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We are using the signals and genes that direct endoderm and pancreas differentiation in the embryo to direct the differentiation of pluripotent embryonic stem (ES) cells into therapeutically important endoderm derivatives, such as insulin-producing beta cells. The goal of this work is to generate cells that could be used in a transplantation-based therapy to treat type 1 diabetes (figure 1 from Spence and Wells, 2007). Embryonic stem cells have enormous therapeutic potential because they are genetically unprogrammed and can form all cell types of the body.
Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells, which have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies.
We have established a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm (DE) formation, FGF / Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation.
The resulting three-dimensional intestinal “organoids” consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells.
Using this culture system as a model to study human intestinal development, we identified that the combined activity of Wnt3a and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data suggest that human intestinal stem cells form de novo during development.
Lastly we determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro.
In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
Lin SC, Wani M, Whitsett JA, Wells JM. Klf5 regulates lineage formation in the pre-implantation mouse embryo. Development. 137(23):3953-63. Dec, 2010.
Spence JR, Mayhew CM, Kuhar M, Tolle K, Vallance JE, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shroyer NF, Wells JM. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2010.
Spence JR, Wells JM. Translational embryology: Using embryonic principles to generate pancreatic endocrine cells from embryonic stem cells. Dev Dyn. 236:3218-27. 2007.
Wells JM. Genes expressed in the developing endocrine pancreas and their importance for stem cell and diabetes research. Diabetes Metab Res Rev. 19:191-201. 2003.
The lineage of the developing pancreas in vivo and in ES cultures. The left panels are mouse embryos at different stages of development (embryonic day 3.5 through 13.5, E3.5-E13.5), and the black bars on the right indicate the equivalent stages in human development. Embryos are oriented with anterior to the left and posterior to the right. The E13.5 stage shows the dissected stomach, pancreas and duodenum with the stomach (s) and dorsal pancreas (dp) to the left and the ventral pancreas (vp) and duodenum (d) to the right. The lacZ staining in the E9.5 and 13.5 embryos shows expression of Pdx1 (Pdx1lacZ/+ animals were from Chris Wright at Vanderbilt University). The lower left panel is a pancreatic islet showing glucagon-expressing a-cells (green) and insulin-expressing b-cells (red). The curved arrows highlight several signaling pathways involved in pancreas development that have been used to direct HESC differentiation into the pancreatic lineage (D’Amour et al. 2005, D’Amour et el. 2006). The middle panel indicates the lineage of the developing endocrine pancreas. The arrowheads in the cell lineage diagram highlight the two separate roles of nodal / activin signaling: to initiate gastrulation and to promote endoderm versus mesoderm fate in a dose-dependent manner.
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Model comparing embryonic intestinal development versus directed differentiation of human PSCs into intestinal tissue in vitro.
a: Schematic of human intestinal development. At the blastocyst stage, the inner cell mass (ICM) gives rise to the entire embryo. The ICM is also the source of embryonic stem cells. At the gastrula stage, the embryo contains the three germ layers including the embryonic / definitive endoderm (yellow). The definitive endoderm forms a primitive gut tube, with the hindgut forming in the posterior region of the embryo. The hindgut undergoes intestinal morphogenesis forming the small and large intestines. b: Schematic of directed differentiation of PSCs into intestinal tissue. PSCs cultured for three days in activin A form definitive endoderm (DE) co-expressing Sox17 and FOXA2. DE cultured for four days in FGF4 and Wnt3a (500ng / ml each) form three-dimensional hindgut spheroids expressing the posterior marker CDX2. Spheroids formed intestinal organoids when grown in three-dimensional conditions that favor expansion and differentiation of intestinal precursors (Matrigel with 500ng / mL R-Spondin1, 100ng / mL Noggin and 50ng / mL EGF. c: Side-by-side comparison of embryonic intestinal development (top) and human intestinal organoid development (bottom). PSCs underwent staged differentiation in a manner that was highly reminiscent of embryonic intestinal development and formed intestinal tissue. Stages of development in c are meant to approximate the ones schematically shown in a and b.
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