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

Abstract

Provided herein is a composition of anorectal transition zone (ATZ) stem cells (including multipotent cells and progenitor cells) for treating anal fistula (e.g., Crohn's disease-related intractable complex perianal fistula or intractable complex perianal fistula of unknown etiology).Also provided is a pharmaceutical composition of anorectal transition zone stem cells (including multipotent cells and progenitor cells) in a pharmacologic acceptable carrier.The cells can be allogeneic or autologous cells, and can be in vitro expanded ATZ (eATZ) stem cells.

Description

[Background technology]

[0001] (background) A transition zone in gastrointestinal tissue exists between two separate epithelial tissue types: the anorectal transitional tissue, which is at the junction between the rectal columnar epithelium, which is of endodermal origin, and the anal stratified squamous nonkeratinizing epithelium, which is of ectodermal origin (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 the basal cell marker p63; cells expressing the simple columnar cell marker cytokeratin 7 (CK7) (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 cells expressing the stem and progenitor cell marker CD34 (McNairn, supra). Further studies in mice used label-retaining cell assays and immunohistochemistry to confirm expression of p63, CK7, CD34, and to identify expression of the pluripotent stem cell marker SOX2, indicating 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] Nearly 85% of fistulas in Crohn's disease patients arise from the anorectal transition zone (also called the dentate line) (Sheikh (2012) "Controversies in fistula in ano", Indian J.Surg., 74(3):217-220). Perianal fistulizing disease is an incurable, debilitating condition affecting up to 40% of patients with Crohn's disease. Perianal fistulas of unknown etiology and those in Crohn's disease patients have a frequency of 1:10,000 in the general population (Eglinton et al. (2012) "The spectrum of perianal Crohn's in a population-based cohort", Dis.Colon Rectum, 55(7):773-777). A tunnel originates at the junction of the rectum and anal canal to form a fistula (Eglinton, supra). The fistula tunnel passes through muscle and fat to open outside the body at the skin of the buttocks. When the fistula becomes exposed to intestinal contents, it sets up a chronic inflammatory state that requires regular removal of tissue debris (called granulation tissue).

[0003] No treatment exists to permanently heal a cleared fistula. Volume-retaining scaffolding products to fill the tract, such as fibrin glue, tend to cause abscess formation (Buchanan et al. (2003) "Efficacy of fibrin sealant in the management of complex anal fistula: a prospective trial", Dis Colon Rectum, 46(9):1167-1174), and porcine plugs, for example, tend to drain spontaneously (Amrani et al. (2008) "The Surgisis AFP anal fistula plug: a new and reasonable alternative for the treatment of anal fistula", Gastroenterol Clin Biol, 32(11):946-948).

[0004] Current treatments reduce symptoms which prolong the period between fistula cleansing. Antibiotics, immunosuppressants, and TNF-α inhibitors provide limited relief while having side effects and unsatisfactory therapeutic outcomes (Kumar and Thompson (2013) "Endoscopic therapy for postoperative leaks and fistulae", Gastrointest. Endosc. Clin. N. Am., 23(1):123-136; Swaminath et al. (2014) "Use of methotrexate in inflammatory bowel disease in 2014: A User's Guide", World J. Gastrointest. Pharmacol. Ther, 5(3):113-121). Injection of allogeneic adipose-derived mesenchymal stem cells (AdMSC or ASC, Alofisel™, Dalvadostrocel) directly into the tissue along the fistula tract demonstrated a 14% additional benefit over placebo control in a single controlled clinical trial, with no statistically significant effect after 1 year follow-up (NICE Technology appraisal guidance [TA556]). [Prior art documents] [Non-patent literature]

[0005] [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 [Non-Patent Document 4] Sheikh (2012) “Controversies in fistula in ano”, Indian J.Surg., 74(3):217~220) [Non-Patent Document 5] Eglinton et al. (2012) “The spectrum of perianal Cronhn's in a population-based cohort”, Dis.Colon Rectum, 55(7):773~777) [Non-Patent Document 6] Buchanan et al. (2003) “Efficacy of fibrin sealant in the management of complex anal fistula: a prospective trial”, Dis Colon Rectum, 46(9):1167–1174 [Non-Patent Document 7] Amrani et al. (2008) “The Surgisis AFP anal fistula plug: a new and reasonable alternative for the treatment of anal fistula”, Gastroenterol Clin Biol, 32(11):946~948 [Non-Patent Document 8] Kumar and Thompson (2013) "Endoscopic therapy for postoperative leaks and fistulae". Gastrointest. Endosc. Clin. N. Am., 23(1):123-136. [Non-Patent Document 9] Swaminath et al. (2014) “Use of methotrexate in inflammatory bowel disease in 2014:A User's Guide”, World J.Gastrointest.Pharmacol.Ther, 5(3):113~121 Summary of the Invention [Means for solving the problem]

[0006] (Abstract) The present invention is based, in part, on the discovery of compositions for use in treating fistulas in subjects in need of such treatment, methods of making compositions useful in treating fistulas, and methods of treating fistulas with such compositions.

[0007] In one aspect, a composition for treating fistula (e.g., anal fistula) is provided herein, the composition comprises isolated anorectal transition zone (ATZ) cells and a pharmacologic carrier. The ATZ cells comprise multipotent stem cells and / or progenitor cells. The cells can be allogeneic or autologous cells. In certain embodiments, the ATZ cells comprise ATZ stem cells that can differentiate into endoderm cells (e.g., express SOX17), mesoderm cells (e.g., express BRACHURY), ectoderm cells (e.g., express PAX6 and / or NESTIN), or combinations thereof. The ATZ stem cells can be in vitro expanded ATZ (eATZ) stem cells.

[0008] In certain embodiments, the ATZ stem cells express one or more of CD34, CD117, and / or CD184, for example, when detected by flow cytometry (e.g., using an antibody or other ligand that binds to the cell surface marker). Alternatively, or in addition, the ATZ stem cells do not express detectable levels of CD45, for example, when detected by flow cytometry. Furthermore, in certain embodiments, the ATZ stem cells express NANOG and / or OCTA4A.

[0009] The ATZ cells can be combined with an exogenous biocompatible scaffold (e.g., a synthetic or biological scaffold). Depending on the situation, the scaffold can include a collagen-based scaffold.

[0010] The ATZ cells can be porcine or human cells.

[0011] In certain embodiments, the composition is cryopreserved. To facilitate its cryopreservation, the composition may include an appropriate cryopreservation medium.

[0012] In another aspect, the present invention provides a pharmaceutical dosage form comprising a composition disclosed herein, optionally disposed in a container or capsule.

[0013] In another aspect, the present invention provides a method for preparing a pharmaceutical composition (e.g., any of the compositions disclosed herein), comprising the steps of: (a) collecting ATZ tissue from a donor (e.g., a porcine donor or a 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 cryopreservation medium and cryopreserving the cell suspension; and (d) combining the cell suspension of step b) or, optionally, step (c) with a pharma- ceutically acceptable carrier.

[0014] The cell suspension comprises ATZ cells, which may comprise multipotent stem cells and / or progenitor cells. In certain embodiments, the ATZ stem cells may differentiate into endoderm cells (e.g., expressing SOX17), mesoderm cells (e.g., expressing BRACHURY), ectoderm cells (e.g., expressing PAX6 and / or NESTIN), or a combination thereof.

[0015] Depending on the situation, the ATZ stem cell expresses one or more of CD34, CD117 and CD184, for example, when detected by flow cytometry.Alternatively, or in addition, the ATZ cell does not express detectable levels of CD45, for example, when detected by flow cytometry.Furthermore, the ATZ stem cell can express NANOG and / or 0CT4A.The ATZ cell can be an allogeneic cell or an autologous cell.

[0016] In certain embodiments, the cells in the suspension (eg, ATZ stem cells) are expanded in vitro.

[0017] Depending on the circumstances, in step (d) of the above method, the cells in the cell suspension are combined with an exogenous biocompatible scaffold (e.g., a synthetic or biological scaffold). In certain embodiments, the scaffold comprises a collagen-based scaffold.

[0018] In another aspect, the invention provides pharmaceutical compositions made by the methods disclosed herein.

[0019] In another aspect, the present invention provides a method of treating a fistula (e.g., anal fistula and / or a refractory fistula) in a subject in need thereof, the method comprising administering to the fistula an effective amount of a pharmaceutical composition or dosage form disclosed herein, thereby treating the fistula.

[0020] In some embodiments, the method closes the fistula, for example with new fibrotic tissue. In some embodiments, the subject has Crohn's disease or has a fistula of unknown etiology. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows isolation of anorectal transition zone 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. [Diagram 2] FIG. 2 shows the isolation of anorectal transition zone crypts from Crohn's disease patient tissue: (a) shows excised anorectal tissue, (b) shows excised mucosal epithelium, (c) shows a brightfield image of freshly isolated ATZ crypts, and (d) shows a brightfield image of freshly isolated ATZ crypts. [Figure 3A] FIG. 3 shows (a) crypt organoid development in porcine gastrointestinal (GI) tissue, and (b) crypt organoid development in rectal and ATZ tissue from a Crohn's disease patient. [Figure 3B] Same as above. [Figure 4A] Figure 4 shows the increased progenitor capacity of porcine ATZ crypt single cell preparations compared to rectal crypts. Figure 4(a) shows the proliferation of porcine ATZ crypts and porcine rectal crypts over 4 weeks. Figure 4(b) shows the increased plating efficiency of crypt organoids derived from ATZ cells over 2 months compared to rectal cells. Cells were isolated and re-plated after 1 month. [Figure 4B] Same as above. [Figure 5A-B]Figure 5 shows protein expression of stem cell and progenitor cell markers in freshly isolated crypts and crypt organoids from pigs and Crohn's disease patients. Figure 5(a) shows CD117 expression in fresh porcine crypt-derived single cells from porcine gastrointestinal (GI) tissue. Figure 5(b) shows CD34 expression in fresh porcine crypt-derived single cells from porcine gastrointestinal (GI) tissue. Figure 5(c) shows double labeling of CD34 and CD117 expression in fresh porcine ATZ crypt cells by flow cytometry. Figure 5(d) shows expression of stem cell and progenitor cell markers in porcine crypt organoids. Small intestine (SI) is shown in the left bar, rectum in the middle bar, and ATZ in the right bar. Figure 5(e) shows expression of the indicated developmental lineage markers in fresh porcine ATZ crypt-derived cells. Figure 5(f) shows expression of stem cell and progenitor cell markers in rectal crypt-derived organoids from Crohn's disease patients. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E-F] Same as above. [Figure 6A] Figure 6 shows mRNA profiling from freshly isolated crypts and crypt organoids of the porcine anorectal transition zone. Figure 6(a): mRNA profiling of freshly isolated porcine ATZ crypt cells. Figure 6(b): mRNA profiling of organoids derived from porcine ATZ crypt cells. [Figure 6B] Same as above. [Figure 7A-B] FIG. 7 shows flow cytometry analysis of stem and progenitor cell markers to evaluate growth medium on day 14 for promoting porcine ATZ crypt stem cell proliferation, where FIG. 7(a) shows CD117 expression, FIG. 7(b) shows Brachyury expression, and FIG. 7(c) shows SOX17 expression. [Figure 7C] Same as above. [Figure 8]Figure 8 shows that porcine ATZ crypt cells generate mature cells of the endodermal lineage. Figures 8(a)-8(d) show bright field images of organoid development from day 1, day 4, day 7, and day 12, respectively. Figures 8(e)-8(h) show DAPI staining and immunocytochemistry images of the nuclei of organoids. Figure 8(e) shows lysosomal expression, Figure 8(f) shows Ki67 expression, Figure 8(g) shows Muc-2 expression, and Figure 8(h) shows CK18 expression. [Figure 9] FIG. 9 shows flow cytometry analysis of endoderm markers in ATZ organoids at days 0, 7, and 14. [Figure 10] Figure 10 shows that porcine ATZ crypt cells generate mature cells of the mesodermal lineage. Figures 10(a)-10(d) show bright field images of vessel-like development, where Figure 10(a) shows undifferentiated ATZ single cells on day 0, Figure 10(b) shows single ATZ cells beginning to form clusters on day 3, Figure 10(c) shows vessel-like structures beginning to form on day 8, Figure 10(d) shows a network of vessel-like structures forming on day 10, Figure 10(e) shows DAPI stained nuclei and immunocytochemistry of endothelial-like cells expressing CD31, and Figure 10(f) shows DAPI stained nuclei and immunocytochemistry of vessel-like cells expressing CD31. [Figure 11] Figure 11 shows that porcine ATZ crypt cells generate mature cells of mesodermal lineage. Figure 11 shows flow cytometry analysis of mesodermal markers Brachyury and CD31 in ATZ organoids at days 0, 7, and 14. [Figure 12A]Figure 12 shows that porcine ATZ crypt cells generate mature cells of ectodermal lineage. Figure 12(a) shows bright field images of keratinocyte development from anal skin and ATZ crypts grown in KFSM. Figure 12(b) shows immunocytochemistry of K14 and K15 markers in anal skin cultures. Figure 12(c) shows immunocytochemistry of K14 and K15 markers in ATZ crypt cultures. Figure 12(d) shows flow cytometry analysis of ectodermal markers PAX6, NESTIN, and CK14 in ATZ cultured cells at days 0, 7, and 14. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13A] FIG. 13 shows bright field images of single porcine small intestinal (SI) crypt cells in media to promote differentiation in three developmental lineages on days 0, 7, and 14, where FIG. 13(a) shows culture in endoderm medium (OGM), FIG. 13(b) shows culture in mesoderm medium (MethoCult), and FIG. 13(c) shows culture in ectoderm medium (KFSM). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14A] Figure 14 shows the results of an in vitro embryoid body assay to test the pluripotency of porcine ATZ crypt single cells. Figure 14(a) shows a bright field image of ATZ embryoid body development. Figure 14(b) shows alkaline phosphatase staining of ATZ embryoid bodies. Figure 14(c) shows immunostaining of ATZ embryoid bodies for the expression of pluripotent stem cell markers SSEA4 and OCT4. Figure 14(d) shows immunostaining of ATZ embryoid bodies with the 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. [Figure 15A]Figure 15 shows the in vitro biocompatibility of ATZ crypt organoids with synthetic scaffold control (PeptiGel or "PG"). Figure 15(a) shows the bright field images of ATZ organoids grown in 2%, 5% and 10% PG or Matrigel (registered trademark) matrix scaffold control and cultured up to day 19, and Figure 15(b) shows the number of ATZ organoids in culture in PeptiGel or Matrigel (registered trademark) matrix and cultured up to day 24. [Figure 15B] Same as above. [Figure 16] Figure 16 shows micrographs of ATZ organoid interaction with PeptiGel, where Figure 16(a) shows Alpha 4 RGD, Figure 16(b) shows Alpha 4 IKVAV, Figure 16(c) shows Alpha 4 YIGSR, Figure 16(d) shows Alpha 4 GFOGER, Figure 16(e) shows Alpha 4 IKVAV+YIGSR, and Figure 16(f) shows Matrigel. [Figure 17] FIG. 17 shows a schematic diagram of the creation and treatment of a porcine perianal fistula. [Figure 18] Figures 18(a)-18(d) show the creation of a perianal fistula for study in a porcine model. [Figure 19] FIG. 19 shows histological evaluation by H&E staining of cross sections of porcine fistula tracts 90 days after fistula treatment, where FIG. 19(a) shows the no treatment control, FIG. 19(b) shows the hydrogel scaffold control (PeptiGel), and FIG. 19(c) shows the ATZ cells+PeptiGel scaffold. [Figure 20] Figure 20 shows an example of histological evaluation of a porcine fistula 90 days after treatment. Figure 20(a) shows ATZ cells + PeptiGel treatment: α-smooth muscle actin immunohistochemistry detects blood vessels. Figure 20(b) shows ATZ cells + PeptiGel treatment. Picrosirius red staining was used for collagen (visualized under polarized light). [Figure 21A-B] FIG. 21 shows three images (a)-(c) of the "no treatment control" fistula tract in the porcine fistula-in-ano model of Examples 13-14. [Figure 21C]Same as above. [Figure 22A-B] FIG. 22 shows three images (a)-(c) of the "no treatment control" fistula tract in the porcine fistula-in-ano model of Examples 13-14. [Figure 22C] Same as above. [Figure 23A-B] FIG. 23 shows three images (a)-(c) of the "Permacol scaffold" only treatment in the porcine fistula-in-ano model of Examples 13-14. [Figure 23C] Same as above. [Fig. 24A-B] FIG. 24 shows three images (a)-(c) of the "ATZ cells + Permacol scaffold" treatment in the porcine fistula-in-ano model of Examples 13-14. [Figure 24C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] (Detailed Description) (definition) As used herein, the following terms and phrases have the meanings indicated below: Unless explained otherwise, 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.

[0023] As used herein, the term "anorectal transition zone" or "ATZ" refers to the tissue sandwiched between the underlying uninterrupted squamous epithelium and dentate (or pectinate) line of the anal epithelium and the overlying uninterrupted columnar epithelium of the rectum. The dentate line is the junction between the upper and lower anal canals. There are many differences between these two regions, including their developmental origin, innervation, venous and arterial supply, and lymphatic supply. Above the dentate line, the anal canal has an endodermal origin and is lined by simple columnar epithelium. Below the dentate line, the anal canal has an ectodermal origin and is lined mainly by stratified squamous epithelium. The ATZ is typically 1 mm to 4 mm and can be easily identified and biopsied by those skilled in the art (see, for example, FIG. 1).

[0024] As used herein, the term "anorectal transition zone cells" or "ATZ cells" refers to a mixed population of cells derived from anorectal transition zone tissue, including crypt, submucosal gland, and other epithelial cells, including multipotent stem cells (i.e., cells that have the ability to self-renew by division and develop into multiple specialized cell types present in a particular tissue or organ), progenitor cells, and other cell types that arise from anorectal transition zone tissue. 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).

[0025] The term "crypt" refers to the crypts of Lieberkuhn, which are structures beneath the surface of the intestinal mucosal lining and contain stem cells that are responsible for the continuous renewal of the intestinal mucosa throughout life.

[0026] As used herein, the term "stem cell" refers to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and proliferate indefinitely to generate more of the same stem cell (i.e., self-renewal).

[0027] 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 the ATZ.

[0028] As used herein, the term "multipotent cells" refers to stem cells that have the ability to differentiate into at least two cell types or lineages, and the term multipotent ATZ cells refers to multipotent cells derived from the ATZ.

[0029] As used herein, the term "pluripotent cells" refers to stem cells that have the ability to differentiate into each of the three major cell groups (i.e., ectoderm, mesoderm and endoderm), and the term pluripotent ATZ cells refers to multipotent cells derived from the ATZ.

[0030] As used herein, the terms "proliferation" and "proliferating" refer to an increase in cell number through mitosis.

[0031] The term "differentiation" refers to the formation of cells that express markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells that cannot further divide or differentiate. For example, in a hematological context, differentiation can be observed in a population of functional cells of a multicellular lineage (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 that cannot further divide or differentiate than the original cells from which they were cultured. The term "terminal differentiation" refers to cells that have become terminally differentiated cells that cannot further divide or differentiate.

[0032] The term "eATZ cells" refers to a cell suspension, for example, in a sterile buffered saline solution, including anorectal transition zone stem cells and progenitor cells that are expanded for stem cell transplantation. The term "expanded" used herein when referring to cells should be considered as having its usual meaning in the art, i.e., cells that are expanded in vitro. eATZ cells can be expanded to provide a cell population that retains at least one biological function of non-expanded ATZ cells, typically the ability to form crypt organoids under standard culture conditions. The expanded cell population can 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, CD184, OCT4, NANOG, SOX17, BRACHURY, PAX6 and NESTIN.

[0033] The term "cell composition" refers to a preparation of cells, which in addition to the cells may include non-cellular components such as cell culture medium (e.g., proteins, amino acids, nucleic acids, nucleotides, coenzymes, antioxidants, metals, etc.). Additionally, the cell 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., as a polymer matrix for encapsulation or a pharmaceutical preparation).

[0034] "Marker" refers to a biological molecule whose presence, concentration, activity, or phosphorylation state can be detected and used to identify a cellular phenotype.

[0035] An "exogenous biocompatible scaffold" is a three-dimensional, porous or fibrous or permeable, volume-retaining biomaterial intended to allow the transport of bodily fluids and gases, and allows cell interaction with minimal inflammation and toxicity. Optionally, such a scaffold is biodegradable. Examples of scaffolds may include biological scaffolds (e.g., laminin-based or collagen-based scaffolds) and synthetic scaffolds (e.g., non-biological polymers). The scaffold may be in the physical form of thread, sheet, paste, powder, or liquid. The scaffold may be used in vitro and in vivo.

[0036] The term "fistula" refers to an abnormal passageway or communication or connection, usually between two internal organs or from an internal organ to the body surface. Examples of fistula include, but are not limited to, perianal fistula, anorectal fistula or anal fistula, cervical fistula, enterovaginal fistula, and rectovaginal fistula. As used herein, the term "anal fistula" includes perianal fistula, anorectal fistula, and anal fistula.

[0037] A single "tract" fistula or simple fistula has one internal opening and one external opening. A "multiple tract" fistula has more than one external opening and / or more than one internal opening. A multiple tract fistula often has separate branches. Each external opening typically represents a tract.

[0038] A "complex perianal fistula" is a perianal fistula tract having one or more of the following: (i) a high intermuscular, transsphincteric, extrasphincteric or suprasphincteric origin; (ii) an external opening; or (iii) associated collections. In some embodiments, the complex perianal fistula may have two or more internal openings and three or more external openings. In some embodiments, the complex perianal fistula may be draining for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer prior to treatment according to the present disclosure.

[0039] As used herein, the phrase "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive or unacceptable toxicity, irritation, allergic response, other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0040] As used herein, the phrase "pharmacologically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle (e.g., a liquid or solid filler, diluent, excipient, or solvent encapsulating material) involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.

[0041] As used herein, the term "phenotype" refers to the observable characteristics of a cell, such as size, morphology, RNA expression, protein expression, or other property.

[0042] As used herein, the term "refractory" refers to resistance to standard treatment. In the case of fistulas described herein, refractory fistulas may include fistulas that are not healed or closed by prior art treatments, including but not limited to administration of antibiotics, administration of immunosuppressants, administration of TNF-α inhibitors, administration of methotrexate, or direct introduction of volume-retaining scaffold products (e.g., fibrin sealants) into the fistula tract, direct introduction of fistula plugs into the fistula tract, direct introduction of adipose-derived mesenchymal stem cells (e.g., Alofisel™, dalvadostrocel) into the fistula tract. In some embodiments, refractory fistulas can be successfully treated by the present invention to promote healing or closure of the fistula.

[0043] Multipotent ATZ Cells and Progenitor ATZ Cells and Organoid Compositions Multipotent ATZ cell and / or precursor ATZ cell described herein can be isolated as single cell (or multicellular) preparation from primary ATZ tissue.ATZ cell described herein (including multipotent ATZ cell and precursor ATZ cell) can be derived from intestinal crypt obtained by biopsy, or can be derived from organoid structure that is ex vivo differentiated from ATZ cell.In some embodiments, the multipotent ATZ cell and precursor ATZ cell are obtained from organoid, which is derived from single primary ATZ cell or dissociated ATZ crypt cell.

[0044] For use in the methods of treatment described herein, the multipotent ATZ cells or precursor ATZ cells are preferably autologous cells derived from human patients or allogeneic cells derived from human donors (preferably histocompatible to reduce or avoid graft-versus-host immune reactions or host-versus-graft immune reactions).Alternatively, the multipotent ATZ cells or precursor ATZ cells can be derived from other mammalian species and modified by methods known in the art (e.g., gene editing to knock out MHC class I and / or MHC class II genes and / or β2-microglobulin genes) to reduce or eliminate alloreactivity.

[0045] In some embodiments, the multipotent ATZ cells or progenitor ATZ cells can be in vitro expanded ATZ (eATZ) cells.

[0046] In some embodiments, the multipotent ATZ cells and progenitor ATZ cells, primary ATZ cells, crypts and / or organoids are combined with exogenous biocompatible scaffolds (e.g., synthetic or biological scaffolds) in vivo or in vitro. In some embodiments, the scaffolds comprise laminin and / or collagen (e.g., Permacol™ paste, Medtronic PLC, Minneapolis, MN). In some embodiments, the scaffolds are Matrigel® matrix (Coming, New York) or functional equivalents.

[0047] In some embodiments, the exogenous biocompatible scaffold is functionalized or non-functionalized. The scaffold can be functionalized with peptides, such as laminin (IKVAV and YIGSR) peptides, fibronectin (RGD) peptides, or collagen (GFOGER) peptides.

[0048] In some embodiments, the exogenous biocompatible scaffold comprises a mixture of functionalized and non-functionalized scaffold components. In some embodiments, the functionalized scaffold component comprises between 0.1% and 10% peptide. In some embodiments, the functionalized scaffold component comprises 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% peptide. In some embodiments, the exogenous biocompatible scaffold comprises between 99.9% and 90% non-functionalized scaffold. In some embodiments, the exogenous biocompatible scaffold comprises 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% non-functionalized scaffold, respectively. In some embodiments, the scaffold component is at least 0.1%, 0.5%, 1%, 5%, or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% functionalized scaffold. In some embodiments, the scaffold is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% non-functionalized scaffold.

[0049] In some embodiments, the non-functionalized scaffold component comprises 98%, 95%, or 90% functionalized scaffold, respectively. In some embodiments, the scaffold component is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% functionalized scaffold. In some embodiments, the scaffold is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% non-functionalized scaffold.

[0050] The functionalized scaffold mixture may also include at least two functionalized scaffolds. The functionalized components of the final scaffold preparation may include 1% to 99% of at least one functionalized scaffold and 99% to 1% of at least another functionalized scaffold in any ratio. For example, the functionalized scaffold components include 50% IKVAV functionalized scaffold and 50% YIGSR functionalized scaffold. Alternatively, the functionalized scaffold component mixture may include 1% IKVAV and 99% YIGSR, or any ratio in between. For example, the functionalized scaffold mixture may be 25% IKVAV and 75% YIGSR. Or in another case, the functionalized scaffold mixture may be 75% IKVAV and 25% YIGSR. In some embodiments, the functionalized scaffold mixture includes a collagen (e.g., GFOGER peptide) scaffold and another functionalized scaffold component. In some embodiments, the mixture of functionalized scaffolds is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% IKVAV functionalized scaffolds. In some embodiments, the mixture of functionalized scaffolds is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% YIGSR functionalized scaffolds. In some embodiments, the functionalized scaffold mixture is at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% collagen functionalized scaffold.

[0051] In some embodiments, the scaffold comprises a functionalized collagen scaffold or a functional equivalent.

[0052] In some embodiments, the scaffold comprises functionalized PeptiGel® Alpha 4 (Manchester BioGel) or a functional equivalent.

[0053] The exogenous biocompatible scaffold may also comprise cell culture medium components. In some embodiments, the scaffold comprises organoid growth medium (OGM), which may be purchased from a commercial supplier (e.g., IntestiCult™, STEMCELL Technologies, Inc., Vancouver, Canada). In some embodiments, the ATZ cells or ATZ crypts may be cultured in OGM. In some embodiments, the ATZ cells or ATZ crypts may be cultured in a feeder-free medium (e.g., mTesR™, STEMCELL Technologies, Inc., Vancouver, Canada) 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 ESC feeder-free medium or iPSC feeder-free medium. In some embodiments, ATZ cells or ATZ crypts cultured in OGM have higher expression of mesodermal lineage markers and endodermal lineage markers compared to ATZ cells or ATZ crypts cultured in such ESC feeder-free medium or iPSC feeder-free medium.The matrix can also contain keratinocyte serum-free medium (KSFM) for differentiation.Such KSFM medium is commercially available from suppliers (e.g., Sigma-Aldrich, St. Louis, MO).

[0054] In another aspect, the present disclosure provides phenotypic characterization of the extracellular and intracellular protein and mRNA expression levels of stem cell and progenitor cell markers in freshly isolated single ATZ cells.ATZ cells can express at least one of CD34, CD117 and CD184 cell surface markers, but do not express detectable levels of CD45, as detected by flow cytometry.In some embodiments, isolated porcine ATZ cells express OCT4 and NANOG.

[0055] ATZ stem cells can also express markers for stem cells of three developmental lineages: endoderm (SOX17), mesoderm (BRACHURY), and ectoderm (PAX6 and NESTIN). In addition, freshly isolated ATZ cells can express markers for pluripotent stem cells (NANOG and OCT4).

[0056] In some embodiments, the ATZ cells and / or ATZ crypts express genes related to endodermal lineage, ectodermal lineage, and mesodermal lineage. In some embodiments, the ATZ cells and / or ATZ crypts express at least one gene marker selected from the group consisting of ALP, BRACHURY, BMP4, CD34, CD117 (KIT), CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, NANOG, PAX6, SOX17, and OCT4. In some embodiments, the ATZ cells and / or ATZ crypts express CD34. In some embodiments, the ATZ cells and / or ATZ crypts express CD117 (KIT) at a higher level than small intestinal crypt cells, colonic crypt cells, and rectal crypt cells. In some embodiments, the ATZ cells and / or ATZ crypts express 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, 7 times or more higher than small intestinal crypt cells, colonic crypt cells, and rectal crypt cells. In some embodiments, the ATZ cells and / or ATZ crypts express CD34. In some embodiments, the ATZ cells and / or ATZ crypts do not express detectable levels of CD45. In some embodiments, the ATZ cells and / or ATZ crypts express CD34 and do not express detectable levels of CD45. In some embodiments, the ATZ crypt expresses at least one gene selected from the group consisting of ALP, BRACHURY, BMP4, CD34, CD117 (KIT), CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, PAX6, and SOX17. In some embodiments, the ATZ crypt expresses the OCT4 gene or the NANOG gene.

[0057] An aspect of the present disclosure provides phenotypic characterization of the extracellular and intracellular protein and mRNA expression levels of stem cell markers and progenitor cell markers in ATZ organoids in culture.In some embodiments, the ATZ organoids express the genes related to endodermal lineage, ectodermal lineage and mesodermal lineage.In some embodiments, the ATZ organoids express at least one protein marker selected from the group consisting of ALP, BRACHURY, CD34, CD117 (KIT), CHGA, CK18, CXCR4, EPCAM, GD2, LGR5, LYS, MUC2, NESTIN and PAX6.In some embodiments, the ATZ organoids express CD34.In some embodiments, the ATZ organoids do not express detectable levels of CD45. In some embodiments, the ATZ organoid expresses at least one gene selected from the group consisting of ALP, BRACHURY, BMP4, CD34, CD117 (KIT), CXCR4, CHGA, CK18, EPCAM, LGR5, LYS, MUC2, NANOG, PAX6, and SOX17.

[0058] In some embodiments, the 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, the ATZ organoid expresses CD34.In some embodiments, the ATZ organoid does not express detectable levels of CD45.In some embodiments, the ATZ organoid expresses CD34 but does not express detectable levels of CD45.

[0059] In some embodiments, the ATZ organoid expresses CD117 (KIT), CXCR4 (CD184) and / or GD2 at high levels compared with small intestinal organoid and rectal organoid.In some embodiments, the 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, the 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, the 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, the ATZ organoid has SOX17 expression that is reduced compared to ATZ crypt cells.In some embodiments, the ATZ organoid has BRACHURY expression that is reduced compared to ATZ crypt cells.In some embodiments, the ATZ organoid has CD31 expression that is reduced compared to ATZ crypt cells.In some embodiments, the ATZ organoid has PAX6 expression that is reduced compared to ATZ crypt cells.In some embodiments, the ATZ organoid has LGR5 that is increased compared to ATZ crypt cells.In some embodiments, the ATZ organoid has CD117 or CXCR4 that is increased compared to ATZ crypt cells. In some embodiments, the ATZ organoid has increased K14 compared with ATZ crypt cell.In some embodiments, the ATZ organoid has increased CD117, CXCR4, LGR5 or K14 expression 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 ATZ crypt cell.In some embodiments, the ATZ organoids have 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, or 7-fold reduced expression of SOX17, BRACHURY, CD31, or PAX6 compared to ATZ crypt cells.

[0060] Protein and nucleic acid expression can be determined by methods known to those skilled in the art.Such methods include, but are not limited to, polymerase chain reaction (PCR), RTPCR, q-RT-PCR, flow cytometry (combined with binding agent (e.g., labeled antibody or labeled ligand 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.

[0061] In some embodiments, the ATZ organoid comprises proliferative cells, secretory cells and cytokeratin.In some embodiments, the ATZ organoid can secrete lysozyme.

[0062] Methods for isolating and generating populations of multipotent ATZ cells and progenitor ATZ cells In another aspect, the present invention provides a method for isolating and generating multipotent ATZ cells and / or precursor ATZ cells.The ATZ cells can be generated by obtaining anorectal transition zone tissue from either biopsied or excised tissue from mammalian subjects (e.g., humans, pigs); washing the tissue with serum-free tissue culture medium (optionally containing antibiotics and / or antimycotics); mincing the tissue; and enzymatically treating or digesting the tissue with collagenase. Digestion can be stopped, for example, by adding protein (e.g., albumin). Crypts can be isolated by vigorously 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 settle or pellet the crypts; the crypts can be resuspended in fresh medium to obtain a population of multipotent ATZ cells and / or precursor ATZ cells.

[0063] Resuspended crypts can be counted under a microscope.Crypt-derived single cell preparations can be made by using needle / syringe physical disruption and / or treatment with mild enzymatic cell dissociation reagent to obtain a population of multipotent ATZ cells and / or progenitor ATZ cells.These cells can then be embedded in an in vitro matrix scaffold (for example, as described above) and cultured with differentiation medium to generate organoids.

[0064] In some embodiments, enzymatic treatment of the minced anorectal transition zone tissue liberates 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.

[0065] The isolated intestinal crypt or multipotent ATZ cell and / or progenitor ATZ cell can then be differentiated into organoids after being placed in a growth medium or scaffold (e.g., laminin, collagen, Permacol™, Matrigel® matrix). Any suitable matrix and growth medium known in the art can be used. In some embodiments, the crypt is placed in a synthetic scaffold. In some embodiments, the scaffold is non-functionalized. In some embodiments, the scaffold is functionalized. Alternatively, the crypt can be further dissociated into single ATZ cells.

[0066] In some embodiments, the isolated ATZ-derived crypt cells have increased multipotency and / or progenitor potential compared to cells derived from rectal crypts.

[0067] In another aspect, the present invention provides for the in vitro growth and maintenance of ATZ stem cells (multipotent cells and / or progenitor cells) by culturing crypts or single cells embedded in scaffolds (e.g., Permacol™, Matrigel® matrix) with specialized intestinal organoid growth medium (e.g., IntestiCult™) that promotes permanent growth and differentiation. Organoid growth medium (OGM) is commercially available, such as IntestiCult™, MethoCult™ (STEMCELL Technologies, Inc., Vancouver, Canada), or keratinocyte serum-free medium (e.g., Sigma-Aldrich, St. Louis, MO). ATZ organoid culture is periodically refreshed by breaking down and replating organoids in organoid growth medium (OGM).

[0068] In another aspect, the present invention provides a method for in vitro expansion of ATZ stem cells (including multipotent cells and / or progenitor cells) in preparation for treating fistula.The method can include repeated subculture of ATZ organoid.The method can further include confirming that collected cells maintain phenotypic characteristics, as determined by, for example, the protein expression level and mRNA expression level of cell surface and intracellular stem cell markers and replating efficiency.Once the characteristics of ATZ cells are confirmed for cell source, biomarkers (for example, cell surface markers (for example, CD34, CD117, and / or CD184)) can be used to determine the appropriate cell number for stem cell therapy.

[0069] Some aspects of the present disclosure provide for the cryopreservation and thawing of ATZ cells after collecting ATZ crypts, single cells, and / or after organoid culture, by resuspending centrifuged cells in serum-free DMSO-containing freezing medium, and gradually lowering its temperature to -80°C or lower.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 freezing medium is removed.In some embodiments, the composition further comprises cryopreservation medium.In some embodiments, the ATZ composition or eATZ composition is cryopreserved.

[0070] (Treatment Method) In another aspect, the present invention provides a method for treating fistula in a subject who needs to treat fistula, comprising administering an effective amount of the pharmaceutical composition or formulation of ATZ cells described herein to the fistula, thereby treating the fistula.For example, an appropriate number of confirmed cells can be combined with a scaffold (e.g., scaffold paste) for injection into the fistula.An exemplary scaffold paste is collagen scaffold paste (Permacol™ paste, Medtronic PLC, Minneapolis, MN).The effectiveness of this treatment is shown in the following examples.

[0071] In some embodiments, the ATZ crypts are cultured ex vivo to generate expanded ATX cells for use in methods of treating fistulas. In some embodiments, the eATZ cells can be cryopreserved after expansion and then thawed prior to treatment.

[0072] In some embodiments, the treatment causes the fistula to heal or close, for example with new fibrotic tissue. In some embodiments, the fistula is a perianal fistula. In some embodiments, the fistula is a refractory fistula. The fistula may be associated with Crohn's disease or may be in an individual with a fistula of unknown etiology.

[0073] In some embodiments, the pharma- ceutically acceptable carrier comprises a biocompatible scaffold (eg, a scaffold described herein above).

[0074] Pharmaceutical Composition In another aspect, a method for preparing a therapeutic or pharmaceutical composition (e.g., a composition described herein) is provided herein. The method includes the steps of: (a) collecting anorectal transition tissue from a patient or donor; (b) enzymatically digesting the transition tissue with an enzyme to prepare a cell suspension; (c) optionally combining at least the cell suspension with a cryopreservation medium and lyophilizing the cell suspension; and (d) combining the cell suspension of step (b) or optionally step (c) with a pharma- ceutical acceptable carrier.

[0075] In one embodiment, the present disclosure provides a pharmaceutical composition comprising adult allogeneic or autologous anorectal transition zone cells (or "ATZ cells") in a pharma- tically acceptable carrier. The ATZ cells can be prepared by a method comprising: (a) collecting anorectal transition tissue from an adult human subject or adult porcine subject; (b) preparing a cell suspension in vitro by enzymatic digestion of anorectal transition tissue; (c) settling / pelleting the cells, then resuspending them in a freezing medium, and cryopreserving the ATZ cells, for example, in liquid nitrogen. Prior to use, the cryopreserved ATZ cells are combined with a pharma-tically acceptable carrier, and then the cell / carrier preparation is injected into the fistula. The carrier can be used to fill the internal shape of the fistula, allowing tissue remodeling and more effective closure of the tract.

[0076] Throughout this specification, when compositions are described as having, containing, or comprising particular components, or when processes and methods are described as having, containing, or comprising particular steps, it is additionally contemplated that there are compositions of the present disclosure that consist essentially of the recited components, or that consist of the recited components, and that there are processes and methods according to this disclosure that consist essentially of the recited processing steps, or that consist of the recited processing steps.

[0077] In this application, when a component or ingredient is said to be included in and / or selected from a list of recited components or ingredients, it should be understood that the component or ingredient can be any one of the recited components or ingredients, or that the component or ingredient can be selected from the group consisting of two or more of the recited components or ingredients.

[0078] Furthermore, it should be understood that the components and / or features of the compositions or methods described herein, whether expressly or impliedly described herein, can be combined in various ways without departing from the spirit and scope of the present disclosure.For example, if a specific compound is mentioned, that compound can be used in various embodiments of the compositions and / or methods of the present disclosure, unless otherwise understood from the context.In other words, although the embodiments are described and shown in this application in a manner that allows a clear and precise application to be described and depicted, it is intended and understood that the embodiments can be combined in various ways or separately without departing from the present teachings and disclosure.For example, it is understood that all features described and shown herein can be applicable to all aspects of the disclosure described and shown herein.

[0079] The phrase "at least one of" should be understood to include each of the listed objects preceding the phrase individually, and to include various combinations of two or more of those listed objects, unless otherwise understood from the context and usage. The phrase "and / or" combined with three or more listed objects should be understood to have the same meaning, unless otherwise understood from the context.

[0080] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing" (including grammatical equivalents thereof) should generally be understood as open-ended and non-limiting (e.g., not excluding additional, unrecited components or steps) unless specifically stated or understood from the context to the contrary.

[0081] When the term "about" is used before a quantitative value, the present disclosure also includes the particular quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from its nominal value, unless otherwise indicated or inferred.

[0082] It should be understood that the order of steps or order of performing certain actions is immaterial so long as the present disclosure remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0083] The use of any and all examples or exemplary language (e.g., "such as" or "including") herein is intended merely to better illustrate the disclosure, and their use does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. EXAMPLES

[0084] (Example) The following examples are merely illustrative of the scope or content of the present disclosure and are not intended to limit the scope or content of the disclosure in any way.

[0085] Example 1. Isolation of intestinal crypts from anorectal tissue This example describes a protocol for isolating intestinal crypts from anorectal tissue derived from pigs and human subjects with Crohn's disease.

[0086] (I. Pig) Examples of healthy adult White Landrace pig tissue from colon to anus (Figure 1(a)) were collected within 2 hours postmortem in AIMV V medium (Thermo Fisher Scientific) containing antibiotic / antimycotic and processed immediately or, in some cases, after overnight storage at 40° C. The tissue was opened longitudinally and cleaned.

[0087] The dentate line was identified between the rectal mucosa and the anal skin (Fig. 1(b)). The tissue was 8-10 cm wide, 1-2 cm high, and pale. The epithelial layer of the dentate tissue (anorectal transition zone) was excised from the rectal mucosa and anal skin (Fig. 1(c)). Enzymatic treatment of the minced anorectal transition zone tissue liberated intestinal crypts (Fig. 1(d-g)) and structures consistent with submucosal glands (Fig. 1(h-i)).

[0088] (II. Crohn's disease patients) From the rectal resection tissue (Fig. 2(a)), the anorectal transition zone was excised (Fig. 2(b)), minced, and enzymatically treated to liberate the crypts (Fig. 2(c-d)).

[0089] Example 2. Crypt-anorectal transition zone organoid development Crypt-anorectal transition zone organoid development was developed from the porcine and human tissues of Example 1.

[0090] Intestinal crypts prepared in Example 1 were embedded in Matrigel® matrix (Coming, New York) with human organoid growth medium (OGM, IntestiCult™, STEMCELL Technologies, Inc., Vancouver, CA) and allowed to develop 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 intestinal, colonic, and rectal tissues (Figure 3(a)). Similarly, organoids derived from the anorectal transition zone and rectum were indistinguishable in patients with Crohn's disease (Figure 3(b)).

[0091] Example 3. Increased progenitor potential of porcine ATZ crypt single cell preparations compared to rectal crypts This example shows that single cell preparations of porcine ATZ crypts compared to rectal crypts have increased progenitor potential over rectal crypts.

[0092] The plating efficiency (organoids formed per cell plated) of single cell preparations of porcine ATZ crypts and porcine rectal crypts was determined for inputs of 500, 1,000 and 2,000 viable crypt cells per well in 24-well flat-well suspension culture plates (Sarstedt) and monitored once a week for 4 weeks. Organoid growth medium (IntestiCult™) was refreshed once at the end of the second week, and Matrigel® matrix was not changed.

[0093] In both groups, only a few organoids were formed from 500 input cells.

[0094] At week 1, the appearance (Figure 4(a)) and frequency (Figure 4(b)) of forming organoids were similar for ATZ and rectal organoids for cultures initiated with 1,000 or 2,000 cells. At week 2, rectal organoid cultures contained more fully differentiated organoids (dark cell clusters), while ATZ organoid cultures contained more undifferentiated viable organoids (sac-like structures) (Figure 4(a)).

[0095] In cultures initiated with 1,000 and 2,000 cells, only few viable organoids were detected at weeks 3 and 4: 3%-4% for the ATZ and 0%-1% for the rectum (Figure 4(b)).

[0096] To determine progenitor capacity after 4 weeks, cultures were harvested and reinitiated with 500, 1,000, or 2,000 viable cells. Organoid growth medium (OGM) was refreshed at week 6 without changing the Matrigel® matrix, and organoid plating efficiency was assessed at weeks 5, 6, and 7 (Figure 4(b)). The highest plating efficiency was observed with 500 re-plated ATZ cells over weeks 5-7 (18%-21%). Plating efficiencies of 1,000 and 2,000 re-plated ATZ cells peaked at week 6 (19% and 15%, respectively) and decreased at week 7 (9% and 5%, respectively). Plating efficiencies of all re-plated rectal cells were <2% over weeks 5-7.

[0097] Taken together, these studies demonstrate increased progenitor potential in porcine ATZ crypt cells compared with rectal crypt cells, consistent with the existence of a long-term repopulating ATZ cell population.

[0098] Example 4. Protein expression of stem cell markers of embryonic lineages in freshly isolated crypts and crypt organoids. For the purposes of describing and illustrating certain examples and embodiments of the present disclosure, protein expression of stem cell markers of embryonic lineages in freshly isolated crypts and crypt organoids derived from pigs and Crohn's disease patients.

[0099] (Freshly isolated pig crypts) Cell surface stem cell markers were examined by flow cytometry on single cell preparations derived from freshly isolated crypts from small intestinal, colonic, rectal, and anorectal tissues. Expression of the stem cell and progenitor cell growth factor receptor KIT (CD117) was expressed at 3- to 5-fold higher levels in ATZ crypt cells compared to small intestinal, colonic, and rectal crypt cells (Figure 5(a)). Crypt cells from these tissues all expressed the stem cell and progenitor cell marker CD34 to various degrees, with ATZ crypt cells expressing the highest levels (Figure 5(b)).

[0100] In double-labeling experiments, 85% of ATZ cells expressed CD34 and 53% of cells expressed CD117; 47% of cells co-expressed CD34 and CD117; of the CD117-expressing cells, 88% co-expressed CD34 (Figure 5(c)).

[0101] (Pig crypt organoids) Cell surface stem cell markers were examined by flow cytometry on single cell preparations of fully differentiated pigeon ovoganoids derived from small intestine (SI), rectum, and anorectal tissues after 10 days of culture. Expression of KIT (CD117), CXCR4 (CD184), and GD2 was increased more than three-fold in ATZ organoids compared to SI and rectum organoids. CD45, a hematopoietic marker, was not detected in any crypt organoids (Figure 5(d)).

[0102] Because KIT and CXCR4 are markers of definitive endoderm and GD2 is a marker of primitive mesoderm, further investigation of the developmental lineage origin of the freshly isolated ATZ crypt cells was not performed. Intracellular protein markers confirmed the expression of transcription factors associated with the endodermal lineage (SOX17), mesodermal lineage (BRACHURY), 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)).

[0103] Collectively, these results indicate that porcine ATZ crypt cell populations express stem and progenitor protein markers for all three developmental lineages, and that endodermal and mesodermal markers were maintained in ATZ crypt organoids.

[0104] (Rectal crypt organoids from Crohn's disease patients) Cell surface expression of CD117 was detected in 80% of single cell preparations of rectal crypt organoids from Crohn's disease patients (Figure 5(f)).

[0105] Example 5. mRNA expression profiling of porcine ATZ crypt cells This example shows mRNA expression profiling of porcine ATZ crypt cells.

[0106] (Fresh porcine ATZ crypts) Transcriptional profiling of fresh porcine ATZ crypt cells confirmed basal expression of markers of mature epithelial cells (EPCAM, LYZ, MUC2, CHGA, CK18); markers of stem cells and progenitor cells (CD34, LGR5); and markers of developing stem cells of endodermal, mesodermal (BRACHURY), and ectodermal (PAX6) lineages; and expression of markers of pluripotent stem cells (NANOG, OCT4A) (Figure 6(a)). No mRNA expression of alkaline phosphatase (ALP) was detected, although the enzyme was detected by immunocytochemistry in Example 9.

[0107] (Porcine ATZ crypt organoids) Transcriptional profiling of ATZ crypt cells cultured in Organoid Growth Medium (IntestiCult™) retained mRNA expression for markers of stem cells of all three developmental lineages (Figure 6(b)). Lower levels of mRNA expression were detected for markers of mature epithelial cells when compared to fresh ATZ cells. mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0108] Collectively, transcriptional profiling of fresh ATZ crypts and organoids confirmed the presence of markers for stem cells of all three developmental lineages.

[0109] Example 6. Culture medium for maintaining and expanding multipotent porcine anorectal transitional stem cells This example demonstrates in vitro culture conditions for maintaining and expanding pluripotent ATZ cells.

[0110] Freshly isolated porcine ATZ crypt single cells were cultured for 14 days in either (1) feeder-free medium (mTeSR™, STEMCELL Technologies, Inc., Vancouver, Calif.) that maintains human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in an undifferentiated state; or (2) standard crypt organoid growth medium (IntestiCult™).

[0111] The frequency of the pluripotent stem cell markers described in Example 4 was evaluated by flow cytometry before (day 0) and after 14 days of culture. CD117, a marker of stem cells and progenitor cells, was expressed in 14% of ATZ crypt cells before (day 0), and increased to 35% in mTeSR and 28% in organoid growth medium (OGM) at day 14 (Figure 7(a)). BRACHYURY, an intracellular marker for mesoderm, was expressed in 97% of ATZ crypt cells before (day 0), and its expression level was maintained at 99% in mTeSR and 81% in OGM at day 14 (Figure 7(b)). SOX17, an intracellular marker for endoderm, was expressed in 34% of ATZ crypt cells before (day 0), and was expressed at <1% in mTeSR medium and 49% in OGM at day 14 (Figure 7(c)).

[0112] These results indicate that OGM may be better at maintaining ATZ crypt cells expressing markers of both endodermal and mesodermal developmental lineages.

[0113] Example 7. In vitro differentiation of porcine anorectal transitional cell-derived crypts into all three developmental lineages This example demonstrates that porcine anorectal transitional cell-derived crypts differentiate in vitro into all three developmental lineages (endoderm, mesoderm, and ectoderm).

[0114] The expression of protein and mRNA markers, respectively, in all three developmental lineages of fresh porcine ATZ crypt stem cells in Examples 4 and 5 led to studies to determine whether the freshly isolated ATZ crypt cells have the capacity to generate mature cells of the endodermal lineage (intestinal mucosa), mesodermal (blood vessels), and ectodermal (keratinocytes).

[0115] Example 7.1. Endoderm Differentiation Potential of Fresh Porcine ATZ Crypts Protein expression levels of mature endoderm cell markers of the intestinal mucosa were assessed after 2 weeks of culturing single cell preparations of fresh ATZ crypts embedded in Matrigel® matrix with OGM to generate organoids. Immunostaining of intact organoids was visualized for expression of lysozyme (LYSOZYME), proliferating cells (KI67), secretory cells (MUC2), and cytokeratin 18 (CK18), which is expressed in gastric epithelial monolayers (Figure 8(a-h)).

[0116] Flow cytometry analysis of fresh ATZ crypt cells showed that SOX17 decreased from 52% on day 0 to 34% on day 14, and KIT (CD117) and CXCR4 (CD184) increased two-fold during culture (Figure 9). The intestinal stem cell marker LGR5 was expressed in 3% on day 0 and increased to 22% on day 14.

[0117] Taken together, these results support that fresh ATZ crypts have the capacity to generate mature cell types derived from the endodermal lineage.

[0118] Example 7.2. Mesoderm differentiation potential of porcine ATZ crypts Protein expression levels of mature mesodermal cell markers were assessed after 2 weeks of culture of fresh ATZ crypt single cell preparations in MethoCult™ to test their ability to generate progeny in the hematopoietic lineage. No hematopoietic colonies were formed in the non-adherent methylcellulose layer.

[0119] However, single ATZ crypt-derived cells (Figure 10(a)) formed clusters in the adherent layer at day 3 (Figure 10(b)), and initiated and formed a network of vessel-like tubular structures at day 10 (Figure 10(c-d)). Immunostaining with CD31 (PECAM), an intracellular marker, was detected in the initiation and formation of a network of vessel-like tubular structures (Figure 10(e-f)). Flow cytometry analysis of fresh ATZ cells showed that BRACHURY expression decreased from 67% at day 0 to 23% at day 7 and further decreased to 7% at day 14; CD31 expression increased from 5% at day 0 to 19% at day 7 and further increased to 37% at day 14 (Figure 11).

[0120] Taken together, these results support that fresh ATZ crypts have the capacity to generate mature cell types derived from the mesodermal lineage.

[0121] Example 7.3. Ectoderm differentiation potential of porcine ATZ crypts 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, MO). Cell clusters developed on day 3, and by day 10, cobblestone-like adherent layers with keratinocyte morphology were formed in anal skin and ATZ cell cultures (Figure 12(a)). Co-expression of keratinocyte markers CK14 and CK15 was detected by immunostaining of anal skin adherent culture cells (Figure 12(b)) and ATZ adherent culture cells (Figure 12(c)).

[0122] Flow cytometry analysis of ATZ adherent cultured cells showed that expression of Pax6, an ectodermal lineage marker, decreased during culture from 18% on day 0 to 6% on day 7 and 5% on day 14 (Figure 12(d)). CK14 expression in ATZ adherent cultured cells increased over time during culture from 3% on day 0 to 18% on day 7 and 42% on day 14 (Figure 12(d)).

[0123] Taken together, these results support that fresh ATZ crypts have the capacity to generate mature cell types derived from the ectodermal lineage.

[0124] Example 8. Absence of multipotent stem cells of all three developmental lineages in porcine small intestinal crypts This example shows that porcine small intestinal (SI) crypts have the capacity to generate mature cells of endodermal, mesodermal, and ectodermal lineages in vitro, using the same methods as described in Example 7 for porcine ATZ crypts.

[0125] Small intestine (SI) single crypt cells were cultured for 2 weeks in organoid growth medium (IntestiCult™) that promotes differentiation into endoderm, organoid growth medium (MethoCult™) that promotes differentiation into mesoderm, and organoid growth medium (KSFM, Sigma-Aldrich, St. Louis, MO). Small intestine (SI) crypt cells generated organoids in organoid growth medium (OGM) as expected (Figure 13(a)). However, when cultured in MethoCult (Figure 13(b)) or KFSM (Figure 13(c)), no proliferation was detected for small intestine (SI) crypt cells on either day 7 or day 14.

[0126] Taken together, these results are consistent with the absence of multipotent stem cells in porcine small intestinal (SI) crypts that are capable of generating cell types of all three developmental lineages, using the assays shown for ATZ crypt cells.

[0127] Example 9. In vitro embryoid body assay to assess pluripotency of porcine ATZ crypt cells This example shows that single cell preparations of fresh ATZ crypts cultured in feeder-free mTeSR™ medium can promote embryoid body formation.

[0128] Freshly plated ATZ crypt single cells at high density generated cobblestone-like adherent layers by day 3; colonies of undifferentiated adherent cells developed by day 7; and differentiated adherent cells generated and migrated from the undifferentiated cell colonies (Figure 14(a)).

[0129] 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.

[0130] Taken together, the results of the in vitro embryoid body assay are consistent with ATZ crypt cells exhibiting pluripotent stem cell-like properties.

[0131] Example 10. Compatibility of porcine ATZ crypt cell growth with synthetic scaffolds This example demonstrates the biocompatibility of porcine ATZ crypt cells growth in organoid growth medium (OGM) with synthetic scaffolds.

[0132] Fresh single cell preparations of porcine ATZ crypt cells were cultured in Organoid Growth Medium (OGM) with 2%, 5%, or 10% synthetic non-functionalized scaffolds ("PG" or PeptiGel® Alpha 2, Manchester BioGel, Chesire, UK) and compared to standard Matrigel® matrix cultures.

[0133] Similar looking crypt organoids were generated by day 19 in all concentrations of PG and Matrigel® matrix (Figure 15(a)). However, the number of viable organoids in PG was reduced compared to organoid growth medium (OGM) at day 24 (Figure 15(b)), indicating that Matrigel® matrix provided additional growth factors for the proliferation of stem and progenitor cells.

[0134] A range of functionalized Alpha 4 PGs were assessed for activity due to the following binding domains: non-functionalized (Figure 16(a)), fibronectin (RGD) (Figure 16(b)), collagen (GFOGER) (Figure 16(c)), and laminin (IKVAV, YIGSR) (Figure 16(d-e)). An increase in the number of ATZ crypt organoids was associated with laminin-functionalized PG in liquid culture (Figure 16(de)), which was similar to the increase in the number of ATZ crypt organoids in Matrigel® matrix (Figure 16(f)).

[0135] Based on these studies, scaffold biocompatibility results with ATZ crypt cells. Equal volumes of IKVAV-functionalized PG and YIGSR-functionalized PG were used as pharmaceutical carriers for the in vivo studies described in Example 11.

[0136] Example 11. A preclinical porcine fistula model to evaluate hydrogel scaffolds supporting allogeneic adult porcine ATZ cells to heal fistulas. This example demonstrates that allogeneic adult porcine ATZ cells in a hydrogel scaffold can be used to treat anal fistulas.

[0137] Using a validated preclinical porcine fistula model previously developed to evaluate surgical and sealant treatments for perianal fistulas (Himpson et al. (2009) "An experimentally successful new sphincter-conserving treatment for anal fistula", Dis. Colon Rectum, 52(4):602-608), we tested the safety and efficacy of treating fistulas with allogeneic adult porcine ATZ cells.

[0138] Three fistulas were mechanically created in each of female white Landcross pigs from the lumen of the anorectal region through muscle and fat to the external skin (Figures 17 and 18). A thread or "seton" was inserted into the fistulas to promote chronic inflammation. After one month, the setons were removed and the fistula tracts were cleaned to remove granulation tissue before treatment.

[0139] Cell preparations of allogeneic porcine ATZ crypts from three pigs were collected prior to this study as described in Example 1 and cryopreserved in cryopreservation medium (CryoStor®, STEMCELL Technologies, Inc., Vancouver, Canada). The viability of the cryopreserved ATZ cells was confirmed prior to the study using the assay described in Example 10.

[0140] Treatment groups for each pig included: (1) no treatment control; (2) pharmaceutical carriers alone (PG-IKVAV and PG-YIGSR ("PG" = PeptiGel® or "PG scaffold")); and (3) ATZ cells + "PG scaffold" (Table 1). The study was terminated on day 90. Anorectal tissues were collected in 10% neutral buffered formalin (NBF). Paraffin-embedded tissues were sectioned for histology and stained with hematoxylin and eosin (H&E) and picrosirius red (PSR). [Table 1]

[0141] Example 12. Histological evidence that allogeneic adult porcine ATZ cells have anti-inflammatory properties and enable tissue remodeling for more effective closure of fistula tracts. This example demonstrates by histological analysis of tissue from the porcine fistula in Example 11 that tissue remodeling leads to closure (healing) of the fistula.

[0142] When treatment was withheld, the luminal side of the fistula tract within the adipose tissue was covered by fibroblast-like cells, indicating that this luminal gap may not close over time (Figure 19(a)).

[0143] For treatment of the fistula tracts with the PG scaffold alone, the fistula tracts within the adipose tissue were filled with inflammatory cells at day 90, indicating that the PG scaffold itself elicited a chronic inflammatory response that persisted throughout the treatment period, with no evidence of tissue remodeling (Figure 19(b)).

[0144] Treatment of the fistula tract with ATZ cells administered with the PG scaffold effectively filled the tract within the adipose tissue with remodeled fibrotic tissue, including signs of immature blood vessels, fibroblasts, smooth muscle cells, and immature adipose tissue (Figure 19(c) and Figure 20).

[0145] In summary, this in vivo study provided histological evidence that adult allogeneic ATZ stem cells and adult allogeneic ATZ progenitor cells (1) reduced PeptiGel® scaffold-mediated chronic inflammation and (2) substantially reduced fistula openings in vascularized, remodeled tissues.

[0146] Porcine ATZ cells in a pharmaceutical carrier exhibit anti-inflammatory properties in this porcine preclinical model similar to those of human adipose-derived mesenchymal stem cells (AdMSC, Alofisel™) used to treat Crohn's disease fistulas.

[0147] However, while ATZ cells in a pharmaceutical carrier in this validated preclinical model closed fistulas at 90 days, Alofisel™ treatment of Crohn's disease patients merely delayed the period between fistula clearance.

[0148] Example 13. A preclinical porcine fistula model to evaluate collagen scaffolds to support gender-mismatched allogeneic adult porcine ATZ cells to heal fistulas. This example demonstrates that allogeneic adult porcine ATZ cells in a collagen scaffold can be used to treat anal fistulas.

[0149] Cell preparations of allogeneic porcine ATZ crypts from one male pig were collected as described in Example 1 and cryopreserved in cryopreservation medium (CryoStor®, STEMCELL Technologies, Inc., Vancouver, Canada) prior to the study. Viability of the cryopreserved ATZ cells was confirmed prior to the study using the assay described in Example 10.

[0150] Three fistulas were mechanically created in one female white Landcross pig from the lumen of the anorectal region through muscle and fat to the external skin as in Example 11 (Figure 17). Setons were inserted into the fistulas to promote chronic inflammation. After one month, the setons were removed and the fistula tracts were cleaned to remove granulation tissue before treatment.

[0151] Pig treatment groups included: (1) no treatment control; (2) pharmaceutical carrier alone (Permacol™); and (3) ATZ cells + Permacol (Table 2). The study was terminated on day 63. Paraffin-embedded tissues were sectioned for histological examination and stained with hematoxylin and eosin (H&E) and picrosirius red (PSR). [Table 2]

[0152] Example 14. Histological evidence that collagen scaffolds support sex-mismatched allogeneic adult porcine ATZ cell tissue remodeling for more effective closure of fistula tracts. This example demonstrates by histological analysis of tissue from porcine anal fistulas in Example 13 that tissue remodeling leads to closure (healing) of the fistula.

[0153] When treatment was withheld, the luminal side of the fistula tract was covered by fibroblast-like cells, indicating that this luminal gap may not close over time (Figure 21(a)). At higher magnification, inflammatory cells, adipocytes, and immature blood vessels were observed (Figure 21(b)). The image in Figure 21(c) is in the same location as the image in Figure 21(a) but stained to highlight newly generated collagen fibers. All collagen features are shown in red. In the area where the untreated fistula tract was closed, highly inflammatory cells and very few blood vessels were detected (Figures 22(b) and 22(c)).

[0154] For treatment of the fistula with Permacol scaffold alone, a fistula was observed in the muscle (Figure 23(a)). At higher magnification, one end of the fistula (closest to the external skin) shows two well-controlled inflammatory nodules (Figure 23(b)). The image in Figure 23(c) is in the same location as the image in Figure 23(a), but stained to highlight the newly generated largen fibers.

[0155] For treatment of the fistula tracts with ATZ cells administered with Permacol, the fistula tracts within the fat show primarily fibrotic tissue with a few inflammatory cells (Figure 24(a)). The image in Figure 24(b) is in the same location as the image in Figure 24(a), but stained to highlight newly generated collagen fibers throughout the filled fistula tract.

[0156] (Note: the dark lines in FIG. 24 (indicated by solid arrows) are folds in the tissue, an artifact resulting from sections not being stretched sufficiently before mounting on slides).

[0157] At higher magnification in Figure 24(a), the internal morphology of the ATZ and Permacol tubes shows considerable regeneration at day 63 (Figure 24(c)), with numerous mature blood vessels containing red blood cells. In addition, there are obvious early muscle bundles containing distinct fiber types (i.e., presumably types I and II) staining in either light or darker shades of pink.

[0158] In summary, this in vivo study provided histological evidence that collagen scaffolds support sex-mismatched allogeneic adult porcine ATZ cell tissue remodeling for more effective closure (healing) of fistulas with mature blood vessels and early muscle bundles, and supported the multilineage differentiation potential of ATZ cells.

[0159] (Incorporated by reference) The entire disclosure of each of the patent and scientific literature referenced herein is incorporated by reference for all purposes.

[0160] (Equivalent) The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the above-described embodiments should be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the above description, and all changes that come within the meaning and scope of the claims are intended to be embraced within the scope of the claims.

Claims

1. A composition comprising isolated anorectal transition zone (ATZ) stem cells and a pharmaceutically acceptable carrier.

2. The composition of claim 1 , wherein the ATZ stem cells comprise progenitor cells.

3. The composition of claim 1 , wherein the ATZ stem cells comprise multipotent stem cells.

4. The composition of claim 1 , wherein the ATZ stem cells are capable of differentiating into endoderm cells, mesoderm cells, ectoderm cells, or a combination thereof.

5. The composition of claim 1 , wherein the ATZ stem cells are in vitro expanded ATZ (eATZ) cells.

6. The composition of claim 1, wherein the ATZ stem cells express at least one marker selected from the group consisting of CD34, CD117, and CD184.

7. The composition of claim 1 , wherein the ATZ stem cells do not express detectable levels of CD45.

8. The composition of claim 1, wherein the ATZ stem cells express at least one of NANOG and OCT4A.

9. The composition of claim 1 , wherein the cells are combined with an exogenous biocompatible scaffold.

10. The composition of claim 9 , wherein the scaffold comprises a synthetic scaffold.

11. The composition of claim 10 , wherein the scaffold comprises a biological scaffold.

12. The composition of claim 11 , wherein the scaffold comprises collagen.

13. The composition of claim 1 , wherein the ATZ stem cells are porcine cells.

14. The composition of claim 1 , wherein the ATZ stem cells are human cells.

15. The composition of claim 1 , further comprising a cryopreservation medium.

16. The composition of claim 1 , which is cryopreserved.

17. A pharmaceutical dosage form having disposed therein a composition according to any one of claims 1 to 16.

18. A method for preparing a composition according to any one of claims 1 to 15, comprising the steps of: (a) preparing a cell suspension by enzymatically digesting ATZ tissue with an enzyme, wherein the ATZ tissue is obtained from a subject; (b) optionally combining at least a portion of the cell suspension with a cryopreservation medium and cryopreserving the cell suspension; and (c) combining the cell suspension of step (a) or, optionally, step (b) with a pharmaceutically acceptable carrier. A method comprising:

19. 19. The method of claim 18, wherein the cell suspension comprises ATZ stem cells.

20. 20. The method of claim 19, wherein the ATZ stem cells comprise multipotent stem cells.

21. 21. The method of claim 20, wherein the ATZ stem cells comprise progenitor cells.

22. 21. The method of claim 20, wherein the ATZ stem cells are capable of differentiating into endoderm cells, mesoderm cells, ectoderm cells, or a combination thereof.

23. 22. The method of claim 21, wherein the ATZ stem cells express at least one marker selected from the group consisting of CD34, CD117, and CD184.

24. 21. The method of claim 20, wherein the ATZ stem cells do not express detectable levels of CD45.

25. The method of claim 20, wherein the ATZ stem cells express NANOG and / or OCT4A.

26. 20. The method of claim 19, wherein the ATZ stem cells in the suspension are expanded in vitro.

27. 20. The method of claim 18, wherein during step (c), the cells in the cell suspension are combined with an exogenous biocompatible scaffold.

28. 28. The method of claim 27, wherein the scaffold is a synthetic scaffold.

29. 28. The method of claim 27, wherein the scaffold is a biological scaffold.

30. 28. The method of claim 27, wherein the scaffold comprises a collagen-based scaffold.

31. 20. The method of claim 19, wherein the subject is a patient with anal fistula and the ATZ stem cells are autologous cells.

32. The method of claim 18, wherein the subject is a donor and the ATZ stem cells are allogeneically derived from the patient with anal fistula.

33. 19. The method of claim 18, wherein the subject is a human.

34. 20. A pharmaceutical composition made by the method of claim 18.

35. 10. The composition of claim 1 for use in treating anal fistula in a patient in need thereof, comprising: A composition characterized by being administered to the anal fistula.

36. 36. The composition of claim 35, wherein administration of the composition closes or heals the anal fistula.

37. 37. The composition of claim 36, wherein the anal fistula is closed or healed by proliferation of fibrotic tissue.

38. 36. The composition of claim 35, wherein the anal fistula is a perianal fistula.

39. 36. The composition of claim 35, wherein the anal fistula is an intractable fistula.

40. 36. The composition of claim 35, wherein the patient has Crohn's disease.

41. 36. The composition of claim 35, wherein the subject has an anal fistula of unknown etiology.

42. A composition according to any one of claims 1 to 16 for use in the treatment of anal fistulas.

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