In vivo models of human small intestine using pluripotent stem cells and methods of making and using same

Abstract

To provide methods of making a vascularized hollow organ derived from a human intestinal organoid (HIO).SOLUTION: Provided is a method of making a vascularized hollow organ, the method comprising a step of a) engrafting a human intestinal organoid (HIO) into an immune compromised organism, preferably a mammal, preferably a mammal having no immune response, preferably a mammal having severe combined immunodeficiency disorder (SCID); where the HIO is obtained from human embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs) where the HIO forms mature intestinal tissue during the engrafting step.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority This application claims priority to and the benefit of U.S. Application No. 62,065,131, filed October 17, 2014, entitled "In vivo Model of Human Small Intestine Using Pluripotent Stem Cells."

[0002] Government Assistance Clause This invention was made with government support under grants DK083325, DK098350, NS080815, and DK092456. The government has certain rights in this invention. [Background technology]

[0003] Due to the complexity of a vascularized hollow organ such as the intestine, the development of an adequate human model for its study and for its replacement after surgery or pathological processes has shown to be a seemingly impossible task. Methods for studying the human intestine have mainly required in vitro culture systems or have relied on animal models to address many translational questions, which do not always translate well to human studies. Classical techniques for primary culture of intestinal epithelium have been largely limited to tissue culture techniques, such as organ culture or intestinal cell lines, which do not recapitulate the hierarchy from stem cells to differentiated cells. Although the recent identification of intestinal stem cells and suitable conditions for human epithelial culture has overcome many of these obstacles, successful in vivo transplantation of epithelial cultures remains challenging due to the need for supporting mesenchyme, as is present in models that expose the host mesenchyme after mucosal injury.

[0004] Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers exciting avenues for the study of embryonic development and disease processes, as well as for pharmaceutical research and as a potential source for therapeutic implants. To date, only limited in vivo models exist for the human intestine, all of which rely on primary epithelial cultures or digested tissue from surgical biopsies containing mesenchymal cells implanted onto biodegradable scaffolds.

[0005] Currently, there is a need in the art for methods to generate models for the study of vascularized hollow organ intestinal biology, particularly the enteric nervous system (ENS). Additionally, there is a need in the art for methods to generate intestinal tissue with a functional enteric nervous system. Currently, methods for studying the human intestine have primarily required in vitro culture systems or have relied on animal models. However, these studies do not always translate well to human studies. Although intestinal stem cell and human epithelial cultures have addressed some of these issues, successful in vivo transplantation of epithelial cultures remains a challenge due to the need for supporting mesenchyme. Summary of the Invention [Means for solving the problem]

[0006] Disclosed is a method for producing vascularized hollow organs from human intestinal organoids (HIOs).HIOs can be derived from human embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs), such that HIOs form mature intestinal tissue.Also disclosed is a method for producing human intestinal tissue that contains functional enteric nervous system (ENS). [Brief description of the drawings]

[0007] [Figure 1]

[0023] Figures 1A-E. HIO transplants engraft in vivo and form mature intestinal tissue. Figure 1A - Schematic depicting the development of HIOs from hPSCs and transplantation under the kidney capsule to generate mature human intestinal tissue. Figure 1B - Two in vitro HIOs at 35 days, consisting of intestinal epithelium (black arrowhead) surrounded by supporting mesenchyme (white arrowhead). Scale bar, 100 μm. Figure 1C - Transplants after 6 weeks (in outline) with complex architecture and established peripheral capillary network. For size comparison, a mouse kidney is shown below the transplants. Scale bar, 5 mm. Figure 1D - Cross-section of transplants at 6 weeks showing intestinal structures with a central lumen. Scale bar, 5 mm. Figure 1E - Close-up of the luminal surface of the transplant showing sheets of villi, each with its own central capillary. Scale bar, 500 μm. n=139 transplants. [Diagram 2]Figure 2A-I. The transplanted intestinal tissue resembles adult intestine and is almost entirely human. Figure 2A-Low and high power imaging of transplanted HIO after H&E staining. Low power imaging shows multiple sites of epithelium, stratified smooth muscle, and peripheral capillaries. Scale bar, 500 μm. High power imaging shows the crypt-villus region and appropriate layers of subepithelial tissue including lamina propria, muscularis mucosa, submucosa, and outer smooth muscle layer. Figure 2B-Alcian blue-periodic acid-Schiff staining of epithelium within the transplant reveals secretory cells within the crypt-villus axis. Black arrowheads point to PAS-labeled Paneth cells present within the crypt base. Figure 2C-All four intestinal lineage cells were present in the transplant, including enterocytes (VIL), goblet cells (MUC2), Paneth cells (LYSO, white arrowheads, scale bar 50 μm), and enteroendocrine cells (CHGA). Additional epithelial staining used E-cadherin (ECAD). Figure 2D - Brush cells are also seen throughout the epithelium as labeled with doublecortin-like kinase 1 (DCLK1). Figure 2E - mMECA-32 staining of mouse host vascular ingrowth. Figure 2F - Edu staining of the epithelial crypt base and active proliferation within the proliferative compartment within the crypt. Figure 2G - Staining for VIM reveals supporting mesenchymal contribution, including layered smooth muscle (white arrowheads) with staining for α-SMA. Merged images show double staining with VIM and α-SMA, showing the pericrypt sheath of supporting ISEMF. Figures 2H-2I - Contribution of human epithelial cells (Figure 2H) and human mesenchymal tissue (Figure 2I) as assessed by HuNuc staining across the entire thickness of the graft. The dotted line separates the graft from the underlying mouse kidney. All scale bars are 100 μm, except where otherwise specified. n=134 grafts. [Diagram 3]Figure 3A-I. Transplanted tissue matures in vivo and resembles mature small intestine. (Figure 3A-D) Immunostaining of transplanted intestinal tissue (in vivo) showing maturation of brush border enzymes including SIM (Figure 3A), DPPIV (Figure 3B), LCT (Figure 3C), and differentiated enteroendocrine cell subtype (GIP) (Figure 3D). Additional epithelial staining was performed with ECAD and CDX2. (Figure 3E-H) For comparison, staining of in vivo HIOs for SIM (Figure 3E), DPPIV (Figure 3F), LCT (Figure 3G), or GIP (Figure 3H) at time points corresponding to transplants. Figure 3I. Relative gene expression of LCT, SIM, DPPIV, and GIP in in vitro HIOs compared to transplanted (Txp) HIOs. Values ​​in the graph represent mean ± sem, *P less than 0.05, **P less than 0.01, t test. In vitro HIO: n = 4, implants (Txp): n = 8. Scale bar, 100 μm. [Figure 4] Figures 4A-I. In mouse hosts, transplanted human intestinal tissue responds to humoral factors after ileocecal resection. (a) Schematic showing the resection experiment in mice with transplanted HIO. Figure 4B. H&E staining of murine epithelium in Sham vs. ICR groups. Comparison of measured villus height (μm, Figure 4C) and percentage of crypt division (Figure 4D) in mouse intestine between Sham and ICR groups. (Figure 4E) H&E staining of transplanted HIO epithelium in Sham vs. ICR groups. Comparison of villus height (Figure 4F) and percentage of crypt division (Figure 4G) in transplanted HIO between Sham vs. ICR groups. (Figure 4H) Immunofluorescence staining using Edu as a marker of intestinal cell proliferation in Sham and ICR groups. ECAD is used to stain the epithelium. (Figure 4I) Comparison of proliferation index (%) between Sham and ICR groups in transplanted HIO. Here, proliferation index = the number of Edu+ cells divided by the total number of cells in the intestinal crypts. Scale bar, 100 μm. Values ​​in the graph represent mean ± sem, *P less than 0.05, **P less than 0.01, ***P less than 0.001, t-test. Sham group: n = 4, ICR group: n = 8. [Diagram 5]Figures 5A-F. Timeline / overview of HIO production, size and engraftment efficiency. (a) Overview showing differentiation induction of HIOs from hPSCs over 35 days. Figure 5B. Photograph of HIOs prior to implantation showing central epithelium and surrounding supporting mesenchyme (outlined). Figure 5C. Relative size of one HIO (outlined) embedded in type I collagen prior to implantation. Figure 5D. Creation of a pocket under the renal capsule using fine forceps. Figure 5E. Photograph of HIOs implanted under the renal capsule. Figure 5F. Table showing engraftment efficiency at 6 week time point after implantation. [Figure 6] HIOs from induced pluripotent stem cells engraft and form mature human intestinal tissue in vivo. Figure 6A. Implants 6 weeks post-implantation using HIOs derived from induced pluripotent stem cells. Figure 6B. Magnified image of the implant showing the luminal surface of the implant with villi and central capillaries. Figure 6C. Magnified H&E of the epithelium within the implant (scale bar 100 μm). Crypt-villus regions were present as well as appropriate layers of subepithelial tissue including lamina propria, muscularis mucosa, submucosa, and stratified outer smooth muscle layer. (Figure 6D-G). All four intestinal lineage cells were present in the implant, including enterocytes (villin-VIL) (scale bar 100 μm) (Figure 6D), goblet cells (mucin-MUC2) (Figure 6E), Paneth cells (lysozyme-LYSO) (Figure 6F), and enteroendocrine cells (chromogranin A-CHGA) (Figure 6G). For additional epithelial staining, E-cadherin (ECAD) or CDX2 were used (all scale bars 50 μm except where specified). [Figure 7] Transmission electron microscopy (TEM) of implanted tissues at 6 weeks. Figure 6A. TEM image of brush border microvilli on the intestinal epithelial surface. In this image, tight and adherens junctions are also seen near the apical surface (scale bar 500 nm). Figure 6B. TEM image of a goblet cell (scale bar 2 μm) with secretory granules (white) containing mucin. Figure 6C. TEM image of an enteroendocrine cell (scale bar 2 μm) with secretory granules (dark) ready for release on the basal side of the cell. Figure 6D. TEM image of smooth muscle within the implant with parallel orientation of smooth muscle fibers (scale bar 10 μm). [Figure 8]Additional epithelial and mesenchymal maturation markers in transplanted versus in vitro HIOs. (Fig. 8A-G) Comparison of relative gene expression of epithelial markers including EpCAM (Fig. 8A), villin-VIL (Fig. 8B), alkaline phosphatase (ALPI) (Fig. 8C), lysozyme (LYZ) (Fig. 8D), chromogranin A (CHGA) (Fig. 8E), mucin (MUC2) (Fig. 8F), and glucose transporter 2 (GLUT2) (Fig. 8G). (Fig. 8H-I) Relative expression of smooth muscle markers desmin (DES) (Fig. 8H) and smooth muscle actin (α-SMA) (Fig. 8I). Values ​​in graphs represent Mean ± sem, ns is non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, in vitro HIO: n = 4, implants (Txp): n = 8. [Figure 9] The transplanted tissues showed mature epithelium maintained by intestinal stem cells. (Fig. 9A-D) An LGR5R5:eGFP BAC reporter ES line was established and used to generate HIOs expressing LGR5-eGFP cells, Fig. 9A. LGR5-eGFP cells (green) were found scattered throughout the epithelium in HIOs in vitro (Fig. 9B). LGR5-eGFP cells (green) were also localized in proliferative crypt base cells (colocalizing with KI67) as expected in transplanted human intestinal tissues (Fig. 9C-D). Fig. 9E. Immunofluorescence staining of stem cell marker ASCL2 (green) localized in the crypt base of the transplants. Fig. 9F. Graphs showed the relative fold change of LGR5 and ASCL2 in HIOs in vitro before transplantation and in the transplants. Fig. 9G. Enteroids were generated from transplanted epithelium to demonstrate the stemness of the tissue. Fig. 9H. Panels show epithelial enteroids derived from the implants at days 1, 5, and 10 after initial seeding and after passage (Fig. 9I-K). Immunofluorescence staining revealed the presence of epithelial cells bearing villin (VIL) and E-cadherin (ECAD) (Fig. 9I), as well as Paneth cells (lysozyme - LYSO) (Fig. 9J) and brush border enzymes (dipeptidyl peptidase 4 - DPPIV) (Fig. 9K) (all scale bars 50 μm). [Figure 10]The transplanted intestinal tissue received its blood supply from murine vascular ingrowth. FIG. 10A. Using a mouse-specific pan-endothelial antibody (mMECA-32), murine blood vessels were revealed just below the surface of the implant (FIG. 10b) as well as in the interior of the implant (white arrowheads, scale bar 50 μm). FIG. 10C. Magnified immunofluorescence image of mMECA-32 stained endothelium (green) with blood vessels within each villi as well as capillary plexus just below the epithelium within the implant. FIG. 10D. Whole mount staining of intra-renal implants following injection of FITC-labeled tomato lectin via the mouse tail vein. Areas 1 and 2 within the outline correspond to subsequent images depicting functional fluorescent murine blood vessels within the implant (scale bar 50 μm). FIG. 10E. Immunofluorescence image depicting murine-specific (mMECA-32) and human-specific (hCD31) staining of the epithelium within the implant. FIG. 10F. Confocal images of whole mount explants showed no connection between murine and human vessels (FIG. 10G). A murine specific lymphatic marker (LYVE-1) showed lymphatic ingrowth from the murine host. (All scale bars 100 μm except where specified). [Figure 11] Intestinal adaptation also occurs in NSG mice after surgical resection and HIO transplantation. Figure 11A. Magnified image of crypt splitting (in outline) in mouse small intestine after resection. (Scale bar 50 μm). Figure 11B. Increased thickness of smooth muscle layer (muscularis) in postoperative ICR tissue (Scale bar 50 μm). Figure 11C. Comparison of murine intestinal crypt depth (μm) between preoperative, sham, and resected (ICR) groups. Figure 11D. Comparison of smooth muscle layer thickness (μm) between preoperative, sham, and resected (ICR) groups. Figure 11E. Magnified image of intestinal epithelium in HIO transplantation in sham mice (Scale bar 50 μm). Figure 11F. Magnified image of crypt splitting in HIO transplantation in ICR mice (Scale bar 50 μm). Values ​​shown in graphs represent Mean ± sem. ns, not significant. *: p less than 0.05, **: p less than 0.01, ***: p less than 0.001, preoperative group: n = 13, sham group: n = 10, ICR group: n = 11. [Figure 12]The transplanted human intestinal tissue maintained digestive and absorptive functions. Figure 12A. Intestinal alkaline phosphatase was active ex vivo (scale bar 100 μm). Figure 12B. FITC-dextran (MW 4,400) injected into the implants in vivo (photographs shown before and after) was significantly increased in murine serum from the first 30 min time point compared to the 4 h time point within each mouse (n=7). Values ​​shown in the graph represent Mean±sem, ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001, paired t-test. Figure 12C. D-Ala-Leu-Lys-AMCA peptide uptake analysis showing peptide transporter (PEPT1) staining within the epithelium of the implants (scale bar 100 μm). Figure 12I. Additional images (n=3 for each group) showing intraepithelial fluorescence after injection of vehicle (DMEM solution), D-Ala-Leu-Lys-AMCA (DMEM + labeled peptide solution), and captopril + D-Ala-Leu-Lys-AMCA (DMEM + labeled peptide solution + competitive inhibitor of transport solution). Fluorescence was observed after injection of labeled peptide (iii), but little to no fluorescence was observed after vehicle injection (ii) or after labeled peptide + captopril injection (iv). [Figure 13] Quality control analysis for iPSCs. Phase contrast image of iPSC colonies cultured in standard feeder-free conditions. FIG. 13B. Immunofluorescence staining of pluripotency markers Oct4 and Nanog in iPSCs. FIG. 13C. G-banded karyotyping showing normal (46,XY) karyotype of subject iPSCs. FIG. 13D. H&E stained sections of iPSC-derived teratomas showed tissues arising from the three embryonic germ layers, namely endoderm, ectoderm, and mesoderm. Control teratomas showed peripheral epithelium (ectoderm) with antibasal nuclei consistent with early teeth, ciliated columnar epithelium (endoderm) consistent with gut, and stellate reticular cells surrounded by chondrocytes (mesoderm). Images are at 400x magnification. [Figure 14]Generation of vagus-like neural crest cells (NCCs). 14A - Schematic representation of the protocol for generating neural crest cells expressing Hox genes. While this method has been reported to generate head NCCs, addition of retinoic acid during the final 48 hours of neurosphere culture induces markers of posterior / vagus-like NCCs, Hox A2, B3, B5, and B7. FIG. 1B - NCCs exhibited stellate morphology and were positive for surface markers HNK-1, p75NTR, and RET. FIG. 14C - Quantitative RT-PCR analysis of regulators of neural plate boundary and neural crest cell specification. FIG. 1D - Treatment of neurospheres with RA for 48 hours results in the formation of NCCs expressing vagus-level HOX genes. The more posterior HOX genes B3, 5, and 7 showed a dose-dependent increase in expression levels (data not shown). Scale bar 100 μM. Values ​​in the graph represent mean ± sem, *P less than 0.01, **P less than 0.001, Student's t test (two-tailed, unpaired), n = 3 biological replicates per condition, data representative of three independent experiments. [Figure 15]Figure 15A-C. In vitro incorporation of NCCs into developing HIOs. Figure 15A - Schematic for incorporation of NCCs into HIOs. HIOs and NCCs were generated separately, combined by low speed centrifugation, embedded in Matrigel, and grown in vitro for 4 weeks. In some cases, HIOs were transplanted into NSG mice and grown in vivo for 6-8 weeks. Figure 15B - In vitro growth of HIOs. Left panel - Brightfield images of 28d HIO+ / -NCCs. Scale bar, 1 mm. Middle panel - Neural (βIII-tubulin) and epithelial (E-cadherin) immunostaining. Right panel - Neuroglial (S100+) and epithelial (E-cadherin) immunostaining. Scale bar, 100 μm. Data are representative of 14 independent experiments combining HIOs with NCCs in vitro. Figure 15C - In vivo growth of HIO+NCCs. HIO+NCCs were transplanted into the renal subcapsular space of NSG mice and grown for 6 weeks. The left panel shows an HIO+NCC that was ∼1 cm in diameter. Scale bar, 1 mm. The middle panel shows neurogenesis adjacent to smooth muscle fibers (desmin+) in a myenteric-like arrangement oriented at right angles to one another in the HIO-NCC, indicating that smooth muscle differentiation occurs in the absence of an ENS. A second layer of desmin+ cells is located submucosally to the epithelium. HIO+NCCs contain nerves (BIII-tubulin+) embedded within the desmin+ smooth muscle layer and with an organization very similar to the myenteric plexus. The right panel shows that neuroglial cells (S100+) are also embedded within the mesenchymal layer of the HIO+NCC, including the submucosa. [Figure 16]FIG. 16A-B. Neuronal diversity and formation of interstitial cells of Cajal (ICC). FIG. 16A. Analysis of different neuronal cell types using neurochemical markers of ENS neurons in HIO+NCC cultured in vitro (upper panel) and after transplantation in vivo (lower panel). Dopaminergic neurons (TH), interneurons (ChAT, 5-HT), sensory neurons (calbindin), excitatory neurons (calretinin), and inhibitory neurons (nNOS) were all found in HIO+NCC transplanted in vivo. In contrast, HIO+NCC in vitro did not contain inhibitory neurons (nNOS), suggesting that they were embryonic in nature. FIG. 16B. Formation of interstitial cells of Cajal (CD177-red) in HIO+NCC in vivo. HIO without NCC did not form neurons (BIII-tubulin-green) and had fewer CD117+ cells. This suggests that NCC may be involved in the differentiation of ICC. [Figure 17] FIG. 17A-D. Live imaging of neural activity in HIO+NCCs. FIG. 17A. Schematic of the protocol for merging NCCs generated from GCaMP6f-expressing PSCs with HIOs generated from H1 PSCs. FIG. 17B. Live imaging of Ca2+ flux in neural crest-derived ENS cells shows rhythmic activity. Snapshots from a 20-minute time-lapse movie showing neural activity in HIO+NCCs. Colored arrows point to cells whose pixel intensity was measured over time and shown in FIG. 17C. FIG. 17C. Numbered snapshots correspond to the arrowheads in FIG. 17B and show rhythmic waves of depolarization of a single neuron in a HIO+NCC culture (only the first three time points are shown in FIG. 17B). The graph measures pixel intensity associated with Ca2+ flux. Line colors correspond to the same colored arrowheads in the snapshots. FIG. 17D. KCl induces waves of calcium efflux. Quantitation of pixel intensities from KCl experiments indicates that KCl induces a fast and broad wave of ENS cell depolarization. [Figure 18]FIG. 18A-C. Formation of 3-dimensional submucosal and myenteric plexuses that control motility. FIG. 18A. Whole mount immunostaining and 3-D imaging of human intestinal and HIO+ENS tissues. En face view of human submucosal and HIO+ENS shows the arrangement of nerves (BIII-tubulin) and glia (S100) into a glial plexus that integrates and is oriented with smooth muscle fibers (desmin). HIO+ENS tissue also contained a glial plexus that is oriented with smooth muscle fibers. FIG. 18B - Formation of intestinal and submucosal plexuses. Left panel - Orthogonal view of in vivo transplanted HIO+NCC shows the close association of glia (S100) with the epithelium (dapi-blue) periphery. Right panel - Side view of whole mount image clearly identifies two plexuses, one associated with the epithelial mucosa (submucosal plexus) and one associated with the outer layer of smooth muscle (myenteric plexus). FIG. 18C. ENS in HIO mediates peristaltic-like contractions. In vivo grown tissues were explanted into Tyrode's solution and subjected to electric field stimulation (EFS). HIO-ENS subjected to high voltage EFS (1 ms pulse at 100V) produced one single contraction (n=2). HIO+ENS subjected to low voltage EFS (1 ms pulse at 50V) produced a sustained train of wave-like contractions (n=5). The sustained train of wave-like contractions was lost when the tissues were incubated in tetrodotoxin (HIO+ENS+TTX, n=2). [Figure 19]FIG. 19A-E. ENS-dependent and -independent control of contractile activity. FIG. 6A. Recordings of spontaneous contractions in explanted HIO and HIO+ENS tissue explants. Phasic contractions were observed after tissue equilibration (without stimulation), suggesting that interstitial cells of Cajal (ICCs) are present in both HIO (n=7) and HIO+ENS (n=7) tissues. FIG. 19B. Recordings showing that inhibition of ICC activity with methylene blue results in loss of contractile activity (n=3). FIG. 19C-Left panel shows representative images of dimethylphenylpiperazinium (DMMP) stimulation in HIO and HIO+ENS. Right panel shows area under the curve (AUC) between DMPP (10 μM) stimulation measured for 2 min before and after stimulation (n=7). FIG. 6D-TXX inhibition of ENS activation. Area under the curve between DMPP stimulation measured for 2 min after stimulation in HIO+ENS followed by tetrodotoxin (TXX, 10 μM) treatment (n=7). FIG. 19E - ENS-dependent relaxation by NO-dependent mechanism. Area under the curve between DMPP stimulation measured for 2 min after stimulation in HIO+ENS followed by NG-nitro-L-arginine methyl ester (L-NAME) treatment (n=7). Values ​​in graphs represent mean±sem, *P<0.05, **P<0.01, Mann&Whitney test. [Figure 20] Characterization of developing neural crest cells. FIG. 20A. Day 6 free-floating neurospheres are approximately 500 μm in diameter and are positive for PAX6, vimentin, and SOX9, characteristic of neural crest cells and the lateral neural plate from which they arise. FIG. 20B. Neurospheres adhered to the fibronectin substrate and produced neural rosettes that were visible by brightfield microscopy. Adherent neurospheres expressed PAX6 diffusely, while vimentin staining was observed at the edges of the neural rosettes where NCCs delaminated. As NCCs migrated away from the rosettes, PAX6 was downregulated, while SOX2 expression was maintained. [Figure 21]Comparison of human and chick NCC behavior. (Figures 21A and 21B) Isolation and culture of head (Hox negative) and trunk (Hox positive) NCC from chick embryos and analysis of Hox gene expression. NCC isolated from the anterior chick neural tube did not express Hox genes, while those isolated from the posterior neural tube were HoxB7 positive. (Figure 21C) Chick head NCCs express the vagus nerve-level Hox genes HoxB7 and HoxB7 when treated with 2 μM RA for 48 hours. Another posteriorizing factor, FGF4, did not affect Hox gene expression. (Figure 21D) Schematic depicting transplantation of human PSC-derived NCC into chick embryos. GFP-labeled human NCCs were injected in ovo between somites into HH10-12 chick embryos. (Figure 21E) Embryos were analyzed at ~HH38. GFP-labeled cells were found to migrate laterally along the neural tube (NT) and colonize the foregut (FG) (Figure 21F). Differentiation of GFP-labeled cells into peripheral nerves (peripherins) (dorsal right, anterior up). [Figure 22] Differentiation potential of head NCC and RA-treated NCC. Figure 22A. In vitro differentiation of human PSC-derived NCC into glial and mesenchymal cells (osteocytes and chondrocytes). Head NCC and RA-treated NCC were equally capable of forming glial cells (peripherin and GFAP), osteocytes (alizarin red), and chondrocytes (alcian blue) in vitro. Figure 22b. Head-like NCC formed pigment epithelium (arrowheads) in vivo and in vitro, suggesting that the ability to form neuroepithelium was maintained. [Figure 23]Generation of human intestinal organoids (HIOs) and expression of NCC migration cues. Figure 23A. Method to generate HIOs by inducing differentiation of human pluripotent stem cells. Figure 23B. Transient expression of differentiation markers at each stage of HIO development. Activin A mediated efficient differentiation into definitive endoderm (FOXA2 and SOX17), WNT activation, and FGF4-induced CDX2 expression in monolayer and free-floating spheroids. Cultivation of spheroids in Matrigel for 4 weeks resulted in the formation of CDX2-expressing HIOs. HIOs were transplanted into the subcapsular space of mice, where after 6 weeks they grew and matured to form intestinal tissue with crypts and villi. Human-specific antibody HuNu indicates that the intestinal epithelium and mesenchyme are of human origin. Figure 23C. HIOs formed in vitro expressed GDNF and EDN3. Values ​​in the graph represent the mean ± sem, *P less than 0.05, **P less than 0.01, t-test (two-tailed, unpaired), n = 3 biological replicates, data are representative of two independent experiments. [Figure 24] Neurons and glia in HIO+NCC cultures are of NCC origin. NCCs were generated from H9-GAPDH-GFP human embryonic stem cells, which broadly express GFP, and merged with HIOs generated from H1 HESCs. HIOs were co-stained with GFP and the pan-neuronal marker PGP9.5 or the glial marker S100. Glial cells in HIO+NCC are of NCC origin, as evidenced by co-expression of GFP and glial markers. [Diagram 25] Ganglion-like structures form in HIO+NCCs grown in vivo (FIG. 24A). In immunostained sections, nerves (BIII-tubulin) were distributed throughout the myenteric plexus. Applicants also observed clusters of nerve cell bodies (HuC / D) that resembled ganglia (FIG. 24B). Whole-mount immunostaining for nerves (BIII-tubulin, frontal view) and nerve cell bodies (HuC / D, lateral view) shows nerve cell bodies organized into ganglion-like clusters in HIO+NCCs. [Figure 26] Spontaneous contraction recordings of transplanted HIO and HIO+NCC tissues. [Figure 27] (FIG. 27A) Left panel shows representative recordings after veratridine stimulation in HIO and HIO+NCC. Right panel shows area under the curve (AUC) between veratridine (3 μM) stimulation measured for 2 min before and after stimulation (n=5). (FIG. 27B) Shows area under the curve between dimethylphenylpiperazinium (DMMP, 10 μM) stimulation measured for 2 min before and after stimulation followed by tetrodotoxin (TXX, 10 μM) treatment (n=3). (FIG. 27C) Shows area under the curve between DMMP stimulation measured for 2 min after stimulation followed by NG-nitro-L-arginine methyl ester (LNAME) treatment (n=3). (d) Left panel, representative recordings after sodium nitropruszide (SNP) stimulation in both HIO (n=4) and HIO+NCC tissues (n=14). Right panel, area under the curve between SNP stimulation measured for 2 min before and after stimulation. Values ​​in the graph represent mean ± sem, Mann & Whitney test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The enteric nervous system (ENS) of the gastrointestinal (GI) tract controls motility, epithelial permeability, and fluid exchange. Disorders in ENS development or function are common, yet human models to study ENS-gut biology are lacking.

[0009] Applicants have generated in vitro human intestinal organoids (HIOs) from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature intestinal epithelium with intestinal stem cells that contribute to crypt-villus structures and stratified human mesenchyme, both of which are supported by mouse vascular ingrowth. Applicants have shown that in vivo transplantation results in significant proliferation and maturation of the epithelium and mesenchyme. This is indicated by the presence of distinct intestinal cell lineages (enterocytes, goblet cells, Paneth cells, brush cells, and enteroendocrine cells), functional brush border enzymes (lactase, sucrose isomerase, and dipeptidyl peptidase 4), and visible subepithelial and smooth muscle layers when compared to in vitro HIOs. Applicants have further shown that the transplanted intestinal tissues exhibit digestive function, as evidenced by permeability and peptide uptake experiments. Transplanted HIO-derived tissue was found to respond to systemic signals from the host mouse after ileocecal resection, suggesting a role for circulating factors in the adaptive response of the intestine.

[0010] Applicants further developed human PSC-derived intestinal tissue with a functional ENS. Using a tissue engineering approach with pluripotent stem cells (PSCs), Applicants recapitulated normal intestinal ENS development by combining PSC-derived neural crest cells (NCCs) with developing human intestinal organoids (HIOs) to generate human intestinal tissue containing a functional ENS.

[0011] Applicants found that NCCs had full differentiation potential in vitro when cultured alone, but differentiated into neural and neuroglial cells when combined with HIOs. NCC-derived ENS neurons were found to self-assemble within the developing intestinal mesenchyme and exhibited neural activity measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures resembling myenteric and submucosal plexuses, formed interstitial cells of Cajal, and had electromechanical coupling that controlled propagating contractions. This is the first demonstration of human PSC-derived intestinal tissue with a functional ENS.

[0012] The enteric nervous system (ENS) is essential for GI motility, secretion, blood flow, epithelial barrier permeability, and fluid exchange. Developmentally, the majority of the ENS arises from vagal neural crest cells (NCCs) derived from the dorsal neural tube, which migrate ventrally and colonize the foregut around the fourth embryonic week in humans. NCCs then proliferate extensively, migrate caudally, and colonize the entire GI tract by the seventh week of human gestation. Failure of enteric NCCs to properly migrate, proliferate, survive, and / or differentiate in the GI tract results in defects in ENS structure and function, resulting in patients with a wide range of enteric neuropathy. Furthermore, enteric neuropathy is common in gastrointestinal disorders such as inflammatory bowel disease, and often occurs secondary to disorders such as Parkinson's disease, diabetes, and age-related degeneration. The lack of a human ENS model system to study the physiopathological processes of enteric neuropathy may explain the surprisingly slow progress in its diagnosis and treatment. Longstanding treatment of patients with Hirschsprung's disease (1:5000 live births), a congenital absence of enteric ganglia, requires surgical resection of the aganglionic intestinal segments, leaving very little normal intestine. The remaining intestine contains ganglia, but it is unclear why many of these patients suffer from recurrent attacks of enteritis and motility disorders. Enteric nerves derived from pluripotent stem cells (PSCs), enteric progenitor cells, or even CNS neural tube cells have been shown to integrate and function in aganglionic chick and murine GI outgrowths, suggesting that a similar approach may work therapeutically in humans.

[0013] Despite the advances being made towards cell-based therapies, much work remains to be done in our understanding of human ENS development and disease. Little is known about the causation of enteric neuropathy, and the mechanisms driving various aspects of human ENS development, such as the formation of neurodiversity, remain unclear. Although there are currently no methods to functionally study human ENS development, recent advances using induced differentiation of human pluripotent stem cells have led to the formation of complex and physiological 3D human organ cultures called organoids. Organoid models have been developed for the intestine, liver, stomach, CNS, thyroid, and lung, to name a few, and have enabled unprecedented studies of human developmental biology and disease. Despite the remarkable complexity of organoids, they lack the cell and tissue types necessary for complete organ function. For example, no organoid system contains an integrated peripheral nervous system.

[0014] As disclosed herein, applicants have demonstrated the development of 3D human intestinal organoids (HIOs) containing a functional enteric nervous system. PSCs were first differentiated into vagus-like neural crest cells (NCCs) and then introduced into developing intestinal cultures at a stage from enterogenesis roughly corresponding to normal colonization of the embryonic gut by ENS. The resulting HIOs may be grown in vitro for less than 8 weeks and have a diverse population of nerves that resemble the developing fetal intestine and are capable of rhythmic and stimulated waves of calcium transitions. When transplanted in vivo, HIOs formed a complex and mature intestinal epithelium with crypts and villi surrounded by submucosal and myenteric smooth muscle layers. HIOs with ENSs formed both submucosal and myenteric neuroglial plexuses. The plexuses contained bundles of neuronal cell bodies with a network of interganglionic fibers that were integrated into the smooth muscle layer. Electric field stimulation of HIOs with ENSs grown in vivo induced propagating motility waves that were blocked with the neurotoxin tetrodotoxin. Organ bath experiments of the explants further demonstrated functional nitrergic neuromuscular coupling in the HIO with the ENS. Thus, Applicants' invention is believed to be the first evidence of in vitro generation of human intestinal tissue with a functional enteric nervous system derived entirely from human pluripotent stem cells.

[0015] In one embodiment, a method for producing a hollow organ that is vascularized is disclosed. In this embodiment, the method can include a) transplanting human intestinal organoids (HIOs) into an immunodeficient organism. The immunodeficient organism can be, for example, a mammal, for example, a mammal that does not have an immune response, for example, a mammal that has severe combined immunodeficiency (SCID). The HIOs can be obtained from human embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs). In one embodiment, during the transplantation step, the HIOs form mature intestinal tissue.

[0016] In one embodiment, the human intestinal organoids (HIOs) can be embedded in collagen. In one embodiment, the collagen can be type I collagen.

[0017] The step of transplanting can include implanting the HIO into the peritoneal capsule of the immunocompromised organism. The step of transplanting can be performed for a period of at least about three weeks, or at least about four weeks, or at least about five weeks, or at least about six weeks. The duration of the step can be determined by one of skill in the art. The step of transplanting can be performed, for example, until the intestinal tissue meets one or more criteria. Such criteria can include, for example, having columnar intestinal epithelium surrounded by supporting mesenchyme, growing 1-3 cm in diameter, forming villi and crypts containing functional intestinal cells, having submucosal and myenteric layers of smooth muscle fibers, or a combination thereof.

[0018] In one embodiment, a method for producing human intestinal tissue containing a functional enteric nervous system (ENS) is disclosed. In this embodiment, the method can include a) contacting vagus-like neural crest cells (NCC) derived from human ES cells and / or iPS cells (iPSCs) with three-dimensional human intestinal organoids (HIOs), and c) transplanting the HIOs in vivo. In one embodiment, the NCCs can be obtained by contacting the human ES cells and / or iPS cells with retinoic acid. In this embodiment, the retinoic acid is contacted with the human ES cells and / or iPS cells in an amount sufficient to cause posteriorization. In some embodiments, the retinoic acid can be contacted with the human ES cells and / or iPS cells for a period of about 1 to about 2 days, or in some embodiments, about 2 days. The retinoic acid contact step can be carried out at the neurosphere stage for a period of about two days, or until sufficient expression of HOXB3, HOXb5, and / or HOXb7 is observed.

[0019] In one embodiment, the transplanting step may be performed for a period of time sufficient to allow detection of nerves and / or glia. In one embodiment, the nerves may have BIII-tubulin. In a further embodiment, the glia have S100. In yet a further embodiment, the nerves and glia may integrate into the smooth muscle layer (desmin+ cells). In one embodiment, the transplanting step may be performed for a period of time sufficient to allow formation of nNOS+ inhibitory nerves.

[0020] In one aspect, human intestinal tissue containing a functional enteric nervous system (ENS) as disclosed herein can be capable of contractile activity.

[0021] Further disclosed is a method of treating a patient in need of replacement of a portion of the gastrointestinal tract, which may comprise replacing a portion of the gastrointestinal tract of the patient with human intestinal tissue produced according to the methods disclosed herein. In a further aspect, a method of determining the effect of a treatment on the human intestinal tract is disclosed, which may comprise contacting the treatment of interest with human intestinal tissue produced according to the methods disclosed herein. The models provided by applicants may be useful for the study of intestinal physiology, disease, and / or translational research.

[0022] Example - Development of an in vivo model of the human small intestine using pluripotent stem cells To establish an in vivo HIO model, applicants generated HIOs from human ESCs or iPSCs as previously described (Spence, et al, Nature 2011; McCracken et al, Nat. Protoc. 2011). The differentiation process took approximately 35 days (Fig. 1A and Fig. 5A) and produced HIOs with columnar intestinal epithelium surrounded by supporting mesenchyme (Fig. 1B and Fig. 5B). Applicants then embedded HIOs in type I collagen and transplanted them under the kidney capsule of immunodeficient nonobese diabetic severe combined immunodeficiency interleukin-2Rγnull (NSG) mice and allowed them to mature and grow for 6 weeks (Fig. 1A and Fig. 5D, 1E). At the time of harvest, HIOs had grown significantly in size, upwards by 50-100 times in volume (Fig. 1C, Fig. 1D, and Fig. 5C and Fig. 6A), and were highly vascularized (Fig. 1C). Notably, 92.4% of the transplanted HIOs successfully engrafted into the renal capsule (Figure 5F). Visual inspection of the tissues showed intestinal morphology with a mucosal-filled lumen and well-developed villous sheets (Figure 1E), each with its own central capillary network (Figure 1D). Histologically, the transplanted tissue resembled native human intestine with a crypt-villus structure and underlying stratified submucosa, including the lamina propria, muscularis mucosa, and submucosal and outer smooth muscle layers (Figures 2A and 6C). The transplanted in vivo tissue appeared more mature and differentiated in comparison to its in vitro counterpart (data not shown), with all major intestinal cell lineages, including enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and brush cells, located within the appropriate regions of the crypt-villus axis (Figures 2B-D and 6D-G). Paneth cells were located within the crypt bases as expected, rather than scattered throughout the epithelium (Fig. 2B, 2C). Transmission electron microscopy (TEM) showed well-developed brush borders with tight junctions, similar to those of in vitro HIOs (Fig. 7A). However, mature goblet cells and enteroendocrine cells, as seen within the epithelium of the implants (Fig. 7B, C), were absent in TEM of in vitro HIOs (data not shown).Within the epithelium, Applicants observed increased relative expression of genes characteristic of epithelial cell types when comparing grafts to in vitro HIOs (Figure 8A-G). Applicants found that blood vessels within the grafts stained positive for mouse-specific pan-endothelial cell antigen (mMECA-32) (Figure 2E). The grafted tissue also actively proliferated within individual crypts as revealed by 5-ethanyl-2'-deoxyuridine (Edu) incorporation (Figure 2F). Using transgenic human LGR5 reporter HIOs to track intestinal stem cells, the inventors found that actively proliferating labeled stem cells were present within the HIOs and localized to the base of the crypts of the grafts (Figure 9A-D). Furthermore, the grafted tissue expressed the stem cell marker ASCL2 (Figure 9E-F), and the inventors were able to generate enteroids derived from the epithelium of the grafts, further indicating the presence of an intestinal stem cell pool (Figure 9G-K). These data suggest that after transplantation in vivo, HIOs undergo substantial maturation into mature intestinal tissue.

[0023] Crosstalk between adjacent mesenchyme and the intestinal epithelium is known to play a major role during GI development. Previously, supporting mesenchyme was shown to develop along the intestinal epithelium of HIOs in vitro and to contain immature populations of subepithelial myofibroblasts, fibroblasts, and smooth muscle cells. Applicants investigated whether HIO mesenchyme also developed into more mature and differentiated cell types after transplantation in vivo. The non-epithelial region of the transplanted tissue stained positive for the mesenchymal marker vimentin (VIM) and contained several stratified epithelial sublayers, including a distinct smooth muscle layer positive for α-smooth muscle actin (α-SMA), indicating a mesenchymal contribution to engraftment (Figure 2G). Furthermore, double staining for α-SMA and VIM demonstrated pericrypt sheaths of intestinal subepithelial myofibroblasts (ISEMFs), known to support the proliferation of human small intestinal epithelial stem cells in vitro and in vivo (Figure 2G). Similarly, the intestinal epithelium has been shown to convey appropriate signals for the development of subepithelial mesenchymal layers, including the lamina propria, ISEMF, and muscularis mucosae, resulting in proper stratification of epithelialized tissue (Figures 2A, 2G). In addition to histological maturation of the smooth muscle layer, the relative expression of desmin and α-SMA was increased in HIOs transplanted in vivo compared to HIOs in vitro (Figures 8H, 8I). TEM of the transplants showed parallel orientation of smooth muscle, similar to that of the adult intestine (Figure 7D). These data are consistent with published reports that the presence of a mesenchymal niche is required for successful transplantation and maturation of human intestinal tissue. For example, the absence of mesenchymal components caused failed engraftment, whereas inclusion of mesenchymal cells along with the epithelium resulted in successful engraftment.

[0024] Because our implants contained a variety of mesenchymal cell types, including endothelial cells, Applicants next investigated which components were of human origin and which were from the host. As expected, the HIO epithelium was entirely of human origin and stained positively for human nuclear antigen (HuNu) (Figure 2H). Furthermore, most of the tissues of mesenchymal origin, including the lamina propria, muscularis mucosa, submucosa, and smooth muscle layer, also stained positively for HuNu (Figure 2I). In contrast, the majority of blood vessels were of mouse origin, as they stained positively for mMECA-32 (Figure 2E). The blood supply of the implanted HIO was supported by mouse blood vessels, as indicated by positive labeling of the endothelium with tail vein injection of FITC-conjugated tomato lectin (Figures 10A-10D). Very few blood vessels of human origin were positive for human-specific pan-endothelial cell antigen in the implants, and such vessels were not connected to the mouse vasculature (Figures 10E, 10F). The lymphatic vessels were of murine origin and stained positive for the mouse-specific lymphatic endothelial hyaluronan receptor 1 (LYVE-1) (Figure 10G).

[0025] Transplantation of PSC-derived pancreatic progenitor cells has been shown to enhance islet cell development in vivo. Applicants analyzed the impact of in vivo growth on the maturity of our 6-week transplants compared to in vitro HIOs at a similar time point (35 days + 6 weeks in vitro) by measuring the expression of several markers of mature epithelium at both the mRNA and protein levels. Applicants observed a significant increase in the mRNA and / or protein expression of markers characteristic of differentiated intestinal cells, including dipeptidyl peptidase 4 (DPPIV), glucose transporter type 2 (GLUT2), sucrose isomaltase (SIM), and villin (VIL), compared to in vitro HIOs (Figures 3A, 3B, 3E, 3F, 3A, Figures 8B, 8G). Additionally, Applicants also examined markers of the brush border enzymes alkaline phosphatase (ALPI) and lactase (LCT), as well as secretory cell types including enteroendocrine (gastric inhibitory peptide (GIP) and chromogranin A (CHGA)), goblet (mucin 2 (MUC2)), and Paneth cells (LYSO). For each of these markers, mRNA and protein expression was also significantly increased in transplanted HIOs (Figures 3C, 3D, 3G, and Figures 8C-8F). Thus, Applicants conclude that in vivo growth promotes maturation of intestinal tissue.

[0026] Given the mature phenotype of in vivo transplanted HIOs, applicants hypothesized that these tissues may have implications as a novel model for studying the in vivo physiology of the human intestine. Applicants therefore investigated whether transplanted HIOs could respond to physiological cues induced by intestinal resection. It has been suggested that humoral factors are involved in the adaptive response of the intestine. In rats parabiotically linked by vascular anastomoses, surgical resection in one rat led to intestinal proliferation in the parabiotic partner.7 However, no model is available to investigate this phenomenon in the human intestine. Here, applicants used a model of ileocecal resection (ICR) in transplanted mice to examine whether circulating humoral factors stimulated in response to intestinal resection could affect transplanted human intestinal tissue in the kidney. Applicants randomized mice bearing HIO grafts at our 6-week post-implantation timepoint into sham (dissection and reanastomosis) or ICR groups and quantified morphometric parameters associated with intestinal adaptation, including crypt depth, villus height, epithelial proliferation, crypt fission, and circular and longitudinal smooth muscle layer (muscularis) thickness (Figure 4A). Adaptation in NSG mice was confirmed with significant differences in villus height, crypt fission (Figures 4B-4D), crypt depth, and smooth muscle layer thickness (Figures 11A-11D) between the sham and ICR groups, while there was no difference in proliferation index between the sham and ICR groups (data not shown). In transplanted HIOs, ICR increased villus height, crypt fission (Figures 4E-4G, and Figures 11E, 11F), and proliferation index (Figures 4H, I) compared with the sham surgery group. These findings support the hypothesis that transplanted HIOs respond to humoral factors. Furthermore, this model may provide insight into the humoral adaptation response after intestinal resection. Functionally, transplanted HIOs expressed active brush border enzymes and exhibited a functional intestinal epithelial barrier as demonstrated by permeability analysis using FITC-dextran (Figures 12A, 12B). Transplanted HIOs were also capable of peptide uptake, reflecting the presence of absorptive function (Figure 12C).

[0027] To our knowledge, this is the first report of the development of a functional model of hPSC-derived human small intestine in vivo. Our study highlights the potential of both ESCs and iPSCs to generate diverse cell types and tissue layers that mature after transplantation. Furthermore, the adaptive response seen in our human grafts after surgical resection in mouse hosts supports the role of humoral factors in this adaptive response and validates the use of our model for further in vivo studies of the human small intestine. Because the HIO serves as a method to "individualize" and bioengineer a functional human intestine, with further translational studies it may also serve as a tool for the eventual treatment of short bowel syndrome and other gastrointestinal disorders.

[0028] method animal Immunodeficient NOD-SCID IL-2Rγnull (NSG) mice aged 8–16 weeks were used in all experiments (obtained from the Comprehensive Mouse and Cancer Core Facility, Cincinnati, Ohio). All mice were housed in the animal facility at Cincinnati Children's Hospital Medical Center (CCHMC). All experiments were performed with approval from the Institutional Animal Care and Use Committee of CCHMC.

[0029] Generation and maintenance of human intestinal organoids Human intestinal organoids were generated and maintained as previously described (Spence et al, Nature 2011, and McCracken et al. Nat. Protoc. 2011). Human embryonic stem cells and induced pluripotent stem cells were grown in feeder-free conditions in 6-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 medium (Stem Cell Technologies). For induction of definitive endoderm (DE), human ES or iPS cells were passaged using Dispase or Accutase (Invitrogen) and plated at a density of 100,000 cells per well in 24-well plates on Matrigel-coated Nunclon surfaces. For cells passaged with Accutase, 10 μM of the compound Y27632 (Sigma) was added to the medium on the first day. Cells were grown until they reached 80-95% confluence. Cells were then treated with 100 ng ml-1 activin A for 3 days as previously described (D'Amour et al. Nat. Biotechnol. 2005). DE were then treated with hindgut induction medium (RPMI 1640, 2 mM L-glutamine, 2% uncomplemented FBS, penicillin-streptomycin, and 100 ng ml-1 activin A) for 4 days with 500 ng ml-1 FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce the formation of mid-hindgut spheroids. Spheroids were then plated in Matrigel (BD) and maintained in intestinal growth medium (Advanced DMEM / F-12, N2, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng ml-1 EGF (R&D) and 100 ng ml-1 Noggin (R&D) to generate human intestinal organoids (HIOs). Medium was changed on day 3 to remove Noggin and once a week thereafter. HIOs were replated in fresh Matrigel every 14 days.

[0030] Generation and characterization of induced pluripotent stem cell lines For iPSC generation, fibroblasts were transduced with recombinant VSV-G pseudotyped polycistronic lentiviral particles co-expressing the reprogramming factors Oct4, Klf4, Sox2, cMyc, and dTomato28. Nucleofected fibroblasts were then plated on hESC-competent Matrigel, and 3–5 days after transduction, MEF medium was replaced with mTeSR1, and cultures were then fed daily with mTeSR1. After ~3 weeks, putative iPSC colonies were identified and exposed to Dispase for 5 min. Individual colonies were manually excised and replated in mTeSR1 on Matrigel-coated dishes. Several lines with typical hESC-like morphology were then independently expanded in mTeSR1 on Matrigel-coated dishes. For passaging, iPSCs were exposed to Dispase for 5 min, washed, and gently ground before replating. iPSC lines were cryopreserved in mFreSR (StemCell Technologies). All cultures were maintained in a 5% CO2 / air environment. All iPSC experiments in this study were approved by the institutional Embryonic Stem Cell Research Oversight (ESCRO).

[0031] For analysis of pluripotency marker expression, iPSC cultures were fixed in PBS containing 3.7% paraformaldehyde for 10 min at room temperature. Cells were then permeabilized with PBS containing 0.5% Triton X-100 for 10 min and incubated in blocking buffer (PBS containing 10% normal donkey serum) for 30 min at room temperature. Antibodies against human Oct4 (Santa Cruz, sc-5279) and Nanog (Abcam, ab21624) were diluted 1:500 in blocking buffer and incubated with the cells overnight at 4°C. After incubation with fluorescently labeled secondary antibodies, cultures were visualized using a fluorescent microscope. 4'6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining (Figures 13A, 13B). Standard G-banded karyotype analysis of iPSCs was performed at the Cytogenetics Laboratory at Cincinnati Children's Hospital Medical Center. At least 20 metaphases were analyzed (Figure 13C). Analysis of in vivo differentiation potential was assessed by teratoma analysis. Undifferentiated iPSCs were injected subcutaneously into immunodeficient NOD / SCID GAMMA C- / - mice. Teratomas formed within 6-12 weeks. Teratomas were excised, fixed, embedded in paraffin, sectioned, and stained with H&E for histological examination (Figure 13D).

[0032] Generation of LGR5:eGFP BAC transgenic reporter hESC lines An LGR5:eGFP bacterial artificial chromosome (BAC) transgenic reporter hESC line was generated. Briefly, BAC RP11-59F15 was obtained from Children's Hospital Oakland Research Institute (http: / / bacpac.chori.org / ) and propagated in SW10535 cells. Single colonies were grown in LB+cam at 32°C and recombinant proteins were induced by incubation at 42°C for 20 min. The recombination cassette consisted of eGFP-FRT-PGKgb2-neo / kan-FRT, a 50 bp homology region in LGR5, and a 20 bp homology region to peGFP-PGKneo. The homology region was chosen to replace the initiator methionine of LGR5 with that of eGFP, followed by a bovine growth hormone polyadenylation signal and a kanamycin / G418 resistance cassette flanked by FRTs upstream and downstream. The recombinant cassette was electroporated into SW105 cells and cells were selected on plates with cam and kanamycin (kan: 50 μg ml-1). Clones were analyzed for correctly targeted LGR5 BAC by PCR, confirmed by sequencing, and nucleofected into single cell suspensions of H9 hESCs using the Amaxa Human Stem Cell Nucleofector Starter Kit. Cells were selected in G418 (200 ng ml-1) for 2 weeks. G418-resistant cells were maintained in antibiotics indefinitely.

[0033] Transplantation of human intestinal organoids NSG mice were maintained on an antibiotic chow diet (275 p.p.m. sulfamethoxazole, and 1365 p.p.m. trimethoprim; test diet). Food and water were provided ad libitum before and after surgery. Single HIOs matured in vitro for 35 days were removed from Matrigel, washed with cold phosphate-buffered saline (DPBS, Gibco), and embedded in purified type I collagen (rat tail collagen, BD Biosciences) 12 hours before surgery to form solidified gel plugs. These plugs were then placed overnight in standard growth medium (Advanced DMEM / F-12, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng ml-1 EGF (R&D). HIOs were then implanted under the renal capsule. Briefly, mice were anesthetized with 2% inhaled isoflurane (Butler Schein), then the left side of the mouse was aseptically preconditioned with isopropyl alcohol and povidone iodine. A small left posterior submucosal incision was made to expose the kidney. A subcapsular pocket was created, and a collagen-embedded HIO was placed into the pocket. The kidney was then returned to the abdominal cavity, and the mouse received an IP lavage of Zosyn (100 mg / kg, Pfizer Inc.). The skin was sealed in two layers, and the mouse received a subcutaneous injection of Buprenex (0.05 mg / kg, Midwest Veterinary Supply). Six weeks after transplantation, the mice were then humanely euthanized or subjected to further experiments.

[0034] Intestinal resection in transplanted mice Male NSG mice implanted with HIOs 6 weeks prior were randomly assigned to ileocecal resection (ICR) or sham surgery. Mice were placed on liquid diet (Jevity 1Cal, Abbott) 24–48 h prior to surgery and switched from antibiotic chow to liquid antibiotics (200 mg sulfamethoxazole and 40 g trimethoprim oral suspension, 5 mL−1, Hi-Tech Pharmacal) (0.3 mg mL−1 trimethoprim) in drinking water for the remainder of the experiment. Surgery was performed under anesthesia as described above, and the abdomen of the mice was incised anteriorly to expose the intestine. In addition to the ileocecal region, an average of 13.6 cm of the most distal small intestine was removed as previously described26. Mice were kept in a 30°C incubator for 48 h after surgery and then euthanized on postoperative day 7.

[0035] Culture of explant-derived enteroids Human enteroids were generated as previously described. Briefly, explants were harvested, dissected, pinned mucosal layer upwards, and then rinsed in cold PBS (Gibco). The tissue was then transferred to PBS containing 2 mM EDTA (EDTA, Sigma-Aldrich) and rocked on ice for 30 min. After EDTA chelation, the tissue was again washed in cold PBS and the crypts were removed from the underlying mucosal tissue and then filtered through a 100 μm cell strainer (Fisher Scientific). Crypts were then pelleted, resuspended in Matrigel (BD Biosciences), and overlaid with intestinal growth medium (Advanced DMEM / F-12, N2, B27, 15 nM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with EGF (50 ng mL-1), Noggin (100 ng mL-1), R-spondin1 (1 μg mL-1) (R&D Systems), 50% Wnt3a-conditioned medium, 1 mM N-acetylcysteine, 10 nM (Leu15)-gastrin, 10 mM-nicotidamide, 10 μM SB202190 (Sigma-Aldrich), 500 nM A-83-01 (Tocris). 2.5 μM thiazovivin, and 2.5 μM CHIR99021 (Stemgent) were added to the medium for the first 2 days. The medium was changed every 3 days. Established enteroids were passaged over time after 7 days of culture by enzymatic (TrypLE Express, Life Technologies) and mechanical dissociation through an 18-gauge needle. Dissociated enteroids were then resuspended in PBS and pelleted before resuspension in Matrigel, culture conditions as described above.

[0036] Intestinal alkaline phosphatase activity Frozen sections were washed in PBS and blocked with donkey serum. Sections were incubated in Permanent Red working solution (Permanent Red substrate + Permanent Red chromogen, Dako) for 30 minutes at room temperature. Sections were then washed in PBS and mounted for imaging.

[0037] In vivo FD4 permeability analysis FITC-conjugated dextran (FD4, 4400 MW, Sigma) was dissolved in sterile water at a final concentration of 20 mg ml-1. Mice bearing 8-week-old implants were then anesthetized as described above, and a left hind subcostal incision was made to expose the left kidney and implant the intestinal tissue. Human implants were then injected with 100 μL of FD4 each. Whole blood was then collected using heparinized hematocrit capillary tubes at time points 30 minutes and 4 hours after injection, and the fluorescence intensity in the murine serum was analyzed using a fluorescence plate reader. The concentration of FITC-dextran was then determined by comparison with a FITC-dextran standard curve when dissolved in water.

[0038] In vivo tomato lectin injection Fluorescein Lycopersicon esculentum (tomato) lectin (Vector Laboratories) was resuspended in PBS at a final concentration of 2 mg ml-1. Eight weeks after HIO implantation, mice were anesthetized as described above and each was injected with 200 μl of tomato lectin via the tail vein. Mice were then humanely euthanized 30 min after injection and tissues were collected for imaging.

[0039] In vivo D-Ala-Leu-Lys-AMCA uptake experiment D-Ala-Leu-Lys-7-amido-4-methylcoumarin (D-Ala-Leu-Lys-AMCA, Sigma) was adjusted to a final concentration of 25 μM in DMEM (Dulbecco's Modified Eagle Medium, Gibco) using a modified method as previously described30. Mice were anesthetized as described above, and a left hind subcostal incision was made to expose the implant. The implant lumen was then injected with 100 μL of D-Ala-Leu-Lys-AMCA, and the mouse was sealed in two layers. Mice were euthanized 30 min after injection, and tissues were collected for analysis. For comparison, implants were also injected with vehicle (DMEM solution) alone or with peptide solution mixed with 1 mM captopril (Sigma), a competitive inhibitor of peptide uptake.

[0040] Tissue processing, immunofluorescence, and microscopy Tissues were fixed in 4% paraformaldehyde (PFA) for 2 hours to overnight. Organoid explants were frozen in OCT and mouse intestinal tissue was embedded in paraffin. OCT sections were blocked with donkey serum (10% serum in 1xPBS + 0.5% Triton-X) for 30 minutes and incubated with primary antibodies overnight at 4°C. Slides were then washed and incubated in secondary antibodies in blocking buffer for 2 hours at room temperature (23°C). Paraffin sections were deparaffinized, subjected to antigen retrieval, and stained similarly to OCT sections. See Table 1 for a list of antibodies and their respective dilutions. Slides were then washed and mounted with DAPI (Life Technologies) using ProLong Gold antifade. Images were captured on a Nikon Eclipse Ti and analyzed using Nikon Elements Imaging Software (Nikon). For transmission electron microscopy (TEM), tissues were fixed overnight in 0.175 M sodium cacodylate buffer pH 7.4 containing 3% glutaraldehyde. Samples were then fixed for 1 hour at 4°C in 0.175 M cacodylate buffer containing 1% osmium tetroxide. Samples were washed and passed through a graded series of ethanol (25, 50, 75, 95, 2x100%). Infiltration was performed with 2x propylene oxide, followed by graded infiltration with LX-112. Samples were placed in a polymerization oven at 37°C overnight and then stored at 60°C for 3 days. To image the TEM sections, a Hitachi H7600 transmission electron microscope was used. For whole mount staining, tissues were treated as above and then cleared in Murray's solution. Imaging was performed with a Nikon A1 confocal microscope.

[0041] [Table 1] TIFF2024156661000002.tif113166

[0042] RNA isolation, and RT-qPCR RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. To synthesize cDNA, a cDNA reverse transcription kit (Applied Biosystems) was used. Taqman (Applied Biosystems) gene expression assays were then performed on triplicate samples using a OneStep cycler (Applied Biosystems). See Table 2 for a list of Taqman probes used.

[0043] [Table 2]

[0044] Morphometric and statistical analyses Tissue sections were stained with hematoxylin and eosin or immunohistochemistry (as disclosed above). All tissue samples were counted blindly. Crypt depth, villus height, and smooth muscle layer (muscularis) thickness were measured for a minimum of 100 well-oriented crypt-villus units or smooth muscle layer compartments per mouse and then averaged using Nikon NIS imaging software. Crypt fission was also calculated similarly, using longitudinal sections to determine the percentage of fissuring crypts out of at least 100 intact crypts per animal. A fissuring crypt is defined as a bifurcated crypt with a bisecting cleft resulting in two (or sometimes more) flask-shaped bases with a shared single crypt-villus junction (Figure 23A,F). Mice were injected with 5-ethynyl-2'-deoxyuridine (Edu) 2 hours before sacrifice, and then the proliferation index was determined by performing routine Edu immunohistochemistry according to the manufacturer's instructions. The proliferation index was determined by calculating the ratio of Edu positive cells to total cells in at least 10 intact crypts. All data are presented as mean+sem. t-tests and two-way ANOVA were performed using Prism (GraphPad). The significance cutoff value determined was P<0.05. No statistical methods were used to predetermine sample size.

[0045] The experiments were not blinded, except for the ICR experiment, in which transplanted animals were randomly assigned to the sham or ICR group. Investigators were not blinded to the assignment during the experiments and outcome assessments, except for the morphometric analysis for the ICR experiment.

[0046] Example - Development of tissue with a functional intestinal system Generation of vagal crest cells Head and trunk (both vagus and sacral) NCCs emanate from separate regions along the AP axis and colonize different regions of the developing embryo. For example, ENSs are derived from vagus NCCs. Therefore, to generate human intestinal organoids with ENSs, applicants hypothesized that vagus NCCs would be a more suitable starting source. Given that numerous methods have been reported to induce differentiation of PSCs into head NCCs,9,10, applicants first began by generating head NCCs in a stepwise manner that mimics normal NCC development (Figure 1, Figure 20). As previously reported, head NCCs derived from PSCs expressed stage-specific molecular markers in a manner similar to head NCCs during embryogenesis, including Sox2, Pax3, and Pax7 in early neuroepithelial stages, and HNK-1, Sox10, Snail2, and vimentin in migrating neural crest cells.

[0047] To generate vagal NCCs originating from more posterior regions of the neural tube, Applicants manipulated signaling pathways known to promote a posterior fate at early stages during the differentiation process. Similar to the observations by Mica et al., Applicants found that 2 days of retinoic acid (RA) at the neurosphere stage caused robust expression of Hox genes, HOXB3, HOXB5, and HOXB7, at the vagal nerve level, but not FGF4 (data not shown) (Figure 14D). Applicants confirmed that the posterior Hox gene hoxb7 was expressed in trunk / vagal NCCs isolated from chick embryos (Figures 21A and 21B). Furthermore, consistent with the known role of RA in patterning vagal NCCs during vertebrate development, RA was able to induce vagal Hox gene expression in chick head NCCs (Figure 21C).

[0048] Differentiation potential of neural crest cells Pluripotent NCCs can form ectodermal derivatives including neural, glial, and melanocytes, as well as head mesodermal lineage cells including osteocytes and chondrocytes. To examine whether NCCs maintained their pluripotent differentiation potential, Applicants induced NCCs to differentiate into various ectodermal mesenchymal lineage cells in vitro (FIG. 22A). Upon growth factor withdrawal, NCCs could differentiate into peripherin+ neural and GFAP+ neuroglial cells in vitro. PSC-derived NCCs could also differentiate into mesodermal lineage cells including osteocytes and chondrocytes, as shown by positive staining for Alizarin Red and Alcian Blue, respectively. Applicants did not observe any significant difference in the in vitro differentiation potential of NCCs due to RA treatment, suggesting that the pluripotency of PSC-derived head and vagus / Hox-positive NCC populations was maintained. Applicants further examined whether PSC-derived NCCs behaved as neural crest cells in the embryonic environment by injecting PSC-derived NCCs into chick embryos. PSC-derived NCCs followed a migratory pathway similar to endogenous NCCs and differentiated into neurons (Figures 21D-21F).

[0049] In vitro generation of human intestinal organoids (HIOs) using NCC-derived neuroglial cells Applicants have previously developed a method for differentiating human PSCs into intestinal organoids following a stepwise differentiation first into definitive endoderm and then into mid / hindgut spheroids that are developed into 3D intestinal tissue (Spence et al., McCracken et al. (Figure 23)). In vitro, HIOs contain stratified mesenchyme containing all major intestinal epithelial cell types, as well as subepithelial myofibroblasts and smooth muscle cells. When transplanted into the renal subcapsular space, HIOs continue to develop and form well-differentiated intestinal tissue with crypts and villi, functional intestinal stem cells, and a smooth muscle layer (Figure 23B).

[0050] HOPs contained most of the epithelial and mesenchymal cell types found in the developing intestine, but no ENS. To incorporate NCC / ENS precursors into developing HIOs, Applicants mechanically aggregated mid / hindgut spheroids with PSC-derived NCCs by low-speed centrifugation and transferred the aggregates into 3D growth conditions for 28 days (Figure 15A). HIOs with or without NCCs were comparable in size (1-2 mm in diameter). However, Applicants detected numerous βIII-tubulin (TUJ1)+ neurons and S100+ glia embedded within the mesenchyme of HIOs combined with NCCs. Applicants detected rare neurons and no glia in HIOs without NCCs in vitro. Furthermore, Applicants confirmed the NCC origin of the glial cells by using NCCs derived from a PSC line that constitutively expressed GFP (Figure 24). The spatial relationships between epithelial, mesenchymal, and neuroglial cells in HIO+NCCs were highly similar to those in human fetal intestine and E11.5 mouse small intestine (data not shown), suggesting that endogenous cues in HIOs help organize developing ENS cells. Consistent with this, HIOs expressed both GDNF and EDN3, two important chemoattractant factors known to regulate NCC migration into intestinal mesenchyme (Figure 23C), suggesting that integration of PSC-derived NCCs into HIO mesenchyme may have utilized normal developmental pathways.

[0051] Maturation of the ENS after in vivo growth Our previous studies, as described above and in Watson et al. 2011, showed that HIOs transplanted into mice and grown in vivo for 6-10 weeks form highly mature intestinal tissue with villi and crypts that are vascularized, grow to 1-3 cm in diameter, and contain functional intestinal stem cells (Figure 17B). Furthermore, HIO mesenchyme matures into submucosal and myenteric layers of smooth muscle fibers (Figure 15C). Analysis of transplanted HIO+PSC-derived NCCs revealed that nerves (βIII-tubulin) and glia (S100) were organized into multiple layers adjacent to the submucosal and myenteric layers of smooth muscle (Figure 15C). Applicants did not detect nerves or glia in transplanted HIOs without NCCs. Although these experiments were performed with a vagus / HOX-positive NCC population, PSC-derived head / HOX-negative NCCs were also capable of engrafting and forming ENS glial cells. However, head NCCs also formed pigment epithelial cells (FIG. 22B), and cartilage (not shown), consistent with having broader multipotency.

[0052] Neurodiversity and function in HIO+NCCs The ENS is a complex network of excitatory and inhibitory neuronal subtypes, as well as interneurons. Examination of neural markers in HIO+NCCs cultured in vitro for 4 weeks suggested a remarkable degree of neurodiversity. Applicants observed tyrosine hydroxylase (TH), calbindin, calretinin, and serotonin (5-HT) positive cells, which are expressed by dopaminergic, excitatory, and interneurons (Figure 3A). However, Applicants did not detect nNOS positive neurons in vitro. In contrast, in vivo transplanted HIO+NCCs had many nNOS+ neurons, suggesting that the ENS had matured during in vivo growth. In transplanted HIO+NCCs, many nerves appeared to be associated with smooth muscle. Furthermore, ENS nerves were associated with cells expressing CD117, a marker for interstitial cells of Cajal (ICC) (Figure 16B). Although ICCs were present in HIOs lacking the ENS, there appeared to be fewer clusters of CD117-expressing cells in HIO-NCCs (data not shown), suggesting a developmental interaction between ENS cells and ICCs.

[0053] The availability of in vitro grown HIOs allowed us to test neuronal functionality using live imaging with the Ca2+ sensor GCaMP6f19. To do this, applicants generated HIOs and incorporated NCCs derived from a PSC line containing GCaMP6 into the HIOs (Figure 17A). Imaging of neuronal activity in HIOs grown in vitro for 8 weeks detected broad, spontaneous, and rapid calcium transients within the neurons. When individual neurons were monitored, there was a clear periodicity of calcium flux in the range of 3-4 sec / neuron (Figures 17B and 17C). To test whether neurons could tolerate synchronous activity, applicants exposed HIOs + GCaMP6-expressing NCCs to KCl (30 mM) and observed large waves of neuronal calcium flux (Figure 17D).

[0054] Formation of glial plexus in HIO+NCCs grown in vivo Histological analysis of ENS nerves and glia suggested that they formed a plexus within the smooth muscle layer of the HIO. However, this conclusion is difficult given the two-dimensional nature of the sectioned tissue. Applicants therefore performed 3D imaging of tissues from HIO+NCC matured in vivo by whole-mount immunofluorescence using nerve, glial, and smooth muscle markers and compared them to human small intestine (Figure 18). The ENS of the human small intestine contained a network of nerves (BIII-tubulin) and glia (S100) integrated into the smooth muscle layer (desmin+ cells). The ENS formed within the developing HIO similarly contained a complex plexus of nerves and glia that appeared to be oriented with the smooth muscle fibers (Figure 18A). Lateral views of 3D images of HIO+NCC specimens clearly showed evidence of a myenteric plexus (Figure 18B) as well as a plexus arranged in the submucosa adjacent to the epithelium (blue-dapi). Applicants also observed neuronal cell body clusters in both sections and 3D imaging (FIG. 25), suggestive of ganglia. Compared to human intestine, ganglia in HIO were smaller and less developed.

[0055] ENS-mediated contractile activity The organization of epithelium, smooth muscle, nerves, and glia in the HIO+ENS tissue was similar to that of the human intestine, but it was unclear whether neuromuscular communication existed in the HIO+ENS tissue. To determine whether the PSC-derived ENS could function in the HIO tissue, applicants explanted the kidney explants in Tyrode's solution and subjected the kidney explants to electric field stimulation (FIG. 19C). HIO tissue without ENS given a single 1 ms pulse at 100 V (high voltage) evoked a single contraction, suggesting direct stimulation of smooth muscle contraction. In contrast, HIO tissue containing ENS given a single 1 ms pulse at 50 V (low voltage) evoked a sustained wave of contraction. To determine whether the contractions were due to ENS activity, applicants blocked nerve activity with tetrodotoxin (TTX), which binds to voltage-gated Na+ channels on the nerve, inhibiting the firing of action potentials. TTX completely inhibited the ability of low-voltage stimulation to evoke movement in the HIO+NCC tissue.

[0056] Neuromuscular connections in HIO+NCC tissue GI motility requires the cooperation of ENS-dependent and -independent contractions and smooth muscle relaxation. To better analyze these processes, applicants isolated tissue slices from HIO and HIO+NCC and analyzed the slices using organ chamber experiments. In the absence of nerve stimulation, applicants observed spontaneous phasic contractions in both HIO and HIO+NCC tissues (FIGS. 19A and 26A), consistent with the presence of ICCs in both conditions (FIG. 16B). Methylene blue, which inhibits ICC activity, abolished the phasic contractions (FIG. 19B). To identify the role of ENS-dependent roles in contraction and relaxation, applicants activated nerves using dimethylphenylpiperazinium (DMPP), a selective α3 nicotinic receptor agonist, and veratridine, which increases nerve excitability by inhibiting Na+ channel inactivation. Pharmacological activation of the ENS induced relaxation in HIO+NCC tissue, but not in HIO alone (FIG. 6C, and FIG. 25A). Applicants confirmed that relaxation was nerve-dependent using TTX. TTX inhibited the ability of nerves to induce muscle relaxation in response to DMPP and / or veratridine (FIG. 19D, and FIG. 27B).

[0057] Applicants aimed to identify the putative neural mediator responsible for ENS-induced smooth muscle relaxation. Because nitric oxide (NO) is a well-known intestinal inhibitory neural mediator, and because nNOS-expressing nerves are abundant in transplanted HIO+NCC tissues (Figure 16), Applicants inhibited NO synthesis using NG-nitro-L-arginine methyl ester (L-NAME). L-NAME pretreatment significantly reduced relaxation after DMPP stimulation, thus indicating that NO was produced by nerves to induce relaxation (Figure 19E and Figure 27C). Applicants further stimulated NO release using sodium nitropruszide (SNP) and observed smooth muscle relaxation in HIP+NCC tissues (Figure 27D).

[0058] method Cell lines and culture conditions Human ES and iPS cells, H1 (WA-01), H9 (WA-09), and WTC11 AAVS1-CAG-GCaMP6f, were maintained on Matrigel (BD Biosciences) in an undifferentiated state without feeders. H9-GAPDH-GFP hESCs were generated by targeting a sequence encoding GFP into the 3'-UTR of the GAPDH gene using standard procedures33 incorporating TALEN-facilitated homologous recombination34. GFP was expressed as a pseudofusion protein with T2A35 immediately adjacent to the C-terminus of GAPDH.

[0059] Cells were fed with mTeSR1 medium and passaged periodically using Dispase II (Gibco). To generate HIOs containing ENS, NCCs and HIOs were generated and merged at the early stage of intestinal differentiation. Briefly, for NCC generation (Figure 1), hPSCs were treated with collagenase IV (500 U / mL) in mTeSR1 for 60 min to detach colonies. Cells were washed to remove collagen, then gently ground and resuspended in neural induction medium on non-TC treated Petri dishes. The neural induction medium was changed daily, and retinoic acid (2 μM) was added on days 4 and 5 for posteriorization. Day 6 free-floating neurospheres were plated on fibronectin (3 μ / cm2) and fed with neural induction medium without RA for 4 days. Free cells were harvested using a brief Accutase treatment (2-3 min) and passaged onto fibronectin or used immediately to merge with HIOs. NCCs were differentiated essentially as described for glial9 and mesenchymal cells36. Applicants generated HIOs using our previous protocol similar to that disclosed above, but notably used the small molecule CHIR99021 instead of WNT3A (Figure 22). After formation of intestinal spheroids, Applicants gently centrifuged spheroids + / - NCCs and embedded them in Matrigel. Cultures were fed with basal intestinal medium (advanced DMEM / F12, 1xB27 supplement, 1xN2 supplement, 10 μM HEPES, 2 mM L-glutamine, 1xPen-Strep) supplemented with 100 ng EGF mL-1 and maintained in vitro for up to 8 weeks.

[0060] In vivo implantation of HIO and electric field stimulation HIO+ / -NCCs were ectopically transplanted into the renal capsule of NOD / SCID-gamma (NSG) mice according to the protocol described above and in Watson et al. Briefly, 4-6 week old HIOs were embedded in collagen and transplanted into the renal subcapsular space. The transplanted HIOs were harvested 6-10 weeks after transplantation and analyzed for neural or glial markers or used for electric field stimulation (EFS) experiments. For EFS, HIOs were explanted into Tyrode's solution and equilibrated for approximately 5 min before stimulation initiation. Electrical stimulation was applied using a Grass S88 Stimulator (Grass Technologies) with a single pulse, 1 ms duration, and settings of 50 or 100 V. HIOs were then incubated for 5 min in 10 μM TTX diluted in Tyrode's solution, rinsed, and placed back into fresh Tyrode's solution. EFS was then repeated. Videos were recorded on a Leica dissecting microscope using Leica Application Suite software and processed with VideoLAN and Handbrake to achieve 16X playback speed.

[0061] Chick Embryo Manipulation Chicken eggs were purchased from Charles River and incubated at 39°C until they reached the desired Hamburger and Hamilton stages. For chicken NCC culture, dorsal neural tubes from the head or trunk regions of HH8 embryos were dissected by Gastromaster and cultured on Matrigel-coated tissue culture dishes with NCC culture medium for 24 hours (BajPai R et al, 2010). After NCCs migrated out of the neural tube explants, the neuroectoderm was peeled off the plate with a tungsten needle. The NCCs left on the plate were then treated with 4ng / ml FGF4 or 2μM retinoic acid for 48 hours. For NCC injection, GFP-labeled human PSC-derived NCCs were harvested and injected intersomitically into HH10-12 chicken embryos. Embryos were harvested for analysis around HH38.

[0062] Immunohistochemistry and microscopy NCC cells, cell monolayers, and day 0 spheroids were fixed in 4% paraformaldehyde (PFA) for 15 min at 23°C, washed, and then stained directly. Four-week in vitro HIOs and in vivo implants were fixed in 4% PFA for 1 h to overnight at 4°C. Tissues were processed, embedded in OCT, sectioned at approximately 10 μm, and mounted on Superfrost Plus slides (Fisherbrand). Frozen sections and cells were treated with 0.25% Triton-X100 for 10 min and then blocked with 5% Normal Donkey Serum (NDS, Jackson Immunoresearch) in PBS for 30 min at 23°C. Primary antibodies diluted in PBS were applied to slides and cells overnight at 4°C, then washed and incubated with secondary antibodies for 2 h at 23°C (see Table 3 for antibodies and dilutions). Slides were mounted using Fluromount-G and images were acquired using a Zeiss ApoTome Imager Z1 or Zeiss LSM510 confocal microscope. Whole mount 3D images were acquired by fixing tissues overnight in 4% PFA at 4°C and then equilibrating in 100% methanol on ice for 1 hour. Tissues were permeabilized with Dent bleach (4:1:1 MeOH:DMSO:30% H2O2) for 2 hours at room temperature before rehydrating with PBS. Tissues were blocked with 5% NDS in PBS for 2 hours at room temperature, incubated with primary antibody in PBS overnight at 4°C, washed, and incubated with secondary antibody in PBS overnight at 4°C. After staining, tissues were dehydrated through a methanol series and cleared in Murray's Clear (2:1 benzoic acid:benzyl alcohol) before imaging on a Nikon A1 inverted confocal microscope. Images were processed using NIS Elements, Bitplane Imaris, Zeiss Axiovision, and Adobe Photoshop CS7 software.

[0063] [Table 3]

[0064] RNA isolation and quantitative real-time PCR Total RNA from cells and organoids was isolated using NucleoSpin RNA Isolation Kit (Macherey-Nagel). Complementary DNA was generated using SuperScript VILO cDNA Synthesis Kit (Invitrogen) immediately after RNA isolation. Both isolation and synthesis were performed according to the manufacturer's protocol. Applicants performed qPCR on a BioRad CFX96 Real-Time PCR Detection System using QuantiTect SYBR Green PCR Kit (Qiagen). Primer sequences were generally obtained from qPrimerDepot (http: / / primerdepot.nci.nih.gov) and can be found in Table 4.

[0065] [Table 4]

[0066] Ex-vivo intestinal motility The transplanted HIO+ / - NCCs were harvested and placed in ice-cold HBSS. Muscle strips (4-6 mm long, 1-2 mm wide) were cut from the transplanted HIO+ / - NCCs. The preparations were suspended vertically in organ baths filled with Krebs solution (17 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, and 11 mM glucose), warmed to 37°C, and gassed with 95% O2 + 5% CO2. After a 60-min equilibration period at an initial tension of 0.5 g, muscle contractile responses were continuously recorded using an 8-chamber tissue organ bath with an isometric force transducer (Radnoti) linked to a computer equipped with LabChart software (AD Instruments). Muscle preparations were stimulated with dimethylphenylpiperazinium (DMPP, 10 μM, Sigma) and veratridine (3 μM, Sigma). Chemical stimuli were applied at 15 min intervals and then washed three times. Tetrodotoxin (TTX, 10 μM, Tocris) or NG-nitro-L-arginine methyl ester (L-NAME, 50 μM, Sigma) was applied 5 min before DMPP stimulation. NOS was inhibited with sodium nitropruside (SNP, 100 μM, Sigma). Methylene blue (50 μM) was used to inhibit ICC activity. The effect of chemical stimulation on tension was assessed by measuring the area under the curve (AUC). Data were expressed as ΔAUC, i.e., the "stimulated" AUC measured 120 s after stimulation minus the "control" AUC measured 120 s before stimulation.

[0067] Consideration Applicants utilized the principles of embryonic intestinal differentiation to engineer human PSC-derived intestinal tissue containing a functional ENS. Human PSC-derived vagus-like NCCs reintegrated with PSC-derived intestinal organoids in 3D growth conditions migrated into the intestinal mesenchyme, self-organized, and differentiated into a range of ENS neural and neuroglial cell types. After transplantation and in vivo growth, NCCs formed complex ganglionic structures and interganglionic fibers that resembled the embryonic development of myenteric and submucosal nerve plexuses. Applicants further demonstrated that NCC-derived ENSs were functionally integrated into intestinal smooth muscle and drove NO-dependent relaxation. The high degree of tissue organization seen in transplanted HIO+NCCs suggests that the tissues engineered by Applicants in vitro possessed the intrinsic information for the coordination of cell migration, proliferation, lineage commitment, and nerve plexus construction that occurs during the embryonic development of the ENS.

[0068] Vagal NCCs, which give rise to the ENS, derive from the more posterior, HOX-positive region of the neural tube. HOXB5, for example, is expressed in human NCCs during gut colonization and is required for the formation of the ENS in mice. Studies in vertebrate embryos have demonstrated the importance of Wnt, FGF, and retinoic acid (RA) signaling in neural patterning and the formation of HOX-expressing NCCs. Applicants observed RA-induced expression of vagal HOX genes A2, B3, B5, and B7, while treatment with FGF4 had little effect (data not shown). Recent findings have observed that activation of Wnt signaling also promotes the formation of NCCs capable of forming melanocytes, a hallmark of vagal / trunk NCCs. Applicants also observed that activation of Wnt signaling can promote HOX gene expression in posterior NCCs, although to a lesser extent than RA (data not shown). At the functional level, PSC-derived head and vagus NCCs were able to integrate into HIOs and form neuroglial lineages. This is consistent with studies performed in chick-quail chimeric embryos, where head NCCs were able to efficiently contribute to the ENS. Applicants observed a difference in that head-like NCC+HIOs also gave rise to pigment cells and chondrocytes, consistent with the broader differentiation potential of head NCCs.

[0069] Applicants routinely observed the formation of a neuroglial plexus in close association with smooth muscle fibers with a structure similar to the myenteric plexus in transplanted HIO+NCC. However, several lines of evidence suggest that the NCC-derived ENS is less mature and more fetal in nature. For example, the development of the submucosal plexus appears delayed in HIO+NCC tissue, which may resemble development in the human fetal intestine, where the submucosal plexus develops 2–3 weeks after the myenteric plexus.26 Another indication of developmental immaturity is that the neuroglial plexus in HIO contained smaller nerve bundles than the adult human intestine, more similar to those observed in the human fetal intestine.16 The immature / fetal nature of HIO+NCC tissue may provide an opportunity to identify specific factors that regulate fetal intestine / ENS maturation. For example, it has recently been shown that the luminal microbiota influences mucosal colonization by glial cells, and the HIO+ / -ENS model may allow for detailed mechanistic analysis of this process. Factors that promote intestinal maturation may be of clinical use in premature infants, who are at high risk of intestinal infections because of their immature mucosa.

[0070] Applicants observed a very high degree of neuronal diversity in HIO+NCC tissues in vitro, including excitatory and interneurons with intrinsic and induced waves of calcium flux, suggestive of neuronal activity. HIO+NCC tissues grown in vivo acquired additional neuronal diversity with nNOS+ inhibitory nerves, known to form at later stages in the developing mouse fetal intestine. Furthermore, neuroglial cells assembled to construct intermyenteric and submucosal nerve plexuses functionally linked with highly differentiated smooth muscle layers. Electric field stimulation (EFS) of these engineered tissues evoked waves of ENS-dependent motility qualitatively similar to peristalsis. However, Applicants also observed intrinsic contractile activity dependent on endogenous ICCs, which were present in both HIO and HIO+NCC. Together, our data suggest that ICCs primarily drive contraction and muscle relaxation is ENS-dependent and mediated by nNOS+ inhibitory nerves present in HIO+NCC tissues. The fact that NOS inhibitory neurons became functional earlier than cholinergic excitatory neurons supports the conclusion that the ENS in HIO is fetal in nature.

[0071] There are several striking differences between rodents and humans with regard to intestinal development, physiology, and disease. For example, crypt development occurs in utero in humans but postnatally in mice. There are differences in the formation of TN neurons during ENS development between mice and humans. Given that HIO+NCCs contain TH-positive neurons in vitro and in vivo, this system may be a unique tool to study the unique developmental features of these cell types. Furthermore, an experimental system to study human TH+ dopaminergic ENS neurons may provide insight into the GI dysmotility symptoms seen in Parkinson's patients caused by the degeneration of this neuronal subtype. Due to the highly tractable nature of this system, it should be particularly useful to study developmental defects of the ENS, such as Hirschsprung's disease. Defects in NCC formation, migration, mesenchymal integration, and proliferation may result in an aganglionic gut compartment. The ability to study the interactions between NCCs and intestinal organoids in vitro, and to observe and manipulate these interactions during the formation of a functional ENS, should enable unprecedented mechanistic dissection of known and novel forms of Hirschsprung's disease in humans.

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[0073] Unless otherwise indicated, all percentages and ratios are calculated by weight.

[0074] Unless otherwise indicated, all percentages and ratios are calculated on the basis of the total composition.

[0075] It should be understood that every maximum numerical limitation provided anywhere in this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation provided anywhere in this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range provided anywhere in this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0076] The dimensions and values ​​disclosed herein should not be understood to be strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm."

[0077] Each document cited herein, including any cross-referenced or related patents or applications, is incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it teaches, suggests, or discloses, either alone or in any combination with any other reference, any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition ascribed to that term in this document shall govern.

[0078] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended in the appended claims to cover all such changes and modifications that are within the scope of this invention.

Claims

1. An in vitro cultured intestinal organoid, comprising enteric neuronal and glial cell types and further comprising a functional enteric nervous system (ENS).

2. The intestinal organoid of claim 1, wherein the neuronal and / or glial cell types are differentiated from vagus-like neural crest cells expressing HOXB3, HOXB5, and HOXB7 and are derived from human embryonic stem cells (ES cells) and / or induced pluripotent stem cells (iPS cells).

3. An intestinal organoid as described in claim 1 or claim 2, wherein the neural cell type expresses tyrosine hydroxylase (TH), calbindin, calretinin, and serotonin (5-HT).

4. An intestinal organoid described in any one of claims 1 to 3, wherein the intestinal organoid is capable of contractile activity.

5. An intestinal organoid as described in any one of claims 1 to 4, wherein the enteric neural cell type exhibits neural activity as measured by rhythmic waves of calcium translocation.

6. An intestinal organoid described in any one of claims 1 to 5, wherein the neural cell type expresses the pan-neuronal marker PGP9.5 and / or βIII-tubulin.

7. An intestinal organoid as described in any one of claims 1 to 6, wherein the neural cell types include dopaminergic neurons expressing TH, interneurons expressing 5-HT and ChAT, sensory neurons expressing calbindin, and / or excitatory neurons expressing calretinin.

8. An intestinal organoid described in any one of claims 1 to 7, wherein the neuronal and glial cell types are integrated into a smooth muscle layer.

9. An intestinal organoid as described in any one of claims 1 to 8, wherein the enteric neuronal and glial cell types are embedded in the mesenchyme of the intestinal organoid.

10. An intestinal organoid described in any one of claims 1 to 9, wherein the glial cell type expresses glial marker S100.

11. An intestinal organoid as described in any one of claims 1 to 10, wherein the neuronal cell type and glial cell type are integrated into a smooth muscle layer (desmin+ cells).

12. An intestinal organoid described in any one of claims 1 to 11, wherein the intestinal organoid is a human intestinal organoid (HIO).

13. Use of the intestinal organoid of any one of claims 1 to 12 for producing human intestinal tissue to treat a patient in need of replacement of a portion of the patient's gastrointestinal tract, wherein the portion is replaced with human intestinal tissue derived from the intestinal organoid.

14. Use of the intestinal organoids described in any one of claims 1 to 12 for determining the effect of a treatment on the intestinal organoids, wherein the treatment is brought into contact with the intestinal organoids.

15. Use of the intestinal organoids described in any one of claims 1 to 12 for producing human intestinal tissue for determining the effect of a treatment on the human intestinal tissue, wherein the treatment is contacted with human intestinal tissue derived from the intestinal organoids.

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