An in vivo model of human small intestine using pluripotent stem cells

Abstract

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

Figure 1

HIOs engraft in vivo to form mature intestinal tissue. (a) Schematic representing development of HIOs from hPSCs and transplantation under kidney capsule to produce mature human intestinal tissue. (b) Two HIOs in vitro at 35 d consisting of intestinal epithelium (black arrowheads) surrounded by supporting mesenchyme (white arrowheads). Scale bar, 100 μm. (c) Engraftment (outlined) after 6 weeks with complex structure and established peripheral capillary network. The mouse kidney is seen below the engraftment for size comparison. Scale bar, 5 mm. (d) Cross-section of engraftment at 6 weeks revealing intestinal structure with central lumen. Scale bar, 5 mm. (e) Magnified luminal surface of engraftment displaying sheet of villi each with its own central capillary. Scale bar, 500 μm. n = 139 translplants.

Figure 2

Engrafted intestinal tissue resembles adult intestine and is almost entirely of human origin. (a) Low- and high-power imaging following H&E staining of engrafted HIO. Low magnification imaging demonstrates multiple areas of epithelium, laminated layers of smooth muscle and peripheral capillaries. Scale bars, 500 μm. High magnification imaging demonstrates crypt-villus domains as well as appropriate layers of subepithelium including lamina propria, muscularis mucosa, submucosa and outer smooth muscle layers. (b) Alcian blue–periodic acid–Schiff staining of epithelium within engraftment revealing secretory lineages within the crypt-villus axis. Black arrowhead points to PAS-labeled Paneth cells present within crypt bases. (c) All four intestinal lineages were present in engraftments including enterocytes (VIL), goblet cells (MUC2), Paneth cells (LYSO; white arrowhead; scale bars, 50 μm) and enteroendocrine cells (CHGA). E-cadherin (ECAD) was used for additional epithelial staining. (d) Tuft cells are also seen throughout the epithelium, as labeled with doublecortin-like kinase 1 (DCLK1). (e) mMECA-32 staining of mouse host vasculature ingrowth. (f) Edu staining of active proliferation within crypt bases and proliferative zones within crypts of epithelium. (g) Staining for VIM reveals the contribution of supporting mesenchyme, including laminated smooth muscle (white arrowheads) with staining of α-SMA. Merged images show dual staining with VIM and α-SMA, revealing a pericryptal sheath of supporting ISEMFs. (h,i) Contribution of human epithelial cells (h) and human mesenchymal tissue (i), as assessed by HuNuc staining through the full thickness of the engraftment. Dotted line separates engraftment from mouse kidney below. All scale bars are 100 μm except where specified otherwise. n = 134 transplants.

Figure 3

Engrafted tissue matures in vivo and resembles mature small intestine. (a–d) Immunostaining of engrafted intestinal tissue (in vivo) revealing maturity of brush-border enzymes including SIM (a), DPPIV (b), LCT (c) and the differentiated enteroendocrine cell subtype (GIP) (d). ECAD and CDX2 were used for additional epithelial staining. (e–h) Staining of HIOs in vitro at comparable time points to transplants for SIM (e), DPPIV (f), LCT (g) or GIP for comparison (h). (i) Relative gene expression of LCT, SIM, DPPIV and GIP in HIOs in vitro as compared to transplanted (Txp) HIOs. Values in graphs represent mean ± s.e.m.; *P < 0.05; **P < 0.01; t-test. HIOs in vitro: n = 4; transplants (Txp): n = 8. Scale bars, 100 μm.

Figure 4

Engrafted human intestinal tissue responds to humoral factors following ileocecal resection (ICR) in the mouse host. (a) Schematic representing resection experiments in mice with transplanted HIOs. (b) H&E staining of murine epithelium in sham versus ICR groups. Comparison of measured villus height (μm; c) and percentage of crypt fission (d) in mouse intestine between sham and ICR groups. (e) H&E staining of engrafted HIO epithelium in sham versus ICR groups. Comparison of villus height (f) and percentage of crypt fission (g) within engrafted HIOs in sham group versus ICR groups. (h) Immunofluorescence staining using Edu as a marker of intestinal cell proliferation in sham and ICR groups. ECAD is used to stain the epithelium. (i) Comparison of proliferative index (%) between sham and ICR groups in engrafted HIOs where proliferative index = number of Edu⁺ cells divided by total number of cells within intestinal crypt. Scale bars, 100 μm. Values in graphs represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; t-test. Sham group: n = 4; ICR group: n = 8.