Cell population with tissue regeneration potential present in the anorectal transition zone, and methods for isolating and using the same

Abstract

Provided herein is the composition of anorectal transition zone stem cells, which comprises multipotentiality and progenitor cells for treating endodermal disease.Also provided is the cell composition of anorectal transition zone stem cells, which comprises multipotentiality and progenitor cells in pharmaceutically acceptable carrier.In one aspect, provided herein is the stem cell composition for treating endodermal tissue or organ, which comprises isolated anorectal transition zone (ATZ) cell and / or ATZ-derived organoid cell.Preferably, isolated ATZ cell comprises isolated ATZ crypt cell, which comprises ATZ multipotential stem cell.

Description

[Background technology]

[0001] background The gastrointestinal transition zone exists between two distinct epithelial tissue types. The anorectal transition zone tissue lies at the junction between the endodermal-derived columnar epithelium of the rectum and the ectodermal-derived non-keratinizing stratified squamous epithelium of the anus (McNairn and Guasch (2011), "Epithelial transition zones: merging microenvironments, niches, and cellular transformation," Eur J Dermatol, 21 (Suppl 2): ​​21-8). Histopathological labeling studies of mouse and human anorectal transition zone tissue have identified cells expressing p63, a marker of basal cells; cytokeratin 7 (CK7), a marker of simple columnar cells (Yang et al. (2015), "Microanatomy of the cervical and anorectal squamocolumnar junctions: a proposed model for anatomical differences in HPV-related cancer risk," Mod Pathol, 28(7): 994-1000); and CD34, a marker of stem and progenitor cells (McNairn, supra). Further studies in mice confirmed the expression of p63, CK7, and CD34 using label-retaining cell assays and immunohistochemistry, and identified the expression of the pluripotent stem cell marker SOX2, supporting the presence of stem and progenitor cells in the anorectal transition zone (Runck et al. (2010), "Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice," Cell Cycle, 9(15): 3039-45).

[0002] Stem cell therapy offers the promise of repairing endodermally derived tissues and organs that have become dysfunctional or damaged due to infection, drug application, or physical trauma. Each of the current sources of cell therapy has limitations in its usefulness. Human embryonic stem cells (ESCs) are pluripotent and give rise to all cells in the body. ESCs have regenerative potential but pose the risk of teratoma formation and autoimmune response induction. Encapsulating ESCs mitigates some of these risks. iPSCs are fibroblasts that are reprogrammed into differentiated cells, but due to the reprogramming method, they have mutagenic potential and pose barriers to long-term transplant survival and functionality. Mesenchymal stem cells (MSCs) have been widely used due to their inherent anti-inflammatory properties, which reduce the risk of autoimmune responses and tumorigenicity. However, because MSCs do not regenerate, their benefits are transient. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] McNairn and Guasch (2011), "Epithelial transition zones: merging microenvironments, niches, and cellular transformation," Eur J Dermatol, 21 (Suppl 2): ​​21-8 [Non-patent document 2] Yang et al. (2015), "Microanatomy of the cervical and anorectal squamocolumnar junctions: a proposed model for anatomical differences in HPV related cancer risk," Mod Pathol, 28(7): 994-1000 [Non-patent document 3] Runck et al. (2010), "Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice," Cell Cycle, 9(15): 3039-45 Summary of the Invention [Means for solving the problem]

[0004] overview The present invention is based in part on the discovery of compositions for use (or suitable for use) in the treatment of endodermally derived tissues or organs in subjects in need thereof, methods of making stem cell compositions useful in treating endodermal tissues or organs, and methods of treating endodermal tissues or organs with such stem cell compositions.

[0005] In one aspect, the present specification provides a stem cell composition for treating endodermal tissue or organ, comprising isolated anorectal transition zone (ATZ) cell and / or ATZ-derived organoid cell.Preferably, isolated ATZ cell comprises isolated ATZ crypt cell, which comprises ATZ multipotent stem cell.Preferably, ATZ-derived organoid cell comprises ATZ multipotent stem cell and ATZ-derived endodermal cell, which can be single cell, cell cluster, two-dimensional cell layer or three-dimensional organoid.Cell can be cultured in suspension, adhesion layer or liquid culture.Composition can comprise multipotent ATZ stem cell and ectodermal precursor ATZ cell.

[0006] The isolated ATZ crypt cells comprise multipotent stem cells and / or progenitor cells that express KIT, LGR5, NANOG, and OCT4A. The multipotent isolated ATZ crypt cells may also express one or more additional stem cell and progenitor cell markers selected from the group consisting of BMP4, CD34, CXCR4, and SOX17. In some embodiments, the multipotent ATZ crypt cells express at least two, three, or four stem cell and progenitor cell markers selected from the group consisting of BMP4, CD34, CXCR4, and SOX17.

[0007] ATZ-derived organoids may express additional endodermal markers, depending on the particular endodermal lineage-specific medium (LSM) used.

[0008] For example, in some embodiments, ATZ-derived crypt cells are grown in pluripotent stem cell MGM (e.g., mTesR™, STEMCELL Technologies, Inc., Vancouver, CA; StemFit® Feeder-Free Stem Cell Culture Media, AMSBIO, Abingdon, UK).

[0009] For example, in some embodiments, ATZ-derived crypt cells are grown in definitive endoderm LSM (e.g., Gibco™ PSC Definitive Endoderm Induction Kit, Thermo Fisher Scientific, Waltham, Massachusetts).

[0010] Isolated ATZ crypt cells or ATZ-derived organoids do not express CD45 and are therefore not of the hematopoietic lineage.

[0011] In some embodiments, ATZ-derived crypt cells grown in intestinal-specific LSM (e.g., IntestiCult™; Human Intestinal Organoid Culture Protocol, Bio-Techne, Minneapolis, Minnesota) may further express ALP.

[0012] In some embodiments, ATZ-derived crypt cells grown in pancreatic-specific LSM (e.g., PancreaCult™; Human Pancreas Organoid Culture Protocol, Bio-Techne) may express one or more additional markers selected from the group consisting of AMY, CK19, FOXA2, GCG, INS, NKX6.1, and SOX9. In some embodiments, ATZ-derived crypt cells grown in pancreatic-specific LSM may express two, three, four, five, six, or seven additional markers selected from the group consisting of AMY, CK19, FOXA2, GCG, INS, NKX6.1, and SOX9.

[0013] In some embodiments, ATZ-derived crypt cells grown in liver-specific LSM (e.g., HepatiCult™; Human Liver Organoid Culture Protocol, Bio-Techne) may express one or more additional markers from the group consisting of AFP, ALB, CK19, EPCAM, FOXA2, HNF4A, and SOX9. In some embodiments, ATZ-derived crypt cells grown in liver-specific LSM may express two, three, four, five, six, or seven additional markers selected from the group consisting of AFP, ALB, CK19, EPCAM, FOXA2, HNF4A, and SOX9.

[0014] In some embodiments, ATZ-derived crypt cells grown in lung-specific LSM (e.g., STEMdiff™ Branching Lung Organoid Kit, or PneumaCult™ Airway Organoid Kit, or PneumaCult™ Apical-Out Airway Organoid Medium, or PneumaCult™ Alveolar Organoid Medium, all from STEMCELL Technologies; Human Lung Organoid Culture Protocols, Bio-Techne; 3dGRO™ Lung Organoid Branching Medium3dGRO™ and Lung Organoid Maturation Medium, Merck) may express one or more additional markers from the group consisting of SOX9, NKX2.1, TMPRSS2, MUC1, VIM, and ACE2. In some embodiments, ATZ-derived crypt cells grown in lung-specific LSM may express two, three, four, five, or six additional markers selected from the group consisting of SOX9, NKX2.1, TMPRSS2, MUC1, VIM, and ACE2.

[0015] ATZ-derived crypt cells grown in thyroid-specific LSM (as directed in the protocol "Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism", PNAS, 118(51): e1-11) may express one or more additional markers from the group consisting of PAX8 and NKX2.1.

[0016] Isolated ATZ crypt cells or ATZ-derived organoids can be combined with exogenous biocompatible scaffolds, such as synthetic scaffolds or biological scaffolds.Depending on the situation, scaffolds can be collagen-based.Alternatively, isolated ATZ crypt cells or ATZ-derived organoids can be combined with matrix materials (such as gel) or pharmaceutically acceptable carriers (such as saline).

[0017] The isolated ATZ crypt cells or ATZ-derived organoids can be porcine cells or human cells.

[0018] The isolated ATZ crypt cells or ATZ-derived organoids can be allogeneic or autologous to the subject in need of treatment.

[0019] Isolated ATZ crypt cells or ATZ-derived organoids can be genetically modified to contain a selection marker (e.g., to detect engraftment), to eliminate cells (e.g., suicide genes), or to reduce host-versus-graft responses (e.g., MHC or B2M knockout).

[0020] In certain embodiments, the composition is cryopreserved. To facilitate cryopreservation, the composition can include an appropriate cryopreservation medium.

[0021] In another aspect, the present invention provides a method for preparing a pharmaceutical composition (e.g., any of the cell compositions disclosed herein), comprising: (a) collecting ATZ tissue from a donor (e.g., a porcine or human donor); (b) enzymatically digesting the tissue to prepare a cell suspension; (c) optionally combining at least a portion of the cell suspension with a medium for cryopreservation and cryopreserving the cell suspension; and (d) combining the cell suspension of step (b) or, optionally, the thawed suspension of step (c) with an exogenous biocompatible scaffold, matrix, or pharmaceutically acceptable carrier.

[0022] Cell suspension comprises isolated ATZ crypt cell and / or ATZ-derived organoid cell, and these cells comprise multipotent stem cells.ATZ crypt cell and / or ATZ-derived organoid cell can also comprise endodermal progenitor cell.In certain embodiments, ATZ cell can be differentiated into endodermal cell (for example, express SOX17).

[0023] In some embodiments, the ATZ stem cells express CD34, CD117 (KIT), and CD184 (CXCR4), e.g., as detected by flow cytometry. Additionally, the ATZ stem cells do not express detectable levels of CD45, e.g., as detected by flow cytometry.

[0024] Furthermore, NANOG and / or OCT4A can be detected in freshly isolated ATZ crypt cells or ATZ-derived organoids.

[0025] In certain embodiments, cells in suspension (eg, ATZ stem cells) are expanded in vitro.

[0026] In some embodiments, in step (d) of above-mentioned method, the cell in cell suspension is combined with exogenous biocompatible scaffold, for example, synthetic scaffold or biological scaffold.In certain embodiments, scaffold comprises collagen-based scaffold.Alternatively, isolated ATZ crypt cells or ATZ-derived organoid cells can be combined with matrix material (for example, gel) or pharmaceutically acceptable carrier (for example, physiological saline).

[0027] In another aspect, the invention provides a cell composition produced by the methods disclosed herein.

[0028] In another aspect, the present invention provides a method for treating damaged or dysfunctional gastrointestinal mucosal tissue (e.g., gastric mucosa or intestinal mucosa) in a subject in need thereof, comprising administering to the damaged or dysfunctional gastrointestinal mucosal tissue an effective amount of a cell composition disclosed herein, thereby treating the damaged or dysfunctional gastrointestinal mucosal tissue.

[0029] In another aspect, the present invention provides a method for treating damaged or dysfunctional pancreatic tissue in a subject in need thereof, comprising administering to the damaged or dysfunctional pancreatic tissue an effective amount of a cell composition disclosed herein, thereby treating the damaged or dysfunctional pancreatic tissue.

[0030] In another aspect, the present invention provides a method for treating damaged or dysfunctional liver tissue in a subject in need thereof, comprising administering to the damaged or dysfunctional liver tissue an effective amount of a cell composition disclosed herein, thereby treating the damaged or dysfunctional liver tissue.

[0031] In another aspect, the present invention provides a method for treating damaged or dysfunctional lung tissue in a subject in need thereof, comprising administering to the damaged or dysfunctional lung tissue an effective amount of a cell composition disclosed herein, thereby treating the damaged or dysfunctional lung tissue. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows the isolation of ATZ crypts from porcine tissue. (a) shows excised colorectal tissue, (b) shows exposed mucosal epithelium, (c) shows excised dentate line, (d) shows freshly isolated ATZ crypts, (e) shows freshly isolated ATZ crypts, (f) shows freshly isolated ATZ crypts, (g) shows freshly isolated bifurcated ATZ crypts, (h) shows freshly isolated ATZ submucosal glands, and (i) shows freshly isolated ATZ submucosal glands.

[0033] [Figure 2] Figure 2 shows the isolation of ATZ crypts from Crohn's disease patient tissue. (a) shows excised anorectal tissue, (b) shows excised mucosal epithelium, (c) shows a bright-field image of freshly isolated ATZ crypts, and (d) shows a bright-field image of freshly isolated ATZ crypts.

[0034] [Figure 3] Figure 3 shows (a) the development of crypt organoids in porcine GI tissue and (b) the development of crypt organoids in rectal and ATZ tissues of Crohn's disease patients.

[0035] [Figure 4] Figure 4 shows the increased precursor potential of single cell preparations of pig ATZ crypts compared to rectal crypts. Figure 4(a) shows the growth of pig ATZ and rectal crypts over 4 weeks. Figure 4(b) shows the increased plating efficiency of crypt organoids derived from ATZ crypt-derived cells over 2 months compared to rectal crypt-derived cells. After 1 month, cells are isolated and re-plated.

[0036] [Figure 5A] Figure 5 shows the protein expression of stem cell and progenitor cell markers in freshly isolated crypts and crypt organoids derived from pigs and Crohn's disease patients. (a) CD117 expression in single cells derived from crypts from pig GI tissue, (b) CD34 expression in single cells derived from fresh crypts from pig GI tissue, (c) double-labeling of CD34 and CD117 expression in fresh pig ATZ crypt cells by flow cytometry, (d) stem cell and progenitor marker expression in pig crypt organoids (small intestine is shown in the left bar, rectum is shown in the middle bar, and ATZ is shown in the right bar), (e) expression of the indicated developmental lineage markers in fresh pig ATZ crypt-derived cells, and (f) stem cell and progenitor marker expression in organoids derived from rectal crypts of a Crohn's disease patient. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above.

[0037] [Figure 6] Figure 6 shows mRNA profiling of freshly isolated crypts and crypt organoids from porcine ATZ. (a) shows mRNA profiling of freshly isolated porcine ATZ crypt cells, and (b) shows mRNA profiling of organoids derived from porcine ATZ crypt cells.

[0038] [Figure 7] Figure 7 shows flow cytometry analysis of stem and progenitor cell markers to evaluate growth media for promoting porcine ATZ crypt stem cell expansion at day 14. (a) shows CD117 expression, (b) shows Brachyury expression, and (c) shows SOX17 expression.

[0039] [Figure 8] Figure 8 shows that porcine ATZ crypt cells generate mature cells of the endodermal lineage. (a)-(d) show bright-field images of organoid development on days 1, 4, 7, and 12, respectively. (e)-(h) show DAPI staining and immunocytochemistry images of organoid nuclei. (e) shows lysosomal expression, (f) shows Ki67 expression, (g) shows Muc-2 expression, and (h) shows CK18 expression.

[0040] [Figure 9] Figure 9 shows flow cytometry analysis of endodermal lineage markers in ATZ organoids at days 0, 7, and 14.

[0041] [Figure 10]Figure 10 shows that porcine ATZ crypt cells generate mature cells of the mesodermal lineage. (a) to (d) show bright-field images of vascular development. (a) shows an undifferentiated single ATZ cell on day 0, (b) shows a single ATZ cell beginning to form clusters on day 3, (c) shows a vascular structure beginning to form on day 8, (d) shows a network of vascular structures forming on day 10, (e) shows DAPI-stained nuclei and immunocytochemistry of endothelial-like cells expressing CD31, and (f) shows DAPI-stained nuclei and immunocytochemistry of vascular-like cells expressing CD31.

[0042] [Figure 11] Figure 11 shows that porcine ATZ crypt cells generate mature cells of the mesodermal lineage, as detected by flow cytometry for mesodermal markers brachyury and CD31 in ATZ organoids on days 0, 7, and 14.

[0043] [Figure 12A] 12 shows that porcine ATZ crypt cells generate mature cells of ectodermal lineage. (a) Brightfield images of keratinocyte development from anal skin and ATZ crypts grown in KFSM, (b) immunocytochemistry for K14 and K15 markers in anal skin cultures, (c) immunocytochemistry for K14 and K15 markers in ATZ crypt cultures, and (d) flow cytometry analysis of ectodermal markers PAX6, NESTIN, and CK14 in ATZ-cultured cells on days 0, 7, and 14. [Figure 12B] Same as above. [Figure 12D] Same as above.

[0044] [Figure 13]13 shows brightfield images of single porcine small intestinal crypt cells in media promoting differentiation into three developmental lineages at days 0, 7, and 14. (a) shows culture in human endodermal medium (IntestiCult™), (b) shows culture in human mesodermal medium (MethoCult™), and (c) shows culture in ectodermal medium (KFSM).

[0045] [Figure 14A] 14 shows the results of an in vitro embryoid body assay examining the pluripotency of single porcine ATZ crypt cells. (a) Brightfield image of ATZ embryoid body development, (b) alkaline phosphatase staining of ATZ embryoid bodies, (c) immunostaining of ATZ embryoid bodies for expression of pluripotent stem cell markers SSEA4 and OCT4, and (d) immunostaining of ATZ embryoid bodies for expression of pluripotent stem cell markers SOX2 and TRA-1-60. DAPI was used for nuclear staining. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above.

[0046] [Figure 15] Figure 15 shows the isolation of porcine pancreatic ducts: (a) excised pancreas; (b) minced pancreatic tissue after enzymatic digestion; (c) isolated pancreatic ducts and single cells.

[0047] [Figure 16A]Figure 16 shows the development of porcine pancreatic duct organoids derived from ATZ and pancreatic tissue grown in PancreaCult™.For ATZ, (a) cell clusters are formed on day 4, filled ring structures are formed on day 7, and compact thick organoids are formed on day 14.For pancreas, (b) cystic organoid structures are formed on day 1 and day 2 after passage, and compact thick organoids are formed on day 4 after passage.Figure 16(c) shows the comparison between cystic organoids derived from ATZ crypts and cystic organoids derived from pancreatic ducts.Figure 16(d) shows the comparison between compact thick organoids derived from ATZ crypts and compact thick organoids derived from pancreatic ducts. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above.

[0048] [Figure 17] Figure 17 shows protein markers detected by immunofluorescence in organoids derived from porcine ATZ crypts grown in PancreaCult™ at days 4 and 7: (a) cytokeratin 19 and (b) insulin.

[0049] [Figure 18] Figure 18 shows the isolation of porcine hepatic stem cells: (a) excised liver; (b) minced liver tissue after enzymatic digestion; (c) isolated liver crypts and single cells.

[0050] [Figure 19A]Figure 19 shows the development of pig liver crypt organoids that are derived from ATZ and hepatic stem cells that are grown in HepatiCult™.For ATZ, (a) cell clusters are formed on the 4th day, filled ring structure is formed on the 7th day, and compact thick organoid is formed on the 14th day.For liver, (b) small cystic organoid structure is formed on the 3rd day, and larger cystic organoid structure is formed on the 1st day and 4th day after subculture. [Figure 19B] Same as above. [Figure 19C] Same as above.

[0051] [Figure 20A] Figure 20 shows protein markers detected by immunofluorescence in organoids derived from porcine ATZ crypts grown in HepatiCult™ at days 4 and 7. (a) Cytokeratin 19, (b) Albumin. [Figure 20B] Same as above. [Figure 20C] Same as above.

[0052] [Figure 21] Figure 21 shows the isolation of ATZ crypts from a patient with idiopathic perianal fistula: (a) a biopsy taken from anorectal tissue, (b) a brightfield image of freshly isolated ATZ crypts, and (c) a brightfield image of freshly isolated ATZ single cell suspension.

[0053] [Figure 22] Figure 22(a-b) shows mRNA expression by qPCR in freshly isolated ATZ crypts from two patients with idiopathic perianal fistula. Ct values ​​were normalized to the housekeeping gene GAPDH.

[0054] [Figure 23]Figure 23 shows the development of organoids in IntestiCult™ from single cell suspensions generated from ATZ crypts of a patient with idiopathic perianal fistula. Cell clusters formed on day 4, organized structures formed on day 7 (both at passage 0), and compact, thick organoids formed on days 7 and 14 at passage 1.

[0055] [Figure 24] FIG. 24 shows the formation of pancreatic organoids obtained by culturing ATZ cells from a patient with idiopathic perianal fistula in PancreaCult™.

[0056] [Figure 25A] Figure 25 shows an in vivo study of donor male pig ATZ cells implanted into the pancreas of a recipient sow. (a) A single-cell suspension of male ATZ donor cells was thawed and mixed with collagen paste. (b) Three sites were injected into the recipient pig pancreas. Figure 25(c) shows suture fixation of the white collagen paste. The animals were sacrificed after 5 weeks. Figure 25(d) shows the excised pancreas (arrow indicates the implantation site). [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above.

[0057] [Figure 26] Figure 26 shows histological staining of pig pancreatic tissue collected in the ATZ cell implantation study in Example 28. The top panel shows picrosirius red staining (dark gray) of collagen fibers, consistent with newly formed matrix. The bottom panel shows fluorescent in situ hybridization (FISH) of the SRY gene, demonstrating the presence of male ATZ donor cells in the recipient pig pancreas.

[0058] [Figure 27A]Figure 27 shows mRNA profiling by RNA-Seq of male donor ATZ crypt cells cultured in IntestiCult™, cells collected from ATZ / Permacol™ implants in the pancreas, and collected control pancreatic tissue from the ATZ implant study in Example 28. (a) mRNA enumeration of NKX6-1, INS, MUC-2 genes, (b) differential gene expression of pancreatic cell type-specific marker genes comparing cells in ATZ / Permacol™ implants with ATZ cells cultured in IntestiCult™, (c) differential gene expression of pancreatic cell type-specific marker genes in ATZ / Permacol™ implants versus control pancreatic tissue. [Figure 27B] Same as above. [Figure 27C] Same as above.

[0059] [Figure 28] Figure 28 shows the formation of the liver organoid that is derived from the ATZ crypt cell of idiopathic patients and that is cultured in HepatiCult™.Ring is formed on the 4th day, and larger cystic organoid is formed on the 2nd and 6th days after passage, and finally, by the 14th day of passage 1, the organoid is formed with full, compact, and thick.

[0060] [Figure 29A] Figure 29 shows an in vivo study in which donor boar ATZ crypt cells were implanted into the liver of a recipient sow. (a) Recipient pig liver was injected into three sites. After five weeks, the animals were sacrificed. (b) The liver was excised, and (c) sections were placed in cassettes and fixed. The implantation site was still visible in the excised tissue (indicated by an arrow in the image). [Figure 29B] Same as above. [Figure 29C] Same as above.

[0061] [Figure 30A]Figure 30 shows histological staining of porcine liver tissue collected in the ATZ implantation study in Example 32. (a) A section of liver in which a complex structure of newly formed large and small vasculatures or ducts was visualized by staining: hematoxylin and eosin (top left panel), collagen fiber staining detected by picrosirius red (dark gray) in the top right panel, and albumin (light gray) in the bottom left panel and in the insert of the bottom right panel.

[0062] [Figure 30B] Liver sections stained with hematoxylin and eosin and picrosirius red in the top panel. In the inserts of the bottom left and right panels, clusters of albumin-producing cells were detected within the ATZ / Permacol™ embedment.

[0063] [Figure 30C] Figure 30(c) shows a liver section distal to the ATZ / Permacol™ implantation site stained with hematoxylin and eosin, in which regenerating-appearing liver tissue is identified in the top panel, and selected regions of interest are highlighted in the bottom panel.

[0064] [Figure 30D] Figure 30(d) shows FISH staining of the SRY male gene (light grey), with the selected region of interest highlighted in the bottom panel.

[0065] [Figure 31A]Figure 31 shows mRNA profiling by RNA-Seq of male donor ATZ crypt cells cultured in IntestiCult™, cells collected from ATZ / collagen implants in the liver, and collected control liver tissue from the ATZ implant study in Example 32. (a) Normalized mRNA counts for SOX9, ALB, and MUC-2 genes; (b) Differential gene expression of hepatic cell type-specific marker genes comparing cells collected from ATZ / Permacol™ in the liver with ATZ cells cultured in IntestiCult™; (c) Differential gene expression of hepatic cell type-specific marker genes comparing cells collected from ATZ / Permacol™ in the liver with control liver tissue. [Figure 31B] Same as above. [Figure 31C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description definition As used herein, the following terms and phrases shall have the following meanings: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0067] As used herein, the term "gastrointestinal mucosa" or "gastrointestinal mucosal epithelium" refers to the epithelial mucosal layer of the gastrointestinal tract, including the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus. "Gastrointestinal mucosa" or "gastrointestinal mucosal epithelium" encompasses the mucosa or mucosal epithelium of the oral cavity, pharynx, esophagus, stomach, intestine, rectum, and anus.

[0068] As used herein, the term "anorectal transition zone" or "ATZ" refers to the intestinal epithelium sandwiched between the continuous squamous epithelium of the anal skin below and the continuous rectal columnar epithelium above at the dentate (or pectinate) line. The dentate line is the junction of the upper and lower anal canals.

[0069] Many differences exist between these two regions, including embryological origin, innervation, venous and arterial supply, and lymphatic supply. Above the dentate line, the epithelium of the anal canal is endodermal in origin and lined by simple columnar epithelium. Below the dentate line, the epithelium of the anal canal is ectodermal in origin and lined primarily by stratified squamous epithelium. The epithelium of the ATZ is generally 1-4 mm wide and can be easily identified and biopsied by one skilled in the art (see, e.g., Figure 1).

[0070] As used herein, the term "anorectal transition zone cells" or "ATZ cells" refers to a mixed population of cells derived from the anorectal transition zone epithelial tissue, including crypts, submucosal glands, and other epithelial cells.

[0071] As used herein, the term "isolated ATZ cells" refers to a mixed population of cells physically isolated from the ATZ epithelium (e.g., from a biopsy sample). If necessary, the ATZ tissue can be physically minced or enzymatically digested to isolate the ATZ cells.

[0072] As used herein, the term "crypt cells" refers to cells of the crypts of Lieberkuhn, which are structures below the surface of the intestinal mucosal layer and contain stem cells that are responsible for the continuous renewal of the intestinal mucosa throughout life.

[0073] As used herein, "ATZ crypt cells" refers to crypt cells of the ATZ, including multipotent stem cells (i.e., cells that have the ability to self-renew by division and develop into the multiple specialized cell types present in a specific tissue or organ), progenitor cells, and mature crypt cells. Multipotent ATZ cells are stem cells that have the ability to differentiate into multiple somatic cell lineages, including endodermal cells (e.g., intestinal mucosa), mesodermal cells (e.g., blood vessels), and / or ectodermal cells (e.g., skin).

[0074] As used herein, the term "isolated ATZ crypt cells" refers to a mixed population of crypt-forming cells (e.g., multipotent stem cells, progenitor cells, fully differentiated or mature cells) that have been physically isolated (e.g., by dissection) from the ATZ epithelium. Alternatively, or in addition, the ATZ crypts can be physically, chemically, or enzymatically dissociated to further isolate the ATZ crypt cells.

[0075] As used herein, the term "stem cell" refers to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and can proliferate indefinitely to give rise to more identical stem cells (i.e., self-renew).

[0076] As used herein, "self-renewal" refers to the process by which stem cells divide to generate more stem cells, perpetuating the stem cell pool throughout life. Self-renewal is division that maintains an undifferentiated state. This requires cell cycle control and, in many cases, maintenance of pluripotency or multipotency, depending on the stem cell.

[0077] As used herein, the term "progenitor cell" refers to a stem cell that has the potential to differentiate into a single cell type or lineage, and the term "progenitor ATZ cell" refers to a progenitor cell derived from an ATZ crypt cell.

[0078] As used herein, the term "multipotent cells" refers to stem cells that have the potential to differentiate into at least two cell types or lineages, and the term "multipotent ATZ cells" refers to multipotent cells derived from ATZ crypt cells.

[0079] As used herein, the term "pluripotent cells" refers to stem cells that have the potential to differentiate into each of the three major cell groups, namely, ectoderm, mesoderm, and endoderm, and the term "pluripotent ATZ cells" refers to multipotent cells derived from ATZ crypt cells.

[0080] As used herein, the term "isolated ATZ stem cells" refers to a mixed population of stem cells (e.g., pluripotent stem cells, multipotent stem cells, and progenitor cells) derived from ATZ crypts that have been isolated (e.g., physically, chemically, or enzymatically dissociated) so that ATZ crypt cells are further isolated from the ATZ crypts. Alternatively, or in addition, ATZ stem cells can be numerically expanded by one or more rounds of replating / passaging compared to other ATZ cells due to their superior self-renewal properties.

[0081] As used herein, the terms "proliferation" and "proliferating" refer to an increase in cell number by mitosis.

[0082] As used herein, the term "differentiation" refers to the formation of cells that are more specialized and express markers known to be associated with cells that are closer to becoming terminally differentiated cells incapable of further division or differentiation.

[0083] For example, in the hematological context, differentiation can be seen in the production of functional cells of multiple cell lineages (e.g., erythrocytes, platelets, granulocytes, macrophages). The term "further" or "higher" differentiation refers to cells that are more specialized and closer to becoming terminally differentiated cells incapable of further division or differentiation than the cells from which the culture began.

[0084] As used herein, the term "terminally differentiated" refers to cells that are incapable of further differentiation and / or further division / proliferation or differentiation.

[0085] As used herein, the term "expanded," when referring to cells, means cells that have been increased in number by proliferation and differentiation in vitro.

[0086] As used herein, the term "minimal growth medium" or "MGM" refers to a cell culture medium that provides the nutrients necessary to maintain ATZ-derived cells without promoting differentiation of multipotent ATZ-derived stem cells to an extent that results in a reduction in the absolute number of multipotent ATZ-derived stem cells.

[0087] As used herein, the term "lineage-specific medium" or "LSM" refers to a cell culture medium that provides the nutrients and growth factors necessary for multipotent ATZ-derived stem cells to undergo differentiation toward cells of a specific lineage, resulting in a decrease in the absolute number of multipotent ATZ-derived stem cells. Lineage-specific differentiation can be partial differentiation (e.g., toward endodermal, mesodermal, or ectodermal precursors) or terminal differentiation (e.g., toward goblet cells, cardiac muscle, or neurons).

[0088] As used herein, the term "eATZ stem cells" refers to expanded populations of ATZ stem cells that can be generated in suspension or adherent 2D culture (e.g., individual cells) (e.g., clumps) or in 3D culture consisting of complex structures (e.g., organoids).

[0089] The expanded population of cells may retain the ability to differentiate into one or more cell types. In some embodiments, the eATZ cells retain at least one marker of ATZ stem cells selected from the group consisting of CD34, CD117 (KIT), CD184 (CXCR4), OCT4, NANOG, SOX17, Brachyury (TBXT), PAX6, and NESTIN.

[0090] Expanded populations of eATZ cells can be differentiated in LSM to generate various organoids for stem cell transplantation, including intestinal organoids or ATZ stem cell-derived pancreatic, liver, or lung organoids.

[0091] As used herein, the term " organoid " refers to the large number of cells that grow in culture, and form three-dimensional structure by self-organization.Organoid structure can be the simple solid mass of cells, hollow mass (for example, cystic organoid), tube or more complicated structure (for example, crypt-like or follicle-like structure).

[0092] As used herein, the term "cellular composition" refers to a preparation of cells, which preparation may include, in addition to cells, non-cellular components, such as cell culture medium, e.g., proteins, amino acids, nucleic acids, nucleotides, coenzymes, antioxidants, metals, etc. Additionally, a cellular composition may have components that do not affect the growth or viability of the cellular components, but are used to present the cells in a particular format, e.g., a polymeric matrix for encapsulation, or as a pharmaceutical preparation.

[0093] As used herein, the term "phenotype" refers to the observable or detectable characteristics of a cell, such as size, morphology, RNA expression, protein expression, or other property.

[0094] As used herein, the term "marker" refers to a biological molecule that can be used to identify the phenotype of a cell by its presence, absence, or concentration, or by its activity, inactivity, or level of activity.

[0095] As used herein, the term "exogenous biocompatible scaffold" refers to a three-dimensional porous, fibrous, or permeable biomaterial intended to provide physical or mechanical support while allowing diffusion or transport of fluids and gases, thereby enabling cell-cell interactions. Preferably, such a scaffold causes limited or minimal inflammation and toxicity. Optionally, such a scaffold is biodegradable. Examples of scaffolds include biological scaffolds (e.g., laminin- or collagen-based scaffolds) and synthetic scaffolds (e.g., non-biopolymers such as PGA, PLA, and PLGA). The scaffold may be in the physical form of a thread, sheet, paste, powder, or liquid. The scaffold can be used in vitro and in vivo.

[0096] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of good medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive or unacceptable toxicity, irritation, allergic response, inflammatory response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0097] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a subject compound from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0098] As used herein, the term "type 1 diabetes" refers to an autoimmune disease that occurs when insulin-producing beta cells are destroyed by the immune system.Insulin is a hormone that cells need to use blood glucose to obtain energy, and it helps regulate the glucose level in the bloodstream.If left untreated, diabetes can cause many complications, including the relatively rapid onset of diabetic ketoacidosis and non-ketotic hyperosmolar coma.Long-term complications include heart disease, stroke, kidney failure, foot ulcers, and eye damage.

[0099] As used herein, the term " type 2 diabetes " refers to the condition characterized by hyperglycemia, relative lack of insulin and insulin resistance.In most cases, symptoms develop slowly, and symptoms can include increased hunger, fatigue, and pain that does not heal.Long-term complications of hyperglycemia include heart disease, stroke, diabetic retinopathy that can lead to blindness, kidney failure, and poor blood flow to limbs that can lead to amputation.

[0100] As used herein, the term "chronic liver disease" refers to several liver diseases, including alcoholic liver disease, non-alcoholic steatohepatitis (NASH), or hepatitis C infection. These diseases can be associated with cirrhosis, a form of irreversible fibrous scarring.

[0101] As used herein, the term "inflammatory bowel disease" is used to describe two long-term conditions involving inflammation of the intestine: ulcerative colitis and Crohn's disease. Ulcerative colitis affects only the colon (large intestine). Crohn's disease can affect any part of the digestive system from the mouth to the anus.

[0102] As used herein, the term "pulmonary disease" refers to many disorders that affect the lungs, such as asthma, chronic obstructive pulmonary disease (COPD), infectious diseases (e.g., influenza, pneumonia, and tuberculosis), occupational lung diseases, and lung cancer.

[0103] As used herein, the "significant level" of stem cell marker can be determined by standard quantitative methods suitable for the marker (for example, quantitative detection of antibody or other ligands that bind to cell surface proteins; quantitative detection of mRNA expression by quantitative PCR; quantitative detection of enzyme catalytic products).For each method, the standard error of measurement or the standard background noise level can be determined.If the measured expression level is not statistically significant at the 5% level for the selected method, the marker is not expressed at a "significant level". Multipotent and progenitor ATZ cell and organoid compositions

[0104] Multipotent and / or progenitor ATZ cell as described herein can be isolated as the preparation of single cell (or multiple cells) from primary ATZ tissue, comprising isolated ATZ crypt cell.The ATZ cell as described herein, including multipotent and progenitor ATZ cell, can be isolated from the ATZ crypt obtained by biopsy, or can be obtained from the organoid structure that is differentiated from the ATZ crypt cell that is isolated ex vivo.In some embodiments, multipotent and progenitor ATZ cell is obtained from the organoid that is derived from single primary ATZ crypt cell or dissociated ATZ crypt cell.

[0105] For use in the treatment methods described herein, multipotent or progenitor ATZ cells are preferably autologous cells from a human patient or allogeneic cells from a human donor (preferably a histocompatible donor to reduce or avoid graft-versus-host or host-versus-graft immune reactivity). Alternatively, multipotent or progenitor ATZ cells may be derived from other mammalian species and can be modified by methods known in the art to reduce or eliminate alloreactivity (e.g., by gene editing to knock out MHC class I and / or class II genes and / or the β2-microglobulin gene).

[0106] In some embodiments, the multipotent or progenitor ATZ cells may be in vitro expanded ATZ (eATZ) cells.

[0107] In some embodiments, multipotent and progenitor ATZ cells, primary ATZ cells, isolated ATZ crypt cells and / or ATZ-derived organoids are combined with exogenous biocompatible scaffolds in vivo or in vitro, for example, synthetic scaffolds or biological scaffolds.In some embodiments, the scaffolds comprise laminin and / or collagen (for example, Permacol™ Paste, Medtronic PLC, Minneapolis, MN).In some embodiments, the scaffolds are Corning™ Matrigel™ Matrix (Corning, Inc., New York; Geltrex™ Growth Factor Basement Membrane Matrix, Thermo Fisher Scientific) or functional equivalents.

[0108] In some embodiments, the scaffold comprises a functionalized collagen scaffold or a functional equivalent.

[0109] The exogenous biocompatible scaffold may also comprise cell culture media components. In some embodiments, the scaffold comprises an endoderm lineage-specific medium (LSM). Endoderm lineage-specific medium (LSM) can be purchased from commercial vendors (e.g., IntestiCult™, Human Intestinal Protocol, Bio-Techne); pancreas (e.g., PancreaCult™); liver (HepatiCult™); lung (e.g., STEMdiff™ Branching Lung Organoid Kit, or PneumaCult™ Airway Organoid Kit, or PneumaCult™ Apical-Out Airway Organoid Medium, or PneumaCult™ Alveolar Organoid Medium, all from STEMCELL Technologies, Inc.; Human Lung Organoid Culture Protocols, Bio-Techne; 3dGRO™ Lung Organoid Branching Medium and Lung Organoid Maturation Medium, Merck).

[0110] In some embodiments, ATZ cells or ATZ crypt cells can be cultured in MGM. In some embodiments, ATZ cells or ATZ crypt cells can be cultured in a feeder-free medium (e.g., mTesR™, STEMCELL Technologies) that maintains human embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs) in an undifferentiated state. In some embodiments, the scaffold comprises such an ESC or iPSC feeder-free medium. In some embodiments, ATZ cells or ATZ crypt cells cultured in LSM have higher expression of mesodermal and endodermal lineage markers compared to ATZ cells or ATZ crypt cells cultured in such ESC or iPSC feeder-free medium. The matrix can also contain keratinocyte serum-free medium (KSFM) for differentiation. Such KSFM medium is commercially available from vendors (e.g., Gibco™ Keratinocyte-SFM Medium, Thermo Fisher Scientific; DermaCult™ Keratinocyte Expansion Medium, STEMCELL Technologies).

[0111] In another aspect, the present disclosure provides phenotypic characterization of extracellular and intracellular protein and mRNA expression levels of stem cell and progenitor cell markers in freshly isolated ATZ cells. ATZ cells can express at least one of CD34, CD117, and CD184 cell surface markers, as detected by flow cytometry, but do not express detectable levels of CD45. In some embodiments, isolated porcine ATZ cells express OCT4 and NANOG.

[0112] ATZ stem cells can also express markers of stem cells of three developmental lineages: endoderm (SOX17), mesoderm (TBXT), and ectoderm (PAX6 and NESTIN). Furthermore, freshly isolated ATZ cells can express markers of pluripotent stem cells (NANOG and OCT4).

[0113] In some embodiments, the ATZ cells and / or crypts express genes associated with endodermal, ectodermal, and mesodermal lineages.

[0114] In some embodiments, the ATZ cells and / or crypts express CD34. In some embodiments, the ATZ cells and / or crypts do not express detectable levels of CD45. In some embodiments, the ATZ cells and / or crypts express CD34 and do not express detectable levels of CD45.

[0115] In some embodiments, the ATZ cells and / or crypts express at least one marker selected from the group consisting of ALP, TBXT, BMP4, CD34, KIT, CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, NANOG, PAX6, SOX17, and OCT4. In some embodiments, the ATZ cells and / or crypts express at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen markers selected from the group consisting of ALP, TBXT, BMP4, CD34, KIT, CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, NANOG, PAX6, SOX17, and OCT4.

[0116] In some embodiments, the ATZ cells and / or crypts express at least one marker selected from the group consisting of ALP, TBXT, BMP4, CD34, KIT, CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, PAX6, and SOX17. In some embodiments, the ATZ cells and / or crypts express at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen markers selected from the group consisting of ALP, TBXT, BMP4, CD34, KIT, CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, PAX6, and SOX17. In some embodiments, the ATZ cells and / or crypts express the OCT4 or NANOG gene.

[0117] In some embodiments, the ATZ cells and / or crypts express CD117 (KIT) at higher levels than crypt cells of the small intestine, colon, and rectum, hi some embodiments, the ATZ cells and / or crypts express CD117 (KIT) at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold or more than crypt cells of the small intestine, colon, and rectum.

[0118] Aspects of the present disclosure provide the phenotypic characterization of the extracellular and intracellular protein and mRNA expression level of stem cell and progenitor cell markers in ATZ organoid under culture.In some embodiments, ATZ organoid expresses the gene related to endoderm, ectoderm and mesoderm lineage.In some embodiments, ATZ organoid expresses at least one protein marker selected from the group consisting of alkaline phosphatase, brachyury, CD34, CD117 (KIT), chromogranin, cytokeratin 18, CD184 (CXCR4), EpCAM, GD2, LGR5, lysozyme, mucin 2, Nestin and PAX6.In some embodiments, ATZ organoid expresses CD34.

[0119] In some embodiments, ATZ organoid does not express detectable level of CD45.In some embodiments, ATZ organoid expresses at least one gene selected from the group consisting of ALP, TBXT, BMP4, CD34, KIT, CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, NANOG, PAX6 and SOX17.

[0120] In some embodiments, ATZ organoid expresses at least one cell surface marker selected from the group consisting of CD31, CD34, CD117 (KIT), CXCR4 (CD184), CK14, CK15, CK18, GD2, SOX2, SSEA4 and TRA-1-60.In some embodiments, ATZ organoid expresses CD34.In some embodiments, ATZ organoid does not express CD45 at detectable level.In some embodiments, ATZ organoid expresses CD34 but does not express CD45 at detectable level.

[0121] In some embodiments, ATZ organoid expresses CD117 (KIT), CXCR4 (CD184) and / or GD2 at higher levels compared with small intestinal organoid and rectal organoid.In some embodiments, ATZ organoid expresses CD117 (KIT) at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times higher compared with small intestinal organoid and rectal organoid.In some embodiments, ATZ organoid expresses CXCR4 (CD184) at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times higher compared with small intestinal organoid and rectal organoid. In some embodiments, ATZ organoid expresses GD2 at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times higher than small intestine organoid and rectum organoid.In some embodiments, in ATZ organoid, SOX17 expression is reduced compared with ATZ crypt cell.

[0122] In some embodiments, in ATZ organoid, TBXT expression is reduced compared with ATZ crypt cell.In some embodiments, in ATZ organoid, CD31 expression is reduced compared with ATZ crypt cell.In some embodiments, in ATZ organoid, PAX6 expression is reduced compared with ATZ crypt cell.In some embodiments, in ATZ organoid, LGR5 is increased compared with ATZ crypt cell.In some embodiments, in ATZ organoid, CD117 or CXCR4 is increased compared with ATZ crypt cell.In some embodiments, in ATZ organoid, K14 is increased compared with ATZ crypt cell.In some embodiments, in ATZ organoid, CD117, CXCR4, LGR5 or K14 expression is increased at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times or 7 times compared with ATZ crypt cell. In some embodiments, the expression of CD31, PAX6, SOX17, or TBXT is reduced by 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, or 7-fold in the ATZ organoids compared to ATZ crypt cells.

[0123] Protein and nucleic acid expression can be determined by methods known to those skilled in the art, including but not limited to polymerase chain reaction (PCR), RTPCR, q-RT-PCR, flow cytometry (combined with binding agents (e.g., labeled antibodies or ligands for cell surface markers)), SDS-PAGE, mass spectrometry, immunoblotting (Western blotting), immunofluorescence microscopy, fluorescence in situ hybridization, or any other techniques known in the art.

[0124] In some embodiments, ATZ organoids comprise proliferating cells, secretory cells and cytokeratin.In some embodiments, ATZ organoids can secrete lysozyme. Methods for isolating and generating populations of multipotent and progenitor ATZ cells

[0125] In another aspect, a method for isolating and producing multipotent and / or progenitor ATZ cells is provided herein. ATZ cells can be produced by obtaining anorectal transition zone tissue from a mammalian subject (e.g., human, pig) either via biopsy or excision, washing the tissue with serum-free tissue culture medium containing antibiotics and / or antimycotics as needed, mincing the tissue, and enzymatically treating or digesting the tissue with collagenase. Digestion can be stopped, for example, by adding a protein such as albumin. Crypts can be isolated by thoroughly stirring or shaking the tissue digest to release the crypts. The medium containing the crypts can be passed through a tissue strainer to remove large debris and centrifuged to sediment or pellet the crypts. The crypts can then be resuspended in fresh medium to obtain a population of multipotent and / or progenitor ATZ cells.

[0126] The resuspended crypts can be counted under a microscope. Single-cell preparations from the crypts can be generated by physical disruption using a needle / syringe and / or treatment with a mild enzymatic cell dissociation reagent to obtain a population of multipotent and / or progenitor ATZ cells. These cells can then be embedded in an in vitro matrix scaffold (e.g., as described above) and cultured in a differentiation medium to generate organoids.

[0127] In some embodiments, the minced ATZ tissue is enzymatically treated to release intestinal crypts and submucosal glands. In some embodiments, the anorectal transition zone tissue is derived from a healthy human donor. In some embodiments, the anorectal transition zone tissue is derived from a diseased human donor.

[0128] The released intestinal crypts or multipotent and / or progenitor ATZ cells can then be placed in a growth medium or scaffold (e.g., laminin, collagen, Permacol™, Corning® Matrigel® Matrix) and then differentiated into organoids. Any suitable matrix and growth medium known in the art can be used. In some embodiments, the crypts are placed in a synthetic scaffold. In some embodiments, the scaffold is not functionalized. In some embodiments, the scaffold is functionalized. Alternatively, the crypts can be further dissociated into single ATZ cells.

[0129] In some embodiments, the isolated ATZ-derived crypt cells have increased pluripotency and / or progenitor potential compared to cells derived from rectal crypts.

[0130] In another aspect, the present invention provides for maintaining in vitro ATZ stem cells (including multipotent and / or progenitor cells) by culturing crypts or single cells embedded in scaffolds (for example, Permacol™, Corning® Matrigel® Matrix) with minimal growth medium (MGM).By using MGM, it is possible to maintain a percentage (+ / -10%) of multipotent ATZ stem cells without promoting terminal differentiation of cells.To be clear, in the mixed culture of ATZ cells, including ATZ-derived crypt cells and / or ATZ-derived organoid cells, there are multipotent ATZ stem cells, ATZ progenitor cells, and partially differentiated or terminally differentiated ATZ cells, and some cells become more differentiated over time.However, if the proliferation rate of multipotent ATZ stem cells is equal to or exceeds the rate of differentiation (and loss of multipotency), ATZ stem cell culture can be maintained indefinitely. While the rate of proliferation of multipotent ATZ stem cells slightly exceeds the rate of differentiation (and loss of multipotency), ATZ stem cell cultures can nevertheless be maintained for several weeks, which is sufficient for the treatment methods described herein.

[0131] Preferably, ATZ organoid culture is periodically refreshed by dividing organoid and re-plating ATZ-derived organoid cell in MGM.By using suitable MGM, it is possible to maintain at least 5%, at least 10% or at least 15% multipotent ATZ stem cells after at least 3 weeks, 4 weeks, at least 5 weeks or at least 6 weeks of organoid culture.For example, the method described herein makes it possible to obtain at least 5% multipotent stem cells after 5 passages, 10% multipotent stem cells after 10 passages, and 10% multipotent stem cells after 20 passages.The percentage of multipotent ATZ stem cells is relative to total ATZ cells.

[0132] In another aspect, the present invention provides a population of cells large enough (e.g., 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 A method for in vitro expansion of ATZ stem cells (including multipotent and / or progenitor cells) to generate ATZ organoids (cells) is provided. The method may include repeatedly passage the ATZ organoids in MGM. Specifically, during expansion, the absolute number of multipotent ATZ-derived stem cells may increase, but the percentage of multipotent ATZ-derived stem cells decreases. For transplantation purposes into damaged or dysfunctional tissues, the absolute number of cells is more relevant in many applications. The expansion method may further include confirming that the collected cells maintain the desired phenotypic characteristics, as determined, for example, by the protein and mRNA expression levels of cell surface and intracellular stem cell markers and replating efficiency. Once the characteristics of ATZ cells for a cell source have been verified, biomarkers such as cell surface markers (e.g., CD34, CD117, and / or CD184) can be used to determine the appropriate cell number for stem cell therapy.

[0133] Examples of commercially available MGM include mTesR™ (STEMCELL Technologies, Inc., Vancouver, CA) and StemFit™ Feeder-Free Stem Cell Culture Media (AMSBIO, Abingdon, UK).In addition, because the rate of proliferation of multipotent ATZ stem cells can equal or exceed the rate of differentiation (and loss of pluripotency), IntestiCult™; Human Intestinal Organoid Culture Protocol (Bio-Techne, Minneapolis, MN) can also be used as MGM at low concentrations, regardless of its name.

[0134] In some embodiments, the mixed culture of ATZ-derived stem cells and ATZ-derived organoids, comprising multipotent ATZ-derived stem cells, can be induced to differentiate towards endodermal tissue by culturing cell in endodermal lineage specific medium (LSM).The mixed population of cells obtained comprises a higher percentage of ATZ-derived endodermal progenitor cells, and can be used for the treatment methods described herein.

[0135] Examples of commercially available endoderm LSM include PancreaCult™ (pancreas); HepatiCult™ (liver); STEMdiff™ Branching Lung Organoid Kit, or PneumaCult™ Airway Organoid Kit, or PneumaCult™ Apical-Out Airway Organoid Medium, or PneumaCult™ Alveolar Organoid Medium, Human Lung Organoid Culture Protocols, Bio-Techne; 3dGRO™ Lung Organoid Branching Medium, 3dGRO™ and Lung Organoid Maturation Medium, Merck (lung).In addition, because the rate of differentiation (and loss of pluripotency) can exceed the rate of proliferation of multipotent ATZ stem cells, IntestiCult™; Human Intestinal Organoid Culture Protocol (intestine) can also be used as LSM at high concentration.

[0136] The present disclosure provides for the cryopreservation and thawing of ATZ cells after collecting ATZ crypts, single cells and / or organoid culture, by resuspending centrifuged cells in serum-free DMSO-containing freezing medium, and gradually lowering the temperature to -80 ° C or less.To re-establish organoid culture, cryopreserved ATZ organoids or single cells are quickly thawed in a 37 ° C water bath, resuspended in pre-warmed tissue culture medium, centrifuged, resuspended in fresh medium, and the freezing medium is removed.In some embodiments, the composition further comprises a cryopreservation medium.In some embodiments, ATZ or eATZ composition is cryopreserved.

[0137] In some embodiments, ATZ-derived endodermal organoids (e.g., intestine, pancreas, liver, lung) are generated from biopsies obtained from patients with tissue or organ dysfunction. Treatment method

[0138] In some embodiments, ATZ crypts are cultured ex vivo to produce eATZ cells for use in methods of treating tissues and organs of endodermal origin. In some embodiments, eATZ cells can be cryopreserved after expansion and then thawed prior to treatment.

[0139] In some embodiments, the pharmaceutically acceptable carrier comprises an exogenous biocompatible scaffold, such as a scaffold described herein above.

[0140] In another aspect, provided herein is a method for preparing a therapeutic or pharmaceutical composition (e.g., a cell composition disclosed herein), the method comprising: (a) collecting ATZ tissue from a patient or donor; (b) enzymatically digesting the ATZ tissue with an enzyme to prepare a cell suspension; (c) optionally combining at least a portion of the cell suspension with a cryopreservation medium and cryopreserving the cell suspension; and (d) combining the cell suspension of step (b) or, optionally, step (c) with a pharmaceutically acceptable carrier.

[0141] In one embodiment, the present disclosure provides a cell composition comprising adult allogeneic or autologous ATZ cells in a pharmaceutically acceptable carrier. The ATZ cells can be prepared by a method comprising: (a) harvesting ATZ tissue from an adult human or pig subject; (b) preparing a cell suspension by enzymatic digestion of the ATZ tissue in vitro; and (c) settling / pelleting the cells, then resuspending them in a freezing medium, and cryopreserving the ATZ cells, for example, in liquid nitrogen. Before use, the cryopreserved ATZ cells are combined with a pharmaceutically acceptable carrier, and the cell / carrier preparation is then used to treat damaged or dysfunctional endodermal tissues (e.g., pancreas, liver). The carrier can be used to provide support for the ATZ cells in endodermal tissues or organs.

[0142] Throughout this specification, when compositions are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is also intended that there are compositions of the present disclosure that consist essentially of or consist of the recited components, and that there are processes and methods according to the present disclosure that consist essentially of or consist of the recited processing steps.

[0143] In this application, when an element or component is said to be included in and / or selected from a list of listed elements or components, it is to be understood that the element or component can be any one of the listed elements or components, or that the element or component can be selected from a group consisting of two or more of the listed elements or components.

[0144] Furthermore, whether expressly or implicitly stated herein, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the present disclosure. For example, when a specific compound is referred to, unless otherwise understood from the context, that compound can be used in various embodiments of the compositions of the present disclosure and / or in the methods of the present disclosure. In other words, within the scope of this application, embodiments are described and depicted so as to enable a clear and concise application to be described and depicted, but it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present teachings and disclosure. For example, it will be understood that all features described and depicted herein may be applicable to all aspects of the present disclosure(s) described and depicted herein.

[0145] The phrase "at least one of" should be understood to include each of the listed objects following this phrase and various combinations of two or more of the listed objects individually, unless otherwise understood from context and usage. The phrase "and / or" in connection with three or more listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0146] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should generally be understood to be open-ended and non-limiting, e.g., not excluding additional, unrecited elements or steps, unless specifically stated otherwise or understood otherwise from the context.

[0147] When the term "about" is used before a quantitative value, the present disclosure also encompasses the specific quantitative value itself unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise specified or inferred.

[0148] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0149] The use of any and all examples or exemplary language herein, e.g., "such as" or "including," is intended merely to better illustrate the present disclosure and not to pose a limitation on the scope of the disclosure, except as claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure. [Example]

[0150] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way. Example 1 Isolation of intestinal crypts from anorectal tissue

[0151] This example describes a protocol for isolating intestinal crypts from anorectal tissue from pigs and from human subjects with Crohn's disease. I. Pig

[0152] Example colon-to-anus tissue samples (Figure 1(a)) from a healthy adult White Landrace pig were collected within 2 hours of sacrifice and collected in AIMV V medium containing antibiotic / antimycotic (Thermo Fisher Scientific, Waltham, Massachusetts) and processed immediately or, in some cases, after overnight storage at 4°C. The tissue was opened longitudinally and cleared of waste material.

[0153] The dentate line between the rectal mucosa and the anal skin was identified (Figure 1(b)). The tissue was 8-10 cm wide, 1-2 cm high, and pale in color. The epithelial layer of the dentate tissue (anorectal transition zone) was excised from the rectal mucosa to the anal skin (Figure 1(c)). Enzyme treatment of the minced anorectal transition zone tissue released intestinal crypts (Figure 1(d-g)) and structures consistent with submucosal glands (Figure 1(h-i)). II. Patients with Crohn's disease

[0154] From the rectal resection tissue (Fig. 2(a)), the anorectal transition zone was excised (Fig. 2(b)), minced, and enzymatically treated to release the crypts (Fig. 2(c-d)). Example 2 Generation of crypt-anorectal transition zone organoids

[0155] Crypt-anorectal transition zone organoids were generated from the porcine and human tissues of Example 1.

[0156] When intestinal crypts prepared in Example 1 were embedded in Corning® Matrigel® Matrix containing human IntestiCult™, they developed into fully branched organoids within 1-2 weeks. The morphology of porcine crypt organoids derived from the anorectal transition zone was indistinguishable from organoids derived from porcine small intestine, colon, and rectum tissue (Figure 3(a)). Similarly, in Crohn's disease patients, organoids derived from the anorectal transition zone were indistinguishable from organoids derived from the rectum (Figure 3(b)). Example 3 Enhanced progenitor potential of single-cell preparations of porcine ATZ crypt cells compared with rectal crypt cells

[0157] In this example, for purposes of describing and illustrating certain examples and embodiments of the present disclosure, we demonstrate that single-cell preparations of porcine ATZ crypt cells have increased progenitor potential compared to rectal crypt cells, as assessed by plating efficiency.

[0158] Plating efficiencies (organoid formation per plated cell) of single-cell preparations of porcine ATZ and rectal crypt cells were determined for inputs of 500, 1,000, and 2,000 viable crypt cells per well in 24-well flat-bottom suspension surface culture plates (Sarstedt, Nuembrecht, Germany) and monitored weekly for 4 weeks. IntestiCult™ was refreshed once at the end of week 2, without replacing the Corning® Matrigel® Matrix.

[0159] In both groups, only a small number of organoids were formed from the input 500 cells.

[0160] At week 1, the appearance (Figure 4(a)) and frequency (Figure 4(b)) of organoid formation were similar between ATZ- and rectum-derived organoids for cultures initiated with 1,000 or 2,000 cells. At week 2, rectum-derived organoid cultures contained more differentiated organoids (dense cell clumps), while ATZ-derived organoid cultures contained more undifferentiated, viable organoids (cystic organoid structures) (Figure 4(a)).

[0161] At weeks 3 and 4, only very few viable organoids were detected in cultures initiated with 1,000 and 2,000 cells: 3–4% for ATZ and 0–1% for rectum (Figure 4(b)).

[0162] After 4 weeks, cultures were harvested and restarted with 500, 1,000, or 2,000 viable cells to determine plating efficiency over an additional 4 weeks. The IntestiCult™ was refreshed at week 6 without replacing the Corning® Matrigel® Matrix. Plating efficiency was assessed at weeks 5, 6, and 7 (Figure 4(b)). The highest plating efficiency was observed from weeks 5 to 7 (18%–21%) when 500 ATZ-derived cells were replated. Plating efficiency peaked at week 6 (19% and 15%, respectively) when 1,000 and 2,000 ATZ-derived cells were replated, and then declined at week 7 (9% and 5%, respectively).

[0163] Plating efficiency for all replated rectum-derived cells was less than 2% over the 5-7 week period.

[0164] Taken together, these studies demonstrate that porcine ATZ crypt cells have increased progenitor potential compared with rectal crypt cells, as determined by plating efficiency, consistent with the existence of a long-term repopulation of the ATZ stem cell population. Example 4 Protein expression of embryonic lineage stem cell markers in freshly isolated ATZ crypts and ATZ-derived crypt organoids

[0165] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example shows protein expression, as detected by immunocytochemistry, of stem cell markers of embryonic lineages in freshly isolated porcine crypts and crypt organoids.

[0166] Freshly isolated porcine ATZ crypts. Single-cell preparations from freshly isolated porcine crypts from small intestine, colon, rectum, and ATZ tissues were examined for cell surface stem cell markers by flow cytometry. Expression of the stem cell and progenitor cell growth factor receptor KIT (CD117) was 3-5-fold higher in ATZ crypt cells compared to crypt cells from the small intestine, colon, and rectum (Figure 5(a)). Crypt cells from all of these tissues expressed the stem cell and progenitor cell marker CD34 to varying degrees, with the highest expression in ATZ crypt cells (Figure 5(b)).

[0167] In double-labeling experiments, 85% of ATZ crypt cells expressed CD34, 53% of cells expressed CD117, 47% of cells co-expressed CD34 and CD117, and among CD117-expressing cells, 88% co-expressed CD34 (Fig. 5(c)).

[0168] Porcine ATZ crypt organoids. Single-cell preparations of fully differentiated porcine crypt organoids derived from small intestine, rectum, and ATZ cells after 10 days of culture were examined for cell surface stem cell markers by flow cytometry. Expression of CD117, CD184, and GD2 was increased more than threefold in ATZ-derived organoids compared to SI- and rectum-derived organoids. The hematopoietic marker CD45 was not detected in any crypt organoids (Figure 5(d)).

[0169] Because KIT and CXCR4 are markers of definitive endoderm and GD2 is a marker of primitive mesoderm, we further investigated the lineage origin of the newly isolated ATZ crypt cells. Intracellular protein markers confirmed the expression of transcription factors associated with the endodermal lineage (SOX17), mesodermal lineage (TBXT), and ectodermal lineage (PAX6 and NESTIN). However, the expression of transcription factors associated with pluripotency (OCT4 and NANOG) was not detected by flow cytometry (Figure 5(e)).

[0170] Taken together, these results indicate that porcine ATZ crypt cell populations express stem cell and progenitor protein markers for all three developmental lineages, and that endodermal and mesodermal markers were maintained in ATZ crypt organoids. Example 5 mRNA expression profiling of porcine ATZ crypt cells

[0171] In this example, mRNA expression profiling of porcine ATZ crypt cells is presented for the purposes of describing and illustrating certain examples and embodiments of the present disclosure.

[0172] Fresh porcine ATZ crypt cells. Transcriptional profiling of fresh porcine ATZ crypt cells confirmed basal expression of markers of mature epithelial cells (EPCAM, LYZ, MUC2, CHGA, CK18); stem and progenitor cell markers (CD34, LGR5); and markers of developmental stem cells of endodermal lineage (SOX17), mesodermal lineage (TBXT), and ectodermal lineage (PAX6); as well as pluripotent stem cell markers (NANOG, OCT4A) (Figure 6(a)). Although alkaline phosphatase (ALP) mRNA expression was not detected, this enzyme was detected by immunocytochemistry.

[0173] Porcine ATZ-derived crypt organoids. Transcriptional profiling of ATZ-derived crypt cells cultured in IntestiCult™ preserved mRNA expression for markers of stem cells across all three developmental lineages (Figure 6(b)). Lower levels of mRNA expression were detected for markers of mature epithelial cells compared to fresh ATZ cells. mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0174] Taken together, transcriptional profiling of fresh ATZ crypts and organoids confirmed the presence of markers of stem cells from all three developmental lineages. Example 6 Culture medium for maintaining and expanding multipotent porcine ATZ stem cells

[0175] This example demonstrates in vitro culture conditions for maintaining and expanding multipotent ATZ cells for the purposes of describing and illustrating certain examples and embodiments of the present disclosure.

[0176] Freshly isolated single porcine ATZ crypt cells were cultured for 14 days in either (1) feeder-free medium (mTeSR™) that maintains human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in an undifferentiated state or (2) human intestinal IntestiCult™.

[0177] Protein expression levels of multipotent stem cell markers described in Example 4 were assessed by flow cytometry before culture (day 0) and after 14 days of culture. The stem cell and progenitor marker CD117 was expressed in 14% of ATZ crypt cells before culture (day 0), increasing to 35% in mTeSR™ and 28% in human IntestiCult™ by day 14 (Figure 7(a)).

[0178] An intracellular marker for mesoderm (Brachyury) was expressed in 97% of ATZ crypt cells before culture (day 0), and by day 14 this expression level was maintained at 99% in mTeSR™ and 81% in human IntestiCult™ (Figure 7(b)).

[0179] SOX17, an intracellular marker for endoderm, was expressed in 34% of ATZ crypt cells before culture (day 0), and by day 14, in less than 1% in mTeSR™ medium and 49% in human IntestiCult™ (Figure 7(c)).

[0180] These results indicate that IntestiCult™ may be preferable for maintaining ATZ crypt cells that express both endodermal and mesodermal lineage markers. Example 7 In vitro differentiation of porcine ATZ cell-derived crypts into all three developmental lineages

[0181] In this example, for purposes of describing and illustrating certain examples and embodiments of the present disclosure, we demonstrate that porcine ATZ-derived crypts differentiate in vitro into all three developmental lineages (endoderm, mesoderm, and ectoderm).

[0182] The expression of protein and mRNA markers in all three developmental lineages of fresh porcine ATZ crypt stem cells in Examples 4 and 5, respectively, led to studies to determine whether freshly isolated ATZ crypt cells have the capacity to generate mature cells of the endodermal lineage (intestinal mucosa), mesodermal lineage (blood vessels), and ectodermal lineage (keratinocytes). Example 7.1 Endodermal differentiation potential of fresh porcine ATZ crypts

[0183] Fresh single-cell preparations of ATZ crypts were embedded in Corning® Matrigel® Matrix containing human IntestiCult™ and cultured for 2 weeks to generate organoids, after which protein expression levels of mature endoderm cell markers in the intestinal mucosa were assessed. Immunostaining of intact organoids was performed to visualize the expression of lysozyme, proliferating cells (KI67), secretory cells (MUC2), and cytokeratin 18 (CK18), which are expressed in the intestinal epithelial monolayer (Figure 8(a-h)).

[0184] Flow cytometry analysis of fresh ATZ crypt cells showed that SOX17 was reduced from 52% on day 0 to 34% on day 14, and that KIT (CD117) and CXCR4 (CD184) increased two-fold during culture (Figure 9). Expression of the intestinal stem cell marker LGR5 increased from 3% on day 0 to 22% on day 14.

[0185] Taken together, these results confirm that fresh ATZ crypts have the capacity to give rise to mature cell types derived from the endodermal lineage. Example 7.2 Mesodermal differentiation potential of porcine ATZ crypts

[0186] Fresh single-cell preparations of ATZ crypts were cultured in MethoCult™ for two weeks, after which protein expression levels of mature mesodermal cell markers were assessed to test their ability to give rise to hematopoietic lineage progeny. No hematopoietic colonies were formed in the non-adherent methylcellulose layer.

[0187] However, single ATZ crypt-derived cells (Figure 10(a)) formed clusters in the adherens layer on day 3 (Figure 10(b)) and initiated and formed a network of vessel-like tubular structures on day 10 (Figure 10(c-d)). Immunostaining for the endothelial cell marker CD31 (PECAM) was detected during the initiation and formation of the network of vessel-like tubular structures (Figure 10(e-f)). Flow cytometry analysis of fresh ATZ cells showed that brachyury expression decreased from 67% on day 0 to 23% on day 7 and further to 7% on day 14, while CD31 expression increased from 5% on day 0 to 19% on day 7 and further to 37% on day 14 (Figure 11).

[0188] Taken together, these results confirm that fresh ATZ crypts have the capacity to give rise to mature cell types derived from the mesodermal lineage. Example 7.3 Ectoderm differentiation potential of porcine ATZ crypts

[0189] Protein expression levels of mature ectodermal cell markers were assessed after 2 weeks of culture of fresh ATZ crypt and anal skin single-cell preparations in keratinocyte differentiation medium (KSFM, Sigma-Aldrich, St. Louis, Missouri). Cell clusters appeared on day 3, and by day 10, a cobblestone-like adherent layer with keratinocyte morphology was formed in anal skin and ATZ cell cultures (Figure 12(a)). Coexpression of keratinocyte markers CK14 and CK15 was detected by immunostaining in anal skin adherent culture cells (Figure 12(b)) and ATZ adherent culture cells (Figure 12(c)).

[0190] Flow cytometry analysis of ATZ-cultured adherent cells showed that expression of PAX6, an ectodermal lineage marker, decreased from 18% on day 0 to 6% on day 7 and 5% on day 14 (Fig. 12(d)). CK14 expression in ATZ-cultured adherent cells increased over time from 3% on day 0 to 18% on day 7 and 42% on day 14 (Fig. 12(d)).

[0191] Taken together, these results confirm that fresh ATZ crypts have the capacity to give rise to mature cell types derived from the ectodermal lineage. Example 8 Absence of multipotent stem cells of all three developmental lineages in crypts of the porcine small intestine

[0192] In this example, for purposes of describing and illustrating certain examples and embodiments of the present disclosure, the same method as described in Example 7 for porcine ATZ crypts is used to demonstrate that porcine small intestinal crypts in vitro have the potential to give rise to mature cells of the endodermal lineage, but not to mature cells of the mesodermal and ectodermal lineages, as expected.

[0193] Single crypt cells from the small intestine were cultured for 2 weeks to promote endoderm (Human IntestiCult™), mesoderm (MethoCult™), and ectoderm differentiation (KSFM, Sigma-Aldrich).

[0194] As expected, organoids (IntestiCult™) were generated from small intestinal crypt cells (Figure 13(a)). However, when cultured in MethoCult™ (Figure 13(b)) or KFSM (Figure 13(c)), no small intestinal crypt cell growth was detected on either day 7 or day 14.

[0195] Taken together, these results are consistent with the absence of multipotent stem cells in porcine small intestinal crypts that can give rise to cell types of all three developmental lineages, using the assays demonstrated for ATZ crypt cells. Example 9 In vitro embryoid body assay to assess pluripotency of porcine ATZ crypt cells

[0196] In this example, for purposes of describing and illustrating certain examples and embodiments of the present disclosure, we demonstrate that single-cell preparations of fresh ATZ crypts cultured in feeder-free mTeSR™ medium can promote embryoid body formation.

[0197] When fresh ATZ crypt single cells were plated at high density, a cobblestone-like adherent layer developed by day 3, colonies of undifferentiated adherent cells emerged by day 7, and differentiated adherent cells emerged and migrated away from the undifferentiated cell colonies (Figure 14(a)).

[0198] Visualization with alkaline phosphatase staining confirmed stem cell-like characteristics consistent with embryoid bodies (Figure 14(b)). Furthermore, pluripotent stem cell markers OCT4 and SSEA4 (Figure 14(c)), as well as SOX2 and TRA-1-60 (Figure 14(d)), were detected by immunocytochemistry on day 5.

[0199] Taken together, the results of the in vitro embryoid body assay are consistent with ATZ crypt cells exhibiting pluripotent stem cell-like properties. Example 10 Isolation of crypts from porcine pancreatic tissue

[0200] This example describes a protocol for isolating ducts from pancreatic tissue.

[0201] An example of tissue from the pancreas of a healthy adult white Landcross pig was collected within 2 hours of sacrifice and placed in AIMV medium (Thermo Fisher Scientific) containing antibiotic / antimycotic (AA) and stored overnight at 4°C before processing (Figure 15(a)). The tissue was diced into 1 cm pieces and transferred to a 50 ml conical tube containing PBS + AA + nystatin solution (Figure 15(b)). The tissue was washed repeatedly until the PBS solution was particle-free. The ducts were released by enzymatic treatment of the pancreatic tissue (Figure 15(c)). Example 11 Establishment and maintenance of organoids derived from porcine ATZ and pancreatic tissue cultured in PancreaCult™

[0202] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how organoids derived from porcine ATZ crypts in Example 1 and pancreatic ducts in Example 10 were cultured in PancreaCult™ to promote the development and maintenance of pancreatic duct organoids.

[0203] The ATZ and pancreatic crypts were separately embedded in Corning® Matrigel® Matrix containing PancreaCult™.

[0204] Small structures formed from ATZ crypts on day 4, filled ring structures formed on day 7, and organoids developed on day 14 (Figure 16(a)). Organoid structures formed from the pancreatic ducts on day 3, and on days 2 and 4 after passaging (Figure 16(b)).

[0205] Although the rates of growth and development differed between ATZ and pancreatic organoids cultured in PancreaCult™, the outer edges and differentiated inner portions of the two organoid structures appeared similar (Figure 16(c)).

[0206] Taken together, these experimental results demonstrate that ATZ crypt cells differentiated into pancreatic organoids in vitro. Example 12 Protein expression of markers during the development of pancreatic organoids derived from porcine ATZ crypts

[0207] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example shows the protein expression of markers detected by immunofluorescence during the development of porcine ATZ-derived pancreatic organoids generated in Example 11.

[0208] Figure 17 shows markers detected by immunofluorescence in ATZ crypt-derived organoids grown in PancreaCult™ on days 4 and 7: (a) cytokeratin 19 (CK19), (b) insulin (INS). Example 13 mRNA expression profiling markers expressed during the development of porcine ATZ crypt-derived pancreatic organoids

[0209] This example shows the mRNA expression of markers detected by gradient qRT-PCR during the development of pancreatic organoids generated in Example 11 for the purpose of describing and illustrating certain examples and embodiments of the present disclosure.

[0210] Transcriptional profiling of organoids derived from porcine ATZ crypt cells cultured in PancreaCult™ revealed genes for master regulators of pancreatic stem and progenitor cell development as well as tissue-specific marker genes for acinar, alpha, beta, and epithelial cells (Table 1). [Table 1]

[0211] The results of transcriptional profiling of CK19 and INS of pancreatic organoids derived from ATZ crypt cells confirmed the results of immunofluorescence described in Example 12.

[0212] Taken together, the results of PCR and immunofluorescence experiments demonstrate that ATZ crypt cells differentiate in vitro into pancreatic cells capable of producing insulin. Example 14 Isolation of stem cells from porcine liver tissue

[0213] This example describes a protocol for isolating stem cells from liver tissue.

[0214] An example of tissue from the liver of a healthy adult white Landcross pig was collected within 2 hours of slaughter and placed in AIMV medium (Thermo Fisher Scientific) containing antibiotic / antimycotic (AA) and stored overnight at 4°C before processing (Figure 18(a)). The tissue was diced into 1 cm pieces and transferred to a 50 ml conical tube containing PBS + AA + nystatin solution (Figure 15(b)). The tissue was washed repeatedly until the PBS solution was particle-free. The liver tissue was treated with enzymes to release the crypts (Figure 18(c)). Example 15 Establishment and maintenance of organoids derived from porcine ATZ crypts and liver tissue cultured in HepatiCult™

[0215] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how organoids derived from porcine ATZ crypts in Example 1 and hepatic stem cells in Example 14 are cultured in HepatiCult™ to promote the development and maintenance of liver organoids.

[0216] ATZ crypts and hepatic stem cells were separately embedded in Corning® Matrigel® Matrix containing HepatiCult™.

[0217] Small ring structures formed from ATZ-derived crypts on day 4, filled ring structures formed on day 7, and organoids developed on day 14 (Figure 19(a)). Small cystic organoid structures formed from liver-derived crypts on day 3, and larger cystic organoid structures formed on days 1 and 4 after passaging (Figure 19(b)). Similar cystic organoid structures with unilateral budding outgrowth were observed for ATZ crypts and hepatic stem cells grown in HepatiCult™ (Figure 19(c)).

[0218] Taken together, these experimental results demonstrate that ATZ crypt cells differentiated into hepatocytes in vitro. Example 16 Protein expression of markers during the development of hepatic organoids derived from porcine ATZ crypts

[0219] This example shows the protein expression of markers detected by immunofluorescence during the development of porcine ATZ-derived liver organoids generated in Example 15 for the purpose of describing and illustrating certain examples and embodiments of the present disclosure.

[0220] Figure 20 shows protein markers detected by immunofluorescence in ATZ crypts and liver organoids grown in HepatiCult™: (a) FOXA2, (b) CK19, (c) ALB. Example 17 mRNA expression profiling markers expressed during the development of porcine ATZ crypt-derived hepatic organoids

[0221] In this example, for the purpose of describing and illustrating certain examples and embodiments of the present disclosure, the mRNA expression of markers detected by PCR during the development of liver organoids generated in Example 15 is shown.

[0222] Transcriptional profiling of organoids derived from porcine ATZ crypt cells cultured in HepatiCult™ revealed genes that are master regulators of hepatic stem and progenitor cell development: FOXA2, SOX9, EPCAM (Table 2).

[0223] The results of transcriptional profiling of FOXA2 in liver organoids derived from ATZ crypt cells confirm the results of immunofluorescence described in Example 15. [Table 2] Example 18 Isolation of stem cells from porcine lung tissue

[0224] This example describes a protocol for isolating stem cells from lung tissue.

[0225] Samples of lung tissue from healthy adult white Landcross pigs were collected within 2 hours of sacrifice, placed in AIMV medium (Thermo Fisher Scientific) containing antibiotic / antimycotic (AA), and stored overnight at 4°C before processing. Lung tissue was diced into 1 cm pieces and transferred to a 50 ml conical tube containing PBS + AA + nystatin solution. The tissue was washed repeatedly until the PBS solution was particle-free and then enzymatically treated to release single cells, including stem cells and progenitors. Example 19 Establishment and maintenance of organoids derived from porcine ATZ crypts and lung tissue cultured in pulmonary LSM

[0226] In this example, for the purpose of describing and illustrating certain examples and embodiments of the present disclosure, the porcine ATZ crypts isolated in Example 1 and the pulmonary stem cells (bronchoalveolar epithelial stem cells and basal cells of airway epithelium) in Example 18 are described as being cultured in lung LSM (e.g., STEMdiff™ Branching Lung Organoid Kit, or PneumaCult™ Airway Organoid Kit, or PneumaCult™ Apical-Out Airway Organoid Medium, or PneumaCult™ Alveolar Organoid Medium, all from STEMCELL Technologies, Inc.; Human Lung Organoid Culture Protocols, Bio-Techne; 3dGRO™ Lung Organoid Branching Medium 3dGRO™ and Lung Organoid Maturation Medium, Merck) to promote the development and maintenance of lung organoids.

[0227] ATZ crypts and pulmonary stem cells are separately embedded in Corning® Matrigel® Matrix containing lung LSM. Example 20 mRNA expression profiling markers expressed during the development of porcine ATZ crypt-derived lung organoids

[0228] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example shows the mRNA expression of markers detected by PCR during the development of lung organoids generated from pig ATZ crypts and lung tissue in Example 19.

[0229] The transcriptional profiling of the organoids derived from pig ATZ crypt cells that are cultured in lung LSM reveals that the genes of the master regulators of lung stem cell and progenitor cell development, and the genes of the proximal and distal branching airway epithelial structures express the group of markers EPCAM, MUC1, NKX2.1, P63, SOX2 or SOX9.When maintained in culture for more than 28 days, the expression level of the markers for more mature lung cells, such as ABCA3, SFTPB and SFTPC, increases. Example 21 Isolation of stem cells from porcine thyroid tissue

[0230] This example describes a protocol for isolating stem cells from thyroid tissue.

[0231] Samples of healthy adult white Landcross pig tissue from the thyroid gland were collected within 2 hours of slaughter, placed in AIMV medium (Thermo Fisher Scientific) containing antibiotic / antimycotic (AA), and stored overnight at 4°C before processing. The thyroid tissue was diced into 1 cm pieces and transferred to a 50 ml conical tube containing PBS + AA + nystatin solution. The tissue was washed repeatedly until the PBS solution was particle-free and then enzymatically treated to release single cells, including stem cells and progenitors. Example 22 Establishment and maintenance of organoids derived from porcine ATZ crypts and thyroid tissue cultured in thyroid LSM

[0232] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how the porcine ATZ crypts isolated in Example 1 and the thyroid stem cells in Example 22 are cultured in thyroid LSM (e.g., as described in the protocol "Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism", PNAS, 118(51): e1-11) to promote the development and maintenance of thyroid organoids.

[0233] ATZ crypts and thyroid stem cells are separately embedded in Corning® Matrigel® Matrix containing thyroid LSM. Example 23 mRNA expression profiling markers expressed during the development of thyroid organoids derived from porcine ATZ crypts

[0234] Transcriptional profiling of organoids derived from porcine ATZ crypt cells cultured in thyroid LSM reveals genes for master regulators of lung stem and progenitor cell development.

[0235] At day 28, elevated expression levels of markers of more mature thyrocytes (e.g., PAX8 and NKX2.1) are observed. Example 24 Isolation of intestinal crypts from anorectal tissue of patients with idiopathic perianal fistula

[0236] This example describes a protocol for isolating intestinal crypts from the anorectal tissue of human subjects with idiopathic perianal fistulas.

[0237] Biopsies from anorectal tissue of patients with idiopathic perianal fistula were collected within 2 hours after sacrifice in AIMV V medium containing antibiotic / antimycotic and processed immediately or, in some cases, after overnight storage at 4°C (Figure 21(a)). Minced ATZ tissue was enzymatically treated to release intestinal crypts (Figure 21(b)), and single cell preparations were prepared (Figure 21(c)). Example 25 qPCR mRNA expression profiling of freshly isolated ATZ crypt cells from patients with idiopathic perianal fistulas

[0238] This example shows mRNA expression profiling by qPCR of ATZ crypt cells from idiopathic perianal fistula patients for the purposes of describing and illustrating certain examples and embodiments of the present disclosure.

[0239] Transcriptional profiling of freshly isolated ATZ crypts from two patients with idiopathic perianal fistulas revealed the expression of markers of pluripotent stem cells (NANOG, OCT4A) and multipotent stem and progenitor cells (BMP4, KIT, CD34, CXCR4, LGR5), as well as a marker of endodermal developmental stem cells (SOX17) (Figure 22(a-b)).

[0240] Combined with the mRNA profiling of porcine ATZ crypts in Example 5, these results confirm that ATZ crypts contain populations of pluripotent and multipotent stem cells. Example 26 Generation of crypt-anorectal transition zone organoids from patients with idiopathic perianal fistula

[0241] Crypt ATZ organoids were generated from human tissue in Example 24.

[0242] Crypts or single cell preparations of ATZ tissue prepared in Example 24 were embedded in Corning® Matrigel® Matrix containing human IntestiCult™ and developed into fully formed organoids in 1-3 weeks ( FIG. 23 ). Cell clumps formed on day 4, organized structures formed on day 7 (both at passage 0), and compact, thick organoids formed on days 7 and 14 at passage 1. Example 27 Establishment and maintenance of organoids derived from ATZ tissue of patients with idiopathic perianal fistula cultured in PancreaCult™

[0243] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how organoids derived from ATZ crypts or single cell preparations from idiopathic perianal fistula patients in Example 24 were cultured in PancreaCult™ to promote the development and maintenance of pancreatic organoids.

[0244] ATZ crypts and single cells were embedded in Corning® Matrigel® Matrix containing PancreaCult™. After one passage, organized, complex organoids formed (Figure 24).

[0245] These results, together with the establishment and maintenance of pancreatic organoids from porcine ATZ crypts and single cells prepared in Example 11, confirm that ATZ crypts have the ability to differentiate into liver organoids in vitro. Example 28 Implantation and recovery of donor porcine ATZ crypt cells delivered as a collagen paste into recipient pig pancreases to test engraftment

[0246] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how single cell preparations of boar ATZ crypts from Example 1 were implanted into the pancreas of healthy sows to test for engraftment and cell expansion.

[0247] The cryopreserved single-cell preparation of boar crypts from Example 1 was thawed, resuspended in AIM V medium, centrifuged, and the cell pellet mixed with collagen paste (Permacol™) and placed in a syringe (Figure 25(a)). An Abbocath Catheter 14F was attached to the syringe containing the ATZ cells in Permacol™, mixed thoroughly, and injected into three locations in the pancreas (ATZ / Permacol™ implants) (Figure 25(b)); secured with sutures (Figure 25(c)). Five weeks later, the pigs were sacrificed, and the ATZ / Permacol implants and control pancreatic tissue were surgically removed (Figure 25(d)). The implant sites were still visible (indicated by arrows in the image). Tissues were sectioned; one half was placed in neutral buffered formalin (NBF) for histological examination, and the other half was placed in RNAlater reagent (Thermo Fisher) for mRNA profiling.

[0248] Pigs transplanted with ATZ cells showed no evidence of inflammation or graft rejection after five weeks. Example 29 Histological staining of pancreatic tissue recovered 5 weeks after implantation of donor pig ATZ crypts into recipient pig pancreases

[0249] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example demonstrates the presence of engraftment of male pig donor ATZ cells in the pancreas of a healthy pig after 5 weeks.

[0250] Histological staining of ATZ cell-embedded sections of the pancreas excised in Example 29 ( FIG. 26 ) shows collagen fiber staining detected by picrosirius red in the top panel (dark gray), consistent with the newly formed support matrix. The bottom panel shows fluorescent in situ hybridization (FISH) of the SRY gene, a sex-determining gene on the Y chromosome, confirmed by detection of male DNA by PCR (not shown), indicating the presence of migrating male ATZ donor cells in the recipient pig pancreas. Example 30 Molecular profiling of pancreatic tissue harvested 5 weeks after implantation of donor porcine ATZ crypt cells into recipient pig pancreases

[0251] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example demonstrates the presence of engraftment and expansion of boar donor ATZ crypt cells in the pancreas of a healthy pig after 5 weeks by molecular profiling.

[0252] mRNA profiling by RNA-Seq was performed on boar donor ATZ crypt cells cultured in IntestiCult™ in Example 28, cells collected 5 weeks after ATZ / Permacol™ implants in the pancreas of the ATZ implant study, and control pancreatic tissue.

[0253] Figure 27(a) shows that genes associated with pancreatic beta cell development (NKX6-1) and insulin production (INS) were detected in ATZ / Permacol™ implants and control pancreatic tissue, but not in ATZ donor cells cultured in IntestiCult™. As expected, expression of MUC-2, a gene encoding a mucin, was detected in ATZ donor cells cultured in IntestiCult™, with only low levels of MUC-2 detected in ATZ / Permacol™ implants and control pancreatic tissue.

[0254] Figure 27(b) shows differential gene expression profiling of human pancreatic cell type-specific marker genes (from Muraro MJ et al. (2016) "A Single-Cell Transcriptome Atlas of the Human Pancreas." Cell Syst. 3(4):385-394.e3) in porcine ATZ / Permacol™ implants compared to donor porcine ATZ cells cultured in IntestiCult™. These data show that ATZ / Permacol™ implants expressed relatively elevated mRNA levels of several pancreatic-associated cell type marker genes (acinar cells, beta cells, and delta and epsilon cells) and also expressed elevated levels of genes associated with tissue remodeling cells (endothelial and mesenchymal cells).

[0255] Figure 27(c) shows differential gene expression profiling of human pancreatic cell type-specific marker genes (from Muraro MJ et al. (2016) "A Single-Cell Transcriptome Atlas of the Human Pancreas." Cell Syst. 3(4):385-394.e3) in porcine ATZ / Permacol™ implants compared to pancreatic control tissue. These data indicate that ATZ / Permacol™ implants expressed relatively low mRNA levels of several pancreatic-related cells (acinar cells, alpha cells, beta cells, and gamma cells) and elevated levels of genes associated with tissue remodeling cells (endothelial and mesenchymal cells), as expected from the initial engraftment of ATZ donor cells.

[0256] Taken together, these mRNA profiling results demonstrated that boar donor ATZ cells, which differentiate into enterocytes in vitro, exhibited the ability to form functional pancreatic cells in vivo, thus supporting the multipotency of ATZ cells, and that ATZ / Permacol™ implants remodeled the liver. Example 31 Establishment and maintenance of organoids derived from ATZ crypts of patients with idiopathic perianal fistula cultured in HepatiCult™

[0257] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example describes how organoids derived from ATZ crypts or single cell preparations from idiopathic perianal fistula patients in Example 24 were cultured in HepatiCult™ to promote the development and maintenance of liver organoids.

[0258] ATZ crypts or single cells were embedded in Corning® Matrigel® Matrix containing HepatiCult™. Ring structures formed at day 4, larger cystic organoids formed at days 2 and 6 after passage 1, and filled, compact, thick organoids formed by day 14 at passage 1 (Figure 28(a-b)).

[0259] These results, together with the establishment and maintenance of liver organoids from porcine ATZ crypts and single cells prepared in Example 15, confirm that ATZ crypts have the ability to differentiate into liver organoids in vitro. Example 32 Implantation and recovery of donor porcine ATZ crypt cells delivered as a collagen paste to recipient pig livers to test engraftment

[0260] This example describes how single cell preparations of boar ATZ crypts from Example 1 were implanted into the livers of healthy sows to test for engraftment and cell expansion, for the purposes of describing and illustrating certain examples and embodiments of the present disclosure.

[0261] The cryopreserved boar crypt single-cell preparation from Example 1 was thawed, resuspended in AIM V medium, centrifuged, and the cell pellet mixed with collagen paste (Permacol™) and placed in a syringe. A large-gauge needle was attached to the syringe containing the ATZ cells in Permacol™, mixed thoroughly, and injected into the pancreas at three locations (ATZ / Permacol™ implants); each injection site was sutured (not shown) (Figure 29(a)). Five weeks later, the pigs were sacrificed, and the ATZ / Permacol implants and liver control tissues were surgically removed (Figure 29(b)). The excised tissues were sectioned; one half was placed in neutral buffered formalin (NBF) for histological examination (Figure 29(c)), and the other half was placed in RNAlater reagent (Thermo Fisher) for mRNA profiling. The implantation site in the excised tissue was still visible (indicated by arrows in the image).

[0262] Pigs transplanted with ATZ cells showed no evidence of inflammation or graft rejection after five weeks. Example 33 Histological staining of liver tissue recovered 5 weeks after implantation of donor pig ATZ crypt cells into the recipient pig liver

[0263] For purposes of describing and illustrating certain examples and embodiments of the present disclosure, this example demonstrates the presence of engraftment of boar donor ATZ cells in the liver of a healthy pig after 5 weeks.

[0264] Histological staining of ATZ / Permacol™ embedded sections of excised liver in Example 32.

[0265] Figure 30(a) shows a liver section in which a complex structure of newly formed large and small vasculatures or tubes is visualized by staining: hematoxylin and eosin (top left panel), collagen fiber staining detected by picrosirius red (dark gray) in the top right panel, and albumin (light gray) in the inserts of the bottom left and bottom right panels.

[0266] Figure 30(b) shows liver sections stained with hematoxylin and eosin and picrosirius red in the top panel. In the inserts of the bottom left and right panels, clusters of albumin-producing cells were detected within the ATZ / Permacol™ embedment.

[0267] Figure 30(c) shows a liver section distal to the ATZ / Permacol™ implantation site stained with hematoxylin and eosin, identifying regenerating-appearing liver tissue in the top panel, with selected regions of interest highlighted in the bottom panel.

[0268] Figure 30(d) shows FISH staining for the SRY male gene (light gray), with the selected region of interest highlighted in the bottom panel. This demonstrates the ability of male ATZ donor cells to migrate from the ATZ / Permacol™ implantation site and give rise to new tissue, as shown in the upper right corner of the top panel. Furthermore, FISH SRY-stained cells demonstrate that the distinct cellular architecture of the new tissue, highlighted in Figure 30(c), is composed of the same cell types as the adjacent established liver.

[0269] Taken together, Figure 30(d) demonstrates that porcine donor ATZ crypt cells have a robust ability to engraft, migrate, and regenerate liver tissue in recipient sows. Example 34 Molecular profiling of pancreatic tissue harvested 5 weeks after implantation of donor porcine ATZ crypt cells into recipient pig livers

[0270] In this example, for purposes of describing and illustrating certain examples and embodiments of the present disclosure, molecular profiling demonstrates the presence of engraftment and generation of new liver tissue after 5 weeks of boar donor ATZ crypt cells in healthy pigs.

[0271] mRNA profiling by RNA-Seq was performed on male donor ATZ crypt cells cultured in IntestiCult™ from the ATZ implant study in Example 32, cells collected 5 weeks after ATZ / Permacol™ implants in the liver, and control pancreatic tissue.

[0272] Figure 31(a) shows that genes associated with hepatic beta cell development (NKX6-1) and insulin production (INS) were detected in ATZ / Permacol™ implants and control pancreatic tissue, but not in ATZ donor crypt cells cultured in IntestiCult™. As expected, expression of MUC-2, a gene encoding a mucin, was detected in ATZ donor cells cultured in IntestiCult™, with only low levels of MUC-2 detected in ATZ / Permacol™ implants and control liver tissue.

[0273] Figure 31(b) shows differential gene expression profiling of human hepatic cell type-specific marker genes (from MacParland, SA, et al. (2018) "Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations." Nat Commun. 2018 Oct 22;9(1):4383) in porcine ATZ / Permacol™ implants in the liver compared to donor porcine ATZ crypt cells cultured in IntestiCult™. These data indicate that ATZ / Permacol™ implants expressed relatively elevated mRNA levels of liver-associated cell type marker genes.

[0274] Figure 31(c) shows differential gene expression profiling of human pancreatic cell type-specific marker genes (from MacParland, SA, et al. (2018) "Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations." Nat Commun. 2018 Oct 22;9(1):4383) in porcine ATZ / Permacol™ implants compared to liver control tissue. These data show that ATZ / Permacol™ implants expressed relatively low hepatocyte mRNA levels and elevated levels of genes associated with tissue remodeling cells (endothelial cells and stellate cells), as might be expected for early regeneration. equivalent

[0275] The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting on the invention described herein. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A cell composition for use in cell transplantation to treat damaged or dysfunctional endodermal tissue in a subject, comprising: A mixed population of ATZ cells comprising multipotent ATZ stem cells and endodermal progenitor ATZ cells in a pharmaceutically acceptable carrier or exogenous biocompatible scaffold. A cell composition comprising:

2. The cell composition of claim 1 , wherein the multipotent ATZ stem cells comprise at least 5% of the total ATZ cells.

3. The cell composition of claim 1 or claim 2, wherein the multipotent ATZ stem cells do not express significant levels of CD45.

4. 1. A method of making a cell composition for use in cell transplantation to treat damaged or dysfunctional endodermal tissue in a subject, comprising: (a) obtaining a sample of ATZ cells that is autologous or allogeneic to the subject; (b) isolating a mixed population of ATZ cells from the sample, the mixed population comprising multipotent ATZ stem cells and endodermal progenitor ATZ cells; (c) expanding the multipotent ATZ stem cells in minimal growth medium (MGM); A method comprising:

5. The method of claim 4, wherein the multipotent ATZ stem cells do not express significant levels of CD45.

6. 6. The method of any one of claims 4 to 5, wherein the multipotent ATZ stem cells express the markers CD34, CD117 and CD184.

7. 7. The method of any one of claims 4 to 6, wherein the multipotent ATZ stem cells express the markers NANOG and OCT4A.

8. 8. The method of any one of claims 4 to 7, wherein the step of expanding the multipotent ATZ stem cells comprises replating the ATZ cells through at least five passages in MGM.

9. 9. The method of claim 4, further comprising the step of (d) culturing the ATZ cells in an endodermal lineage-specific medium (LSM) to promote at least partial differentiation of the multipotent ATZ stem cells, thereby generating an expanded population of endodermal progenitor ATZ cells.

10. 10. The method of claim 9, wherein the damaged or dysfunctional endodermal tissue is pancreatic tissue and the LSM comprises pancreatic-specific LSM.

11. The method of claim 10, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of ATZ stem cell-derived pancreatic organoid cells.

12. The method of claim 9, wherein the damaged or dysfunctional endodermal tissue is liver tissue and the LSM comprises liver-specific LSM.

13. The method of claim 12, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of liver organoid cells derived from ATZ stem cells.

14. The method of claim 9, wherein the damaged or dysfunctional endodermal tissue is intestinal mucosa tissue and the LSM comprises intestinal mucosa-specific LSM.

15. The method of claim 14, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of intestinal mucosal organoid cells derived from ATZ stem cells.

16. The method of claim 9, wherein the damaged or dysfunctional endodermal tissue is intestinal mucosa tissue and the LSM comprises intestinal mucosa-specific LSM.

17. The method of claim 16, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of intestinal mucosal organoid cells derived from ATZ stem cells.

18. 1. A method of treating damaged or dysfunctional endodermal tissue in a subject, comprising: (a) obtaining a sample of ATZ cells that is autologous or allogeneic to the subject; (b) isolating a mixed population of ATZ cells from the sample, the mixed population comprising multipotent ATZ stem cells and endodermal progenitor ATZ cells; (c) expanding the multipotent ATZ stem cells within the mixed population in minimal growth medium (MGM); (d) introducing a therapeutically effective amount of the multipotent ATZ stem cells in the mixed population into the damaged or dysfunctional tissue in the subject; A method comprising:

19. 1. A method of treating damaged or dysfunctional endodermal tissue in a subject, comprising: (a) obtaining a sample of ATZ cells that is autologous or allogeneic to the subject; (b) isolating a mixed population of ATZ cells from the sample, the mixed population comprising multipotent ATZ stem cells and endodermal progenitor ATZ cells; (c) expanding the multipotent ATZ stem cells within the mixed population by culturing the mixed population in minimal growth medium (MGM); (d) culturing the mixed population in an endodermal lineage-specific medium (LSM) to promote at least partial differentiation of multipotent ATZ stem cells within the mixed population to generate an expanded population of endodermal progenitor ATZ cells within the mixed population; (e) introducing a therapeutically effective amount of the multipotent ATZ stem cells and endodermal progenitor ATZ cells into the damaged or dysfunctional tissue in the subject; A method comprising:

20. 20. The method of any one of claims 18 to 19, wherein the multipotent ATZ stem cells do not express significant levels of CD45.

21. 21. The method of any one of claims 18 to 20, wherein the multipotent ATZ stem cells express the markers CD34, CD117 and CD184.

22. 22. The method of any one of claims 18 to 21, wherein the step of expanding the multipotent ATZ stem cells comprises replating the ATZ cells through at least five passages in MGM.

23. 20. The method of claim 19, wherein the damaged or dysfunctional endodermal tissue is pancreatic tissue and the LSM comprises pancreatic-specific LSM.

24. 24. The method of claim 23, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of ATZ stem cell-derived pancreatic organoid cells.

25. 20. The method of claim 19, wherein the damaged or dysfunctional endodermal tissue is liver tissue and the LSM comprises liver-specific LSM.

26. The method of claim 25, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of liver organoid cells derived from ATZ stem cells.

27. 20. The method of claim 19, wherein the damaged or dysfunctional endodermal tissue is lung tissue and the LSM comprises lung-specific LSM.

28. 28. The method of claim 27, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of ATZ stem cell-derived lung organoid cells.

29. 20. The method of claim 19, wherein the damaged or dysfunctional endodermal tissue is intestinal mucosa tissue and the LSM comprises intestinal mucosa-specific LSM.

30. The method of claim 29, wherein the LSM promotes the differentiation of multipotent ATZ stem cells to generate a population of intestinal mucosal organoid cells derived from ATZ stem cells.

31. A cell composition comprising a mixed population of ATZ cells, including multipotent ATZ stem cells and endodermal progenitor ATZ cells, in a pharmaceutically acceptable carrier or exogenous biocompatible scaffold.

32. 32. The cell composition of claim 31, wherein the multipotent ATZ stem cells comprise at least 5% of the total ATZ cells.

33. 33. The cell composition of claim 31 or claim 32, wherein the multipotent ATZ stem cells do not express significant levels of CD45.

34. A cell composition according to any one of claims 31 to 33 for use as a medicament.

35. A cell composition for use in a method for treating damaged or dysfunctional endodermal tissue in a subject, the cell composition comprising isolated ATZ cells and / or ATZ-derived organoid cells.

36. The cell composition for use of claim 35, wherein the isolated ATZ cells and / or ATZ-organoid cells comprise ATZ crypt cells, such as ATZ multipotent stem cells.

37. The cell composition for use described in claim 35 or claim 36, wherein the isolated ATZ cells and / or ATZ-organoid cells comprise endodermal progenitor ATZ cells.

38. 38. A cell composition for use according to any one of claims 35 to 37, comprising a pharmaceutically acceptable carrier or an exogenous biocompatible scaffold.

39. A cell composition for use according to any one of claims 35 to 38, wherein the subject is a human.

40. 40. The cell composition for use according to any of claims 35 to 39, wherein the damaged or dysfunctional tissue is gastrointestinal mucosal tissue.

41. 40. A cell composition for use according to any of claims 35 to 39, wherein the damaged or dysfunctional tissue is pancreatic tissue.

42. 40. A cell composition for use according to any of claims 35 to 39, wherein the damaged or dysfunctional tissue is liver tissue.

43. 40. The cell composition for use according to any of claims 35 to 39, wherein the damaged or dysfunctional tissue is lung tissue.

44. The cell composition for use according to any one of claims 35 to 43, wherein the isolated ATZ cells and / or ATZ-derived organoid cells are allogeneic to the subject.

45. The cell composition for use described in any of claims 35 to 44, wherein the isolated ATZ cells and / or ATZ-derived organoid cells are autologous to the subject.

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