Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

Abstract

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 1. FGF4 and Wnt3a act synergistically in a temporal and dose-dependent manner to specify stable posterior endoderm fate

ActivinA (100ng/ml) was used to differentiate H9-HES cells into definitive endoderm (DE). DE was treated with the posteriorizing factors FGF4 (50, 500ng), Wnt3a (50, 500ng) or both for 6, 48 or 96 hours. Cells were placed in permissive media for 7 days and expression of foregut markers (ALB, PDX1) and the hindgut marker (CDX2) were analyzed by RT-qPCR (a) and immunofluorescence (b-d). Controls DE was grown for identical lengths of time in the absence of FGF4 or Wnt3a. High levels of FGF4+Wnt3a for 96 hours gave resulted in stable CDX2 expression and lack of foregut marker expression. Error bars are S.E.M (n=3). Significance is shown by; * (p<0.05), ^ (p<0.001), # (p<0.0001).

Figure 2. Morphogenesis of posterior endoderm into three-dimensional, hindgut-like spheroids

a, 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 hindgut endoderm (E) and adjacent mesenchyme (M) are CDX2 positive (green). (FG – foregut, HG – hindgut).

Figure 3. hESCs and hiPSCs form 3-dimensional intestine-like organoids

a, 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 NEUROG3

28 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.