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Nature
2011 Feb 03;4707332:105-9. doi: 10.1038/nature09691.
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Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
Spence JR
,
Mayhew CN
,
Rankin SA
,
Kuhar MF
,
Vallance JE
,
Tolle K
,
Hoskins EE
,
Kalinichenko VV
,
Wells SI
,
Zorn AM
,
Shroyer NF
,
Wells JM
.
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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 that 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. Here we establish 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 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 indicate that human intestinal stem cells form de novo during development. We also 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. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
Figure 2. Morphogenesis of posterior endoderm into three-dimensional, hindgut-like spheroidsa, Bright field images of DE cultured for 96 hours in media, FGF4, Wnt3a or FGF4+Wnt3a. FGF4+Wnt3a cultures contained 3D epithelial tubes and free-floating spheres (black arrows) b, CDX2 immunostaining (Green) and nuclear stain (Draq5 - blue) on cultures shown in a. c, Bright field image of hindgut-like spheroids. d-f, Analysis of CDX2, basal-lateral laminin and E-Cadherin expression demonstrate an inner layer of polarized, cuboidal, CDX2+ epithelium surrounded by non-polarized mesenchymal CDX2+ cells. g, CDX2 expression in an e8.5 mouse embryo (sagittal section). Inset is a magnified view showing that both hindgutendoderm (E) and adjacent mesenchyme (M) are CDX2 positive (green). (FG – foregut, HG – hindgut).
Figure 3. hESCs and hiPSCs form 3-dimensional intestine-like organoidsa, A time course shows that intestinal organoids formed highly convoluted epithelial structures surrounded by mesenchyme after 13 days. b-e, Intestinal transcription factor expression (KLF5, CDX2, SOX9) and cell proliferation on serial sections of organoids after 14 and 28 days (serial sections are b and c, d and e). f and g, Expression of KLF5, CDX2, and SOX9 in mouse fetal intestine at e14.5 (f) and e16.5 (g) is similar to developing intestinal organoids. The right panels show separate color channels for d, e and g. h, i and j, whole mount in situ hybridization of 56 day old organoids showing epithelial expression of Sox9 (h) and restricted “crypt-like” expression of the stem cell markers Lgr5 (i) and Ascl2 (j). Insets show sense controls for each probe.
Figure 4. Formation and function of intestinal cell types and regulation of enteroendocrine differentiation by NEUROG328 day iPSC-derived organoids were analyzed for a, villin (VIL) and the goblet cell marker mucin (MUC2), b, the paneth cell marker lysozyme (LYSO) or c, the endocrine cell marker chromogranin A (CGA). d, Electron micrograph showing an enterocyte cell with a characteristic brush border with microvilli (inset). e, Epithelial uptake of the fluorescently labeled dipeptide d-Ala-Lys-AMCA (arrowheads) indicating a functional peptide transport system. f-h, Adenoviral expression of Neurog3 (pAd-NEUROG3) causes a 5-fold increase in CGA+ cells compared to a GFP control (pAd-GFP); (n=4 biological samples);*(p=0.005). i-k, Organoids were generated from hESCs that were stably transduced with shRNA-expressing lentiviral vectors. Compared to control shRNA organoids, NEUROG3 shRNA organoids had a 95% reduction in the number of CgA+ cells; (n=3 for shRNA controls and n=5 for Neurog3-shRNA); *(p=0.018). Error bars for h,k are S.E.M.
Baker,
Differentiation in three dimensions.
2011, Pubmed
Baker,
Differentiation in three dimensions.
2011,
Pubmed
Barranco,
Stem cells: Intestinal tissue generated in vitro.
2011,
Pubmed
Basma,
Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.
2009,
Pubmed
Cai,
Directed differentiation of human embryonic stem cells into functional hepatic cells.
2007,
Pubmed
Cao,
Intestinal lineage commitment of embryonic stem cells.
2011,
Pubmed
D'Amour,
Efficient differentiation of human embryonic stem cells to definitive endoderm.
2005,
Pubmed
D'Amour,
Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.
2006,
Pubmed
Dessimoz,
FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo.
2006,
Pubmed
Gracz,
Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro.
2010,
Pubmed
Groneberg,
Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1.
2001,
Pubmed
Haveri,
Transcription factors GATA-4 and GATA-6 in normal and neoplastic human gastrointestinal mucosa.
2008,
Pubmed
Jenny,
Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium.
2002,
Pubmed
Kosinski,
Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development.
2010,
Pubmed
Kroon,
Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.
2008,
Pubmed
Lee,
Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity.
2002,
Pubmed
López-Díaz,
Intestinal Neurogenin 3 directs differentiation of a bipotential secretory progenitor to endocrine cell rather than goblet cell fate.
2007,
Pubmed
Mayhew,
Converting human pluripotent stem cells into beta-cells: recent advances and future challenges.
2010,
Pubmed
McLin,
The role of the visceral mesoderm in the development of the gastrointestinal tract.
2009,
Pubmed
,
Xenbase
McLin,
Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development.
2007,
Pubmed
,
Xenbase
Ootani,
Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.
2009,
Pubmed
Ormestad,
Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production.
2006,
Pubmed
Sato,
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.
2009,
Pubmed
Song,
Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.
2009,
Pubmed
Spence,
Translational embryology: using embryonic principles to generate pancreatic endocrine cells from embryonic stem cells.
2007,
Pubmed
Torihashi,
Gut-like structures from mouse embryonic stem cells as an in vitro model for gut organogenesis preserving developmental potential after transplantation.
2006,
Pubmed
Touboul,
Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.
2010,
Pubmed
Wang,
Mutant neurogenin-3 in congenital malabsorptive diarrhea.
2006,
Pubmed
,
Xenbase
Wells,
Early mouse endoderm is patterned by soluble factors from adjacent germ layers.
2000,
Pubmed
Zhang,
Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.
2009,
Pubmed
Zorn,
Vertebrate endoderm development and organ formation.
2009,
Pubmed
,
Xenbase
de Santa Barbara,
Development and differentiation of the intestinal epithelium.
2003,
Pubmed
van der Flier,
Transcription factor achaete scute-like 2 controls intestinal stem cell fate.
2009,
Pubmed
