessary to convert endoderm into pancreatic progenitors and insulin expressing cells into maturely functional β cells.
Studies of growth factor regulation of pancreas development in embryo models may provide important insights for directing hESC differentiation towards the pancreatic lineage (Wells, J. M. & Melton, D. A. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 127, 1563-1572 (2000)). For example, it was demonstrated in a chick-quail chimera system that BMP4 induces PDX1 expression in uncommitted endoderm and noggin blocks PDX1 expression in committed endoderm (Kumar, M. et al., Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev. Biol. 259, 109-122 (2003)). However, hESC differentiation is a multifactorial process, in which numerous factors influence the transition from pluripotency toward a differentiated cell lineage. Moreover, recent studies with hESCs have begun to focus on the differentiation of definitive endoderm as a first step toward development of pancreatic lineage cells. Others have reported on Activin A induction of definitive endoderm from hESCs (see D'Amour, K. A., et al. (2005)). However, pancreatic lineage cells were not induced by this protocol. Furthermore, preliminary results testing Activin A (at 5 ng/ml, 50 ng/ml, or 100 ng/ml) in serum-free media suggest that this treatment alone cannot induce pancreatic cell differentiation. This is not surprising given that it has been demonstrated that, in the absence of feeder cells, Activin A can maintain pluripotency of hESCs (Beattie, G. M. et al., Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489-495 (2005)). Other hESC studies evaluating pancreatic differentiation have either been inconclusive as to the origin of insulin staining cells or required a period of in vivo growth in undefined conditions (Brolen, G. K. et al., (2005)).
Recent improved techniques reported for culturing hESCs into cells of the pancreatic lineage, such as that disclosed in U.S. Patent Application Publication No. 2011/0081720, illustrate the ability to produce pancreatic cell types for research and therapeutic uses. Thus, reproducible culture methods utilizing defined components that promote islet differentiation from human pluripotent stem cells have been shown. However, advances in our understanding of extrinsic signaling events controlling the formation of definitive endoderm and regional specification of the pancreas are leading to new methodologies for directed differentiation of stem cells into cells of the pancreatic lineage. Subtle differences in media growth factor concentrations, timing and/or sequence of growth factor introduction, and length of incubation with particular growth factors may induce pluripotent stem cells to differentiate into many different cell lineages. Moreover, the types and concentrations of supporting extracellular matrix components may further affect the differentiation of pluripotent stem cells. Therefore, how these influences are orchestrated will likely determine the fate of pluripotent stem cells cultured in vitro.