Patch transplantation of stem cells / precursors into solid organs

Abstract

Compositions and methods are provided for transplanting cells into solid organs (particularly internal organs) via a grafting strategy. These methods and compositions can be used to repair diseased organs or to establish experimental models of host disease states. The methods involve the attachment of a patch graft containing donor cells to the surface of a tissue or organ. The donor cells can be a mixture of stem cells / progenitors that support early lineage-stage mesenchymal cells. The patch graft promotes donor cell migration into the host organ and supports successful integration of the donor and host cells to repair the diseased organ.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefit of U.S. Application No. 16 / 422,086, filed on 24 May 2019. Furthermore, this application relates to International Patent Application PCT / US2018 / 036960, filed on 11 June 2018, which claims priority of U.S. Provisional Patent Application No. 62 / 518,380, filed on 12 June 2017, and No. 62 / 664,694, filed on 30 April 2018. The contents of these applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing filed electronically in ASCII format, which is incorporated herein by reference in its entirety.

[0003] This invention generally relates to the field of cell transplantation or tissue engraftment. More specifically, to transplantation from solid organ to solid organ or tissue, particularly to internal organs. This invention relates to compositions and methods that provide strategies for the rapid transplantation, engraftment, and integration of cells into solid organ tissues to treat diseases of solid organs or to establish disease model systems. A representative example of this possibility is cell therapy for the treatment of liver or pancreatic diseases. [Background technology]

[0004] There is an unmet need for transplantation strategies involving solid organ-derived cells, hematopoietic cells, mesenchymal stem cells, or strategies different from those used in skin. Transplantation of hematopoietic and mesenchymal cells, typically derived from single-cell suspensions, is conventionally performed via vascular channels and relies on the activation of adhesion molecules at the relevant target site due to microenvironmental signaling, a process called "homing." Methods used in skin employ transplantation techniques using cells and / or tissues applied directly to the target site.

[0005] Transplantation of cells derived from solid organs other than skin has traditionally been done via vascular routes or by direct injection into the tissue. This approach is not logical because adhesion molecules on cells derived from solid organs are constantly activated, leading to rapid (seconds) cell aggregation that can generate life-threatening embolisms. Even when embolisms are well managed to minimize health risks, cell engraftment efficiency is low, at less than 20% for adult cells and even lower (<5%) for stem cells / progenitors. Most transplanted cells die or are transported to ectopic sites where they can survive for months, generating tissue in inappropriate locations and potentially having clinically adverse effects. The small percentage of cells that engraft at the target site integrate slowly, taking weeks to months to reconstruct critical parts of the tissue.

[0006] When cells are coated with hyaluronan and delivered via vascular pathways through hyaluronan clearance in tissue (e.g., liver), there are improvements in engraftment in the liver and minimization of ectopic cell delivery. However, this improvement is still less efficient than using transplantation strategies and, importantly, still allows for delivery of cells to ectopic sites.

[0007] Grafting solid organs is challenging to design due to concerns about the size, shape, and complexity of the organ structure, in addition to dynamic mechanical forces. Therefore, there remains a need to improve methods for cell engraftment into solid organs. This disclosure addresses this need and provides relevant advantages. [Overview of the Initiative]

[0008] Described herein are novel patch graft compositions and methods for transplanting cells into tissues and solid organs.

[0009] In one embodiment, the present disclosure relates to a method for engrafting cells onto a solid organelle of an object that requires them, the method being This involves bringing the patch graft into contact with a solid organ. The patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a biomaterial having first viscoelastic properties, and the biomaterial facilitates contact between at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both, to promote engraftment between cells of a solid organ. This includes clearly indicating that at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both are engrafted among the cells of a solid organ.

[0010] In some embodiments, demonstrating that at least a portion of the aforementioned epithelial cells are engrafted among the cells of the solid organ is demonstrated by measuring the secretion level from the solid organ or the metabolic effect of the solid organ in a biological sample obtained from the subject.

[0011] In another aspect, the disclosure relates to a method for engrafting cells onto a solid organelle of an object that requires them, the method being This involves bringing the patch graft into contact with a solid organ. The patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a hydrogel layer having first viscoelastic properties, and the hydrogel facilitates the migration of at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both, from the patch across the outer surface of a solid organ, thereby facilitating contact. This includes demonstrating that at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both are migrating along the outer surface of the solid organ.

[0012] In some embodiments, explicit indication includes measuring a parameter or a change in a parameter that indicates the physiological effect on the target brought about by migrating cells.

[0013] In some embodiments, the patch graft further includes a backing that facilitates the migration of at least a portion of the mixture of epithelial and mesenchymal cells to a solid organ.

[0014] In some embodiments, at least a portion of the mixture of epithelial and mesenchymal cells migrates across a substantial width of the solid organ and distributes throughout the solid organ.

[0015] In some embodiments, the solid organ is an endodermal organ.

[0016] In some embodiments, the solid organ is an endodermal organ including the liver, pancreas, intestine, lung, bile duct, thymus, thyroid, parathyroid, and the urogenital sinus regions of the prostate and vagina.

[0017] In some embodiments, the endodermal organ includes the liver and engraftment is accompanied by remodeling of Glisson's capsule.

[0018] In some embodiments, the present disclosure provides a method of further generating a combination of (i) engrafted epithelial and mesenchymal cells and (ii) host cells.

[0019] In some embodiments, the method of the present disclosure generates functional liver parenchymal cells.

[0020] In some embodiments, the parenchymal cells include hepatocytes and cholangiocytes.

[0021] In some embodiments, the endodermal organ includes the pancreas and engraftment is accompanied by remodeling of the pancreatic capsule and pancreatic tissue near the transplantation site.

[0022] In some embodiments, the method of the present disclosure generates functional pancreatic cells.

[0023] In some embodiments, the functional pancreatic cells include acinar cells and islets.

[0024] In some embodiments, the pancreas secretes at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide at increased levels.

[0025] In some embodiments, the pancreas exhibits a metabolic effect of reducing blood glucose levels.

[0026] In some embodiments, the pancreas exhibits a metabolic effect of increased glucose tolerance.

[0027] In some embodiments, the pancreas secretes digestive enzymes or bicarbonate fluids at increased levels.

[0028] In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or a combination thereof.

[0029] In some embodiments, the methods of the present disclosure result in an increase in the levels of metabolites derived from digestive enzymes secreted by the pancreas.

[0030] In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or a combination thereof.

[0031] In some embodiments, the methods disclosed herein result in improved digestion.

[0032] In some embodiments, the liver secretes fluids rich in urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonates, or blood clotting factors.

[0033] In some embodiments, the biological samples obtained from the subjects show a decrease in at least one level of cholesterol, blood glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase.

[0034] In some embodiments, the patch includes a backing arranged over a hydrogel containing a mixture of epithelial cells and mesenchymal cells.

[0035] In some embodiments, the backing is used to connect the hydrogel layer to the host organ.

[0036] In some embodiments, at least one or both of the epithelial cells and mesenchymal cells are early lineage stage cells.

[0037] In some embodiments, early lineage mesenchymal cells (ELSMCs) include angioblasts, endothelial precursors, astrocytes, or a combination thereof.

[0038] In some embodiments, early lineage stage epithelial cells (ELSE), ELSMC, or both are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS).

[0039] In some embodiments, the epithelial cells are mature, and the mesenchymal cells are ELSMCs.

[0040] In another aspect, the disclosure relates to introducing, restoring, increasing, or improving the functionality of a diseased, impaired, or dysfunctional solid organ, and the method includes contacting the diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells under conditions that promote engraftment of epithelial cells and mesenchymal cells, and demonstrating the introduction, restoration, increasing, or improving the functionality of the diseased, impaired, or dysfunctional solid organ.

[0041] In some embodiments, explicit representation includes measuring the level of secreted or metabolite products or effects in a biological sample obtained from the subject.

[0042] In some embodiments, the methods of the present disclosure further include specifying that at least a portion of the mixture of epithelial cells and mesenchymal cells is distributed among the cells of a host organ.

[0043] In some embodiments, the exposed surface of the patch graft includes a covering that inhibits the adhesion of the patch graft to nearby organs and tissues.

[0044] In some embodiments, the solid organs include endodermal organs.

[0045] In some embodiments, the endoderm organs include regions derived from the urogenital sinuses of the liver, pancreas, intestines, lungs, bile ducts, thymus, thyroid gland, parathyroid gland, or prostate or vagina.

[0046] In some embodiments, the solid organ includes the pancreas, and an increase in the secretion level of at least one of the following is measured: insulin, c-peptide glucagon, somatostatin, or pancreatic polypeptide.

[0047] In some embodiments, the solid organ includes the pancreas, and a decrease in blood glucose levels is measured.

[0048] In some embodiments, the solid organ includes the pancreas, and increased glucose tolerance is demonstrated.

[0049] In some embodiments, the solid organ includes the pancreas, and an increase in the levels of digestive enzymes or bicarbonate fluid is explicitly indicated.

[0050] In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase.

[0051] In some embodiments, the solid organ includes the pancreas, and an increase in the level of products derived from digestive enzymes secreted by the pancreas is measured.

[0052] In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase.

[0053] In some embodiments, the solid organs include the pancreas, demonstrating improved digestion.

[0054] In some embodiments, the solid organ includes a liver, and the secretions include fluids rich in urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonates, blood clotting factors, or a combination thereof.

[0055] In some embodiments, the solid organ includes the liver, and the metabolic effect is a reduction in the levels of one or more of the following: cholesterol, blood glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase.

[0056] In some embodiments, the solid organ includes the liver, and the subject suffers from type 1 tyrosinemia.

[0057] In some embodiments, the metabolic effect is a decrease in tyrosine or alpha-fetoprotein levels.

[0058] In another aspect, the disclosure relates to a method for treating a subject diagnosed with a condition at least partially resulting from having a disease, disorder, or dysfunction of a solid organ, the method being (i) Contacting a diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells, (ii) Enabling epithelial cells and mesenchymal cells to migrate to and distribute among the cells of the host solid organ, (iii) A method comprising demonstrating that the negative effects of a diseased, impaired, or dysfunctional solid organ are mitigated in the subject of treatment.

[0059] In some embodiments, explicit representation includes measuring the level of secreted or metabolite products or effects in a biological sample obtained from the subject.

[0060] In some embodiments, the migration and distribution phases result in the alleviation of disease, impairment, or dysfunction.

[0061] In some embodiments, the solid organs are endoderm organs.

[0062] In some embodiments, the endoderm organs include the liver, pancreas, intestines, lungs, bile ducts, thymus, thyroid gland, parathyroid gland, and urogenital sinus regions of the prostate or vagina.

[0063] In some embodiments, the endodermal organ is the pancreas, and the subject suffers from diabetes.

[0064] In some embodiments, an increase in the level of at least one of the following is measured: insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide.

[0065] In some embodiments, a decrease in blood glucose levels is clearly demonstrated.

[0066] In some embodiments, an increase in glucose tolerance is explicitly demonstrated.

[0067] In some embodiments, the subjects include mammals.

[0068] In some embodiments, the mammal is a human.

[0069] In another aspect, the present disclosure relates to a patch graft comprising a mixture of epithelial cells and mesenchymal cells and one or more biomaterial layers, wherein the layers are at least a) A first inner layer for contacting solid organs, exhibiting first viscoelastic properties, incorporating a mixture of epithelial cells and mesenchymal cells, supporting the ability of epithelial and mesenchymal cells to produce secretory matrix metalloproteinases (MMPs), and promoting the viability and immaturity of the aforementioned epithelial and mesenchymal cells, b) A backing that optionally provides a barrier to cells migrating in directions other than solid organs, and exhibits a second viscoelastic property, c) Optionally, a third outer layer of covering or material near the patch graft to minimize adhesion of the patch graft to the inner wall and / or inner surface of the body cavity, including organs. and Includes The aforementioned viscoelastic properties are determined by measuring the fluidic properties and expressed in Pascals (Pa). ru.

[0070] In some embodiments, the epithelial cells include early lineage stage epithelial cells (ELSE) and the mesenchymal cells include early lineage stage mesenchymal cells (ELSMC), or the epithelial cells and mesenchymal cells are at a later lineage stage but are equivalent to each other.

[0071] In some embodiments, ELSMCs include angioblasts, endothelial precursors, astrocytes, or a combination thereof.

[0072] In some embodiments, ELSE and / or ELSMC are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS).

[0073] In some embodiments, the epithelial cells are in the late lineage stage, the mesenchymal cells are early lineage mesenchymal cells (ELSMCs), or the mesenchymal cells are in the late lineage stage and the epithelial cells are early lineage epithelial cells (ELSEs).

[0074] In some embodiments, the second viscoelastic property (expressed in Pa) has a higher value than the first viscoelastic property.

[0075] In some embodiments, one or more biomaterial layers comprise a hydrogel, which further comprises minimally sulfated or non-sulfated glycosaminoglycans.

[0076] In some embodiments, the non-sulfated glycosaminoglycan includes hyaluronan.

[0077] In some embodiments, the hyaluronane comprises thiol-modified hyaluronane, and its gelation by disulfide crosslinking is induced in the presence of polyethylene glycol diacrylate (PEGDA).

[0078] In some embodiments, the fluidity is determined, at least in part, by the initial concentrations and stiffness of thiol-modified hyaluronan and PEGDA before gelation, and by the final stiffness of the hydrogel after gelation, which is achieved by the precise ratio of the volumes of thiol-modified hyaluronan and PEGDA.

[0079] In some embodiments, the first inner layer exhibits a first viscoelasticity ranging from about 50 Pa to about 150 Pa.

[0080] In some embodiments, the optional backing includes a hyaluronan hydrogel layer exhibiting viscoelasticity from about 600 to about 800 Pa.

[0081] In some embodiments, an optional third outer layer comprises a hyaluronan hydrogel layer having viscoelastic properties ranging from about 200 to about 300 Pa.

[0082] In some embodiments, the backing includes silk.

[0083] In some embodiments, the silk backing includes refined fibroin of silkworm silk woven into the scaffold, including Seri-Silk® or Contour Seri Silk®.

[0084] In some embodiments, epithelial cells include bile dendritic stem cells (BTSCs), and mesenchymal cells include early lineage mesenchymal cells (ELSMCs).

[0085] In some embodiments, ELSMCs include angioblasts and their direct offspring, endothelial precursors, astrocyte precursors, or a combination thereof.

[0086] In some embodiments, angioblasts express CD117, CD133, and VEGFr, but do not express CD31.

[0087] In some embodiments, the endothelial cell precursor expresses CD133, VEGFr, CD31, and von Willebrand factor.

[0088] In some embodiments, the astrocyte precursor expresses CD146, ICAM-1, and alpha-smooth muscle actin (ASMA), and is negative for vitamin A.

[0089] In some embodiments, a mixture of epithelial cells and mesenchymal cells is produced by selectively repeating a panning procedure to remove cells that adhere to a tissue culture dish or surface within about 15 to 30 minutes at 37°C, thereby depleting a cell suspension of mature mesenchymal cells.

[0090] In some embodiments, the remaining cell suspension is cultured on a low-adhesion dish in serum-free medium until multiple organoids are formed by the self-organization of epithelial and mesenchymal cells.

[0091] In some embodiments, the serum-free medium comprises a basic medium (copper-free, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, fatty acid-free, fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg / ml transferrin / Fe, 5 μg / ml insulin, and a mixture of purified free fatty acids that exist in complex with highly purified, fatty acid-free albumin).

[0092] In some embodiments, the serum-free medium further contains 10 μg / mL of high-density lipoprotein.

[0093] In some embodiments, multiple organoids are formed after approximately 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0094] In some embodiments, multiple organoids are, a) At least one marker selected from the pluripotent gene group consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1, b) At least one marker selected from the endodermal transcription factor group consisting of Sox9, Sox17, Pdx1, HNF4 alpha, HNFB1, and ONECUT2, c) A BTSC that is positive for at least one marker selected from the group of stem cell / precursor-related surface markers consisting of one or more isoforms of EpCAM, NCAM, LGR5, CD44, CXCR4, sodium-iodine cotransporter (NIS), CD49 (integrin A6), CD29 (integrin B1), and integrin B4; BTSCs are negative for markers of mature hepatocytes or pancreatic cells, including P450, aquaporins, enzymes involved in bile production, amylase, and digestive enzymes.

[0095] In some embodiments, one or more biomaterial layers include recombinant MMPs.

[0096] In some embodiments, one or more biomaterial layers include cells that have been engineered to express MMPs. [Brief explanation of the drawing]

[0097] The patent or application file contains colored drawings. Copies of the publication of the patent or application containing the colored drawings will be provided by the Office upon request and payment of the necessary fees.

[0098] [Figure 1-1] Figures 1A to 1D show: A) A schematic diagram of the estimated processes and time required for organoid preparation, patch graft assembly, and surgical procedure. B) Donor cells for use in stem cell patch grafts were isolated from a cell suspension of biliary dendritic tissue derived from transgenic pigs possessing green fluorescent protein (GFP) linked to H-2B (ACTB-IRES-pH2B-eGFP). The cells were prepared as organoids (suspension aggregates of epithelial cells and their mesenchymal cell partners) in serum-free Kubota medium and low-adhesion culture dishes. Biliary dendritic stem cell (BTSC) organoids and their early lineage mesenchymal cell (ELSM) partners (angioblasts and precursors that become endothelial and astrocytes) are shown in contrast to phase-contrast microscopy images showing the expression of the transgene GFP69. Histological images of paraffin-embedded, sectioned, and hematoxylin / eosin-stained stem cell organoids are shown. (d) Magnified images of BTSC and ELSMC organoids. C) Immunofluorescence (IF) images clearly showing the expression of stem cell, liver, and pancreatic markers, indicating that these cells are precursors of both liver and pancreas, as shown in multiple prior publications and summarized in reviews. D) A representative example of a qRT-PCR assay to evaluate the expression of various genes in organoids and indicate that the cells are stem cells or early precursors. Mature hepatocytes derived from piglet liver were used as a control. [Figure 1-2] Same as above. [Figure 2-1]Figures 2A-2E show a schematic diagram of a patch graft attached to a pig liver, with the graft composition shown at the bottom. The graft consists of early lineage cells, both epithelial and mesenchymal, necessary for the production of matrix metalloproteinases (MMPs), which are major regulators of cell motility, migration, and engraftment. The graft structure comprises layers of biomaterial and cells attached to the target site. For cell engraftment, the matrix component of the graft biomaterial adjacent to the target site must be soft (approximately 100 Pa), such as hyaluronan hydrogel. The culture medium must be devoid of serum, growth factors, and cytokines that affect donor cell differentiation and should be adapted to the survival and proliferation of early lineage cells such as stem cells / precursors. The backing should have sufficient tensile strength for use in surgical procedures, but its effect on donor cell differentiation should be neutral (type I collagen should be avoided). The backing is impregnated or coated with a more rigid 10× hydrogel (approximately 700 Pa) to act as a barrier directing donor cell migration towards the target tissue and minimizing adhesion. After attachment to the target site, a 2× HA hydrogel, which is sufficiently fluid to coat or coat the outer surface, is added to further minimize adhesion. × indicates the stiffness of the cross-linked hydrogel. B) A graft attached to the host liver. C) A schematic diagram of the graft showing the layers that make up the graft composition. D) An assay to experimentally evaluate the fluid properties (shear and compressive mechanical forces) of a specific hydrogel layer. E) Analysis of the fluid properties of a three-layer hyaluronane hydrogel. [Figure 2-2] Same as above. [Figure 3-1]Figures 3A–3C show A) Masson's trichrome stained images of patch grafts one week later. Masson's trichrome distinguishes collagen (blue), cytoplasm (red), and nucleus (dark purple). This was used to distinguish Glisson's sheath (usually adjacent to the hepatic lobules) and adhesion on the outer surface of the graft. In the low-magnification image (1), the trichrome staining strongly revealed that the host tissue was often observed to separate from the backing and graft in the first week after transplantation, which is thought to be due to the secretion of MMPs. The hepatic lobules adjacent to the graft show decolorization, which was later found to be caused by the penetration of hyaluronan into the tissue. A higher-magnification image (2) of a section following (1), stained hematoxylin / eosin, shows evidence of extensive remodeling areas, where Glisson's sheath appears altered and absent. Higher-magnification images of two areas within the remodeling zone highlight the inflammatory response (a) and hepatic lobule remodeling (b). B) Trichrome staining of the patch graft after 3 weeks. Hyaluronan was reabsorbed, and the reduction in MMP expression allowed donor cells to mature. Inflammation was significantly reduced, and a tissue band (2) between the graft and the liver parenchyma and a recovery of the liver's histological structure remained. C) A porcine BTSC / ELSMC graft attached to the liver of an NRG / FAH mouse maintained for 30 days without nitisinone. Hematoxylin / eosin staining. [Figure 3-2] Same as above. [Figure 4-1]Figures 4A–4E show: A) Low-magnification images of patch grafts on the surface of pig liver one week later. Black areas constitute the hyaluronan regions of the graft. Donor GFP± cells (with pink nuclei; white arrows indicate regions with numerous donor GFP-BTSCs / ELSMCs) were visualized by labeling with an antibody against GFP and secondarily with an antibody linked to the red fluorescent probe Alexa594. Host nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI). Host tissue (a) spreads within the hyaluronan (HA: black background) of the graft; tissue adjacent to the backing contains organoids in places, but most donor cells were dispersed within cells that are easily identified as having pink nuclei. There is no evidence of the normal structure of Glisson's sheath in this area constituting the region of remodeling. B) Engraftment and migration of donor cells were rapid. Within one week, all identified donor cells were located within the host liver, both near the graft site and on the opposite side of the liver lobe (estimated distance from the graft site is approximately 1.5 cm, a significant distance for donor cells to migrate in one week). Donor cells (pink nuclei) are shown near lobules of mature host hepatocytes (dark green due to lipofuscin autofluorescence) at a distance of approximately 1.5 cm from the graft site side of the liver lobe. C) Maturation of donor cells to adult fate occurred in parallel with HA reabsorption. The region containing donor GFP+ cells (single cells with pink nuclei) near dark green (lipofuscin autofluorescence) host hepatocytes (a) is expanded and is easily distinguishable from mature donor (b) hepatocytes (pink nuclei) and the limited lineage derived from donor GFP+ stem cells. Some GFP-labeled cytoplasmic staining was observed, particularly in the first week post-transplant, but nuclear staining gradually increased by the second week (see Figures 4D-4E). In other IHC assays (data not shown), the bright green color of cells surrounded by plates of both host and donor hepatocytes was confirmed by the IHC assay to be a mixture of mesenchymal cells (epithelial and astrocytes). The bright green autofluorescence derived from these cells was easily distinguishable from the dark deep green of lipofuscin. D) Examination of porcine liver one week after patch transplantation.Immunohistochemical testing for pancytic keratin (pCK) and Sox9, as well as immunofluorescence (IF) staining, were performed on consecutive 3 μm sections using Sirius Red staining, i.e., an azo dye that stains collagen. At the patch graft site, the grafted donor cells were associated with hepatic lobules. In the upper panel (original magnification = 5×), the patch graft consists of mesenchymal and epithelial pCK+ cells (arrows). The middle panel provides a higher magnification (20x). The epithelial cells exhibit the immunophenotype typical of biliary dendritic stem cells (BTSCs), expressing bile cytokeratin (pCK) and the endodermal stem cell marker Sox9. GFP labeling in these cells was clearly present in the nucleus. BTSCs within the patch graft are arranged in cell rows reconstructing bile ducts (arrows) and are directly continuous with the hepatocyte plate of the adjacent hepatic lobule (arrowhead). Hepatocytes in the lobules are negative for pCK and Sox9. In the lower panel (original magnification = 20×), immunofluorescence staining for GFP allows for the identification of individual graft cells and their progeny. Hepatocytes in lobules adjacent to the patch graft are GFP-positive, partially in the nucleus and partially intracellularly, indicating that these were donor cells integrated into the host liver parenchyma. At the interface between the patch graft and the hepatic lobules, pCK+ / GFP+ tubules are directly continuous with GFP+ / pCK- cells within the lobules (arrowheads), suggesting the maturation of transplanted cells leading to their hepatocyte fate. E) Examination of porcine liver two weeks after patch transplantation. IF staining revealed that GFP+ cells were present in lobules away from the graft site and co-expressed mature hepatocyte markers such as hepatocyte nuclear factor (HNF) 4α and albumin. GFP labeling was mainly in the nucleus in donor cells. Separate or fused channels were included. Nuclei are shown in blue (DAPI). Original magnification: 40×. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1]Figures 5A–5P show the rescue of NRG / FAH mice from type I tyrosinemia using a porcine BTSC / ELSMC patch graft. Evidence of donor cell entry into the liver is shown in Figure 3C. The porcine BTSC / ELSMC patch graft was surgically applied to the livers of NRG / FAH mice, followed by regular watering (withdrawal of nitisinone). Figure 11 shows the survival figures of the mice along with their body weight. The experimental subjects with the patch graft survived and gained weight after 30 days, while the controls began to lose weight after 2 weeks and had to be euthanized by day 17. The kidneys of the animals with the patch graft containing cells were normal (Figure 5N). Figures 5A–5B show the livers of positive control NRG / FAH mice in a host treated with Nitison (NTBC), visualized by low and high magnification images of H&E stained sections of the liver. Figures 5C-5D) show negative control mice with cell-free patch grafts, visualized by low- and high-magnification images of H&E-stained sections of NRG / FAH mouse liver that were not treated with NTBC, and evaluated on day 16. Figures 5E-5F) show low- and high-magnification images of liver sections of NRG / FAH mouse liver patches grafted with porcine BTSC / ELSMC (see also Figure 3C). Figures 5G-5H) show low- and high-magnification images of grafted livers stained for GFP (using an antibody against GFP conjugated to Novo Red). Note that GFP+ cells are present throughout the liver, and GFP is mainly localized in the nucleus (Figure 5H). Figure 5I shows an image illustrating the localization of GFP linked to histone H2B. Note that GFP (linked to histone H2B) expression is present in both the nucleus and cytoplasm of some cells. Figure 5P shows an immunofluorescence image showing weak cytoplasmic labeling by an antibody against histone H2B. Figures 5J-5K show low and high magnification images of control sections prepared without primary antibody. Figures 5L-5M show controls for testing mouse liver (NRH / FAH) patches transplanted with porcine BTSC / ELSMC. Figure 5L shows the negative control for goat antibody. Figure 5M shows the negative control for rabbit antibody.Figure 5N shows images of kidneys obtained from NRH / FAH mice with patch grafts on the liver, (1) a graft without cells, and (2) a graft containing BTSC / ELSMCs. Figure 50 shows the results for porcine fumaacetoacetate hydrolase (FAH) in mouse livers (NRH / FAH) patch-grafted with porcine BTSC / ELSMCs, as shown in Figure 5P. Note that the histone H2B is mainly found in the nucleus, but is also found in the cytoplasm of some cells. [Figure 5-2] Same as above. [Figure 6-1]Figures 6A–6H show isoforms of secreted and membrane-bound MMPs expressed by both epithelial stem cells / precursors and their partners ELSMCs, compared to those in various mature liver cells. Quantification of expression levels showed that the membrane-bound morphology was similar in both stem cells / precursors / ELMCs and mature epithelial / mesenchymal partners. Note the comparison in (D). In contrast, the secreted morphology was expressed at very high levels in stem cells / precursors / ELMCs and at low or very low levels in mature cell types. The adult cell populations analyzed were isolated from suspensions of piglet liver and biliary dendritic tissue and consisted of CD45+ cells (hematopoietic cells), CD146+ cells (astrocytic cells), CD31+ cells (endothelial cells), and EpCAM+ / CD45- cells (differentiated precursors and diploid hepatocytes and cholangiocarcinomas). BTSCs were isolated from biliary dendritic cells by the protocol described in the Methods section. Figure 6A) shows MMP expression in BTSCs. Figure 6B) shows MMP expression in astrocytes and endothelium. Figure 6C) shows MMP expression in 2N (diploid) hepatocytes. Figure 6D) shows representative MMPs compared between adult cell types and BTSCs. Figure 6E shows representative MMP expression in the remodeling region using BTSC / ELSMC grafts. The compartment adjacent to the BTSC / ELSMC patch graft is stained with trichrome and shows the remodeling region (in brackets). Figure 6F) shows a representative image of the IHC assay for MMP1 (Novo-red+). Methyl green was used as the background stain. Figure 6G) shows the area stained with MMP2 (Novo-red+). Hematoxylin was used as the background stain. Within the hepatic lobules, the remodeling process included plates or cords (rust-colored) of MMP2+ cells that had transitioned to complete disappearance of the lobular structure. With HA clearance, the lobular structure reappeared. [Figure 6-2] Same as above. [Figure 7]Figure 7 shows schematic examples of engraftment and integration phenomena in the liver and pancreas. Patch grafts were attached to the liver via sutures at the corners of the patches. To minimize adverse reactions by the pancreas (e.g., autolysis), the patch grafts were secured to the pancreas either completely with surgical glue at the corners of the patches (mice), or one end of the patch was sutured to the duodenum, and the other end was covered with surgical glue at the corner to conceal the pancreas (pig). [Figure 8] Figures 8A and 8B show a comparison between transgenic pig liver control and wild-type pig liver control. Both are unstained frozen sections and were imaged for green fluorescence. The high-magnification image of a frozen section of liver obtained from a transgenic pig (8A) shows punctate GFP+-H2B ​​fluorescence due to nuclear localization. However, note the variation in fluorescence levels between different cells. In contrast, the frozen section obtained from the liver of a wild-type pig (8B) shows green fluorescence originating from the autofluorescence of lipofuscin and other components, and is clearly cytoplasmic. [Figure 9] Figures 9A and 9B show the remodeling zone near the graft site. During the first two weeks after transplantation, the area adjacent to the graft undergoes extensive remodeling of the Glisson's sheath and the basal lobular structure of the liver parenchyma. See also Figure 3. Images were created using higher magnification images of specific areas of the remodeling zone at 1 week (9A) and 2 weeks (9B) after graft transplantation. Staining: Hematoxylin / eosin. [Figure 10] Figure 10 shows adverse conditions associated with patch grafts with a specific backing. These adverse conditions include sites of necrosis, adhesions, and cholestasis, with cholestasis being found to occur when the graft was placed too close to several ducts, resulting in occlusion of those ducts due to swelling. [Figure 11]Figure 11 shows a chart of survival rate and body weight in NRG / FAH mice that were patch-grafted with porcine BTSC / ELSMC and then deprived of the drug nitisinone (NTBC). The chart begins with animals after drug deprivation. Animals with patch grafts (n3) remained stable and actually gained body weight over the 30-day trial. In contrast, controls (n2) were initially stable but began to decline within about two weeks. They needed to be euthanized by day 16. [Figure 12] Figures 12A–12E illustrate adverse conditions associated with patch grafts with a specific backing. Adverse reactions included necrosis (12A), discoloration (12B), and adhesion (12C). A difficulty that proved independent of the biomaterial and composition of the graft was skin infection (12D, 12E). Skin infection demonstrated common problems resulting from immunosuppression. Infection was observed only in the skin and not within the peritoneum or associated with the graft. [Figure 13] Figures 13A–13C show frozen sections obtained from the livers of NRG / FAH mice that received a porcine GFP+ BTSC / ELSMC patch graft (Figure 13A). Mice were evaluated one month after transplantation. Sections were stained with DAPI, but not with the primary antibody. The overall pale green background is due to the autofluorescence of various components of the liver. Bar: 500 μm. Mice that received a porcine BTSC / ELSMC patch graft and were evaluated one month later. Figure 13B shows a frozen section showing host cells (blue nuclei and yellow / green cytoplasm) interspersed in tissue predominantly dominated by donor cells (red / purple nuclei). Bar: 500 μm. Figure 13C shows a higher magnification image of 13B with labels used to indicate mouse cells interspersed in tissue predominantly dominated by donor (porcine, GFP+) cells. Bar: 100 μm. [Figure 14]Figure 14 shows the rapid distribution and integration of donor cells throughout the host tissue. The schematic diagram shows regions of piglet liver sampled one week after surgical application of a GFP+BTSC patch graft to the piglet liver. DNA PCR assays for GFP expression in different regions of the piglet liver were prepared. The agarose gels show the results of the DNA PCR assays for GFP expression in each of these zones. [Figure 15] Figures 15A to 15D show data obtained from serological tests in mice treated with patch grafts containing BTSC / ELSMC mouse organoids derived from DS-red mice. The control group received cell-free grafts. The patch grafts were attached to the pancreas of NRG / Akita mice, a mouse model of type 1 diabetes. There were 3 mice in the control group (untreated or treated with cell-free biomaterial), and 4 and 5 diabetic mice treated with patch grafts. A) shows blood glucose levels in untreated diabetic mice (circle), diabetic mice treated with graft biomaterial only (square), and diabetic mice treated with patch graft-containing cells (triangle). B) shows serum levels of C-peptide in untreated diabetic mice (see the first column on days 0, 7, 14, and 21), diabetic mice treated with graft biomaterial alone (see the second column on days 0, 7, 14, and 21), and diabetic mice treated with patch-graft-containing cells (see the third column on days 0, 7, 14, and 21). C) shows serum insulin levels in untreated diabetic mice (see the first column on days 21 and 28), diabetic mice treated with graft biomaterial alone (see the second column on days 21 and 28), and diabetic mice treated with patch-graft-containing cells (see the third column on days 21 and 28). D) shows blood glucose levels in normal mice (triangle), diabetic mice treated with graft biomaterial alone (circle), and diabetic mice treated with patch-graft-containing cells (square). [Figure 16]Figures 16A and 16B show representative light microscope images of mouse pancreatic sections stained with an antibody against insulin (brown) and counterstained with hematoxylin (same magnification, bar = 100 μm). (A) Diabetic mice treated with a patch graft containing BTSC / ELSMC organoids prepared from DS-red mice. (B) Diabetic mice treated with a cell-free graft. Note the large number of cells that do not express insulin. Although insulin protein is evident in the control, it is not functional as shown in the serological assay (Figure 15). Immunohistochemistry was prepared on tissue one month after patch graft transplantation. [Figure 17] Figures 17A–17C show Akita / NRG mice conjugated with patch grafts of BTSC / ELSMC organoids prepared from DS-red mice. A) is a representative micrograph showing an island or patch region containing immunoperoxidase (brown)-stained neurogenin 3 (NGN3), counterstained with hematoxylin, at initial magnification 40×, bar=100μm. Diabetic mice treated with organoids in a patch graft. B) shows antibody-positive organoids (arrows) within and around the patch region. C) shows pancreatic islets from Akita / NRG diabetic mice treated with a cell-free patch graft. Note that NGN3 expression is minimal or absent. [Figure 18] Figures 18A and 18B show representative micrographs of island or patch regions containing immunoperoxidase (brown)-stained neurogenin 3, counterstained with hematoxylin, at initial magnification 40×, bar = 100 μm. (A) shows diabetic mice conferred with a patch graft containing BTSC / ELSMC organoids prepared from DS-red mice. Several DS-red-positive stained cells (indicating donor cells) can be observed in the pancreatic parenchyma (black arrows). (B) Some antibody-positive organoids are seen, indicated by narrow portions (brown arrows). Note: Broader (blue) arrows point to silk fibers within the graft. [Figure 19]Figures 19A and 19B show evidence of engraftment of GFP+ transgenic pig-derived BTSC / ELMC organoids into wild-type pig pancreases up to one week after surgery. The images show low-magnification images of pig pancreatic sections, and sections of the piglet pancreas and surrounding tissue one week after attachment of a patch graft containing GFP+ piglet BTSC / ELSMC organoids to the pancreas of a wild-type piglet. The images show staining with DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) and immunofluorescence contrasting insulin (A) and amylase (B). Insulin staining was performed using an antibody conjugated to a red fluorescent probe, and GFP staining was performed using an antibody with a green fluorescent probe. Note that the cells also migrate into the submucosa of the duodenum, where Brunner's gland is located. A) shows the visibility of numerous GFP+ donor-derived cells near the area where the patch graft was placed. The nuclei stained with DAPI appeared blue. Insulin expression, a characteristic feature of pancreatic islet beta cells, was identified using an anti-insulin antibody conjugated to a red fluorescent probe. Endogenous (host) islet beta cells in the recipient pancreas appear red in the upper part of the pancreas. Donor-derived islet beta cells have a red / purple nucleus derived from the merging of blue for DAPI and green for GFP, and a red / yellow cytoplasm derived from insulin staining. These are observed in the lower part of the pancreas proximal to the patch graft placement site. This low-magnification image clearly shows the degree of engraftment in the pancreas, as well as engraftment into the submucosa of the duodenum and the location of Brunner's gland (presumably the starting point of the cellular network contributing to organogenesis of the liver and pancreas). Bar: 1 mm. B) shows immunofluorescence staining of amylase in serial sections obtained from the same tissue block as in Figure 18A. Amylase (green) is detected mainly in the pancreatic acinar tissue, as well as in the mucosal layer and the lumen of the duodenum. Insulin (red) does not overlap with amylase (green). This staining, when combined with the staining shown in Figure 19A, suggests that the majority of GFP+ donor-derived cells are differentiated to a pancreatic acinar-like fate. Bar: 1 mm. [Figure 20-1]Figures 20A–20C show pancreatic sections obtained from three GFP+ BTSC / ELSMC patch graft recipients (7 days post-transplant), stained with DAPI (blue) and immunofluorescence for insulin, other islet hormones, and GFP. The inventors observed GFP+ cells in the pancreatic parenchyma and near the patch graft site in all recipients. In particular, GFP+ cells appeared at a considerable distance from the patch graft site and appeared to be well integrated into the recipient pancreatic parenchyma. The patch graft material (SERI silk) exhibited some degree of autofluorescence in different channels and was still visible 7 days post-transplant. Bar: 2 mm. Figure 20A is a higher magnification of Figure 19A. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 21-1] Figures 21A–21C show higher magnification images corresponding to the sections in Figures 20A–20C. Figures 21A–21C show evidence of coexistence of endogenous host islet beta cells (insulin+ / GFP-: red cytoplasm) and donor-derived beta cells (insulin+ / GFP+: purple nucleus and red / orange cytoplasm) in the pancreas 7 days after transplantation of the GFP+BTSC / ELSMC patch graft. The inventors observed donor-derived and endogenous host islet-beta cells in all cases. The majority of GFP+ cells exhibited a phenotype consistent with that of acinar cells. GFP+ cells organized to form tubular structures can be seen at the bottom of Figure 21A. Islet cells showed GFP expression in the nucleus even at 1 week, while GFP expression in acinar cells at 1 week was cytoplasmic. As observed in Figures 4A–4E, GFP cytoplasmic staining was present in the first week after patch graft attachment to the liver, and these images show GFP staining in the cytoplasm of acinar cells (blue nucleus and green cytoplasm). In the liver, this became complete GFP staining in the nucleus by approximately two weeks after transplantation. Ongoing studies are evaluating whether this also occurs in pancreatic acinar cells. Bar: 50 μm. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 22] Figure 22 shows schematic line drawings of different stem cell subpopulations. [Modes for carrying out the invention]

[0099] Described herein are novel patch graft compositions and methods for transplanting cells into tissues and solid organs.

[0100] The embodiments described herein will be further described below. However, the embodiments of this disclosure may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so as to make this disclosure thorough and complete and fully communicate the scope of the invention to those skilled in the art. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated in their entirety by reference.

[0101] definition Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the context of this application and the related aforementioned technical field, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Terms not expressly defined below should be interpreted according to their general meanings.

[0102] The implementation of this technology shall, unless otherwise instructed, employ conventional techniques in tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the scope of skills in this field. For example, Sambrook and Russell eds. (2012) Molecular Cloning: A Laboratory Manual, 4rd edition; the series Ausubel et al. eds. (2012) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. al.(1995)PCR 2:A Practical Approach;Harlow and Lane eds.(2014)Antibodies,A Laboratory Manual,2d edition;Freshney(2011)Culture of Animal Cells:A Manual of Basic Technique,6th edition;Gait ed.(1984)Oligonucleotide Synthesis;USPatent No.4,683,195;Hames and Higgins eds.(1985)Nucleic Acid Hybridization;Anderson(1999)Nucleic Acid Hybridization;Hames and Higgins eds.(1984)Transcription and Translation;Immobilized Cells and Enzymes (IRL Press(1986));Perbal(1984)A Practical Guide to Molecular Cloning;Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental See Immunology. .

[0103] Unless otherwise indicated by the context, it is particularly intended that the various features of the present invention described herein may be used in any combination. Furthermore, this disclosure also assumes that in some embodiments, features or combinations of features described herein may be excluded or omitted. For illustrative purposes, where this specification states that a complex comprises components A, B, and C, it is particularly intended that A, B, or C, or any combination thereof, may be omitted and discarded individually or in any combination.

[0104] For example, all numerical values, including ranges for pH, temperature, time, concentration, and molecular weight, are approximations that change (+) or (-) in increments of 1.0 or 0.1 as needed, or in variations of + / - 15%, 10%, 5%, or 2%. It should be understood that, although not always explicitly stated, the term "approximately" precedes all numerical values. Also, although not always explicitly stated, it should be understood that the reagents described herein are illustrative only, and their equivalents are known in the art.

[0105] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to also include the plural forms unless the context otherwise explicitly indicates.

[0106] As used herein, the term "about" when referring to a measurable value such as a quantity or concentration means encompassing variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0107] When used to describe any selection of components, ranges, dosage forms, etc. disclosed herein, terms such as “acceptable,” “effective,” or “sufficient” are intended to indicate that the aforementioned components, ranges, dosage forms, etc. are suitable for the disclosed purpose.

[0108] Furthermore, as used herein, “and / or” means any and all possible combinations of one or more related enumerated items, as well as the absence of any combination when interpreted as a binary choice ("or").

[0109] As used herein, the term “contains” is intended to mean that compositions and methods include the enumerated elements but do not exclude others. As used herein, the transitional phrase “essentially consisting of” (and grammatical variation) is to be interpreted as encompassing the enumerated materials or steps and those that “do not substantially affect the basic and novel properties” of the enumerated embodiments. See re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasized in the original text); see MPEP § 2111.03. Thus, as used herein, the term “essentially consisting of” should not be interpreted as equivalent to “contains.” “Consists of” is to mean that elements of other components in excess of trace amounts and substantial method steps for administering the compositions disclosed herein. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0110] As used herein, the term “effective dose” or “effective amount” means an amount sufficient to treat a medical condition or state (e.g., liver disease or pancreatic disease). An effective dose may be administered in one or more doses, topical application, or in a single dose. Such delivery depends on many variables, including the duration for which individual dose units are used, the bioavailability of the composition, and the route of administration. However, it is understood that a particular amount of a composition for any particular patient will depend on various factors, including the activity of the particular drug used, the patient’s age, weight, health status, sex, and diet, the timing of administration, the amount of excretion, the combination of compositions, the severity of the specific disease being treated (e.g., liver disease or pancreatic disease), and the form of administration.

[0111] The terms “equivalent” or “biological equivalent” are used interchangeably when referring to specific molecules, biomaterials, or cellular materials that have minimal homology while maintaining the desired structure or function.

[0112] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. Gene expression levels may be determined by measuring the amount of mRNA or protein in a cell or tissue sample, and furthermore, the expression levels of multiple genes may be determined to establish an expression profile for a particular sample.

[0113] As used herein, the term “functional” may be used to modify any molecule, biological material, or cellular material with the intention of achieving a specific effect.

[0114] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides can have any three-dimensional (3D) structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.

[0115] Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure may be conferred before or after the assembly of the polynucleotide, if present. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by binding with labeling components. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any aspect of this art that is a polynucleotide encompasses both a double-stranded body and each of two complementary single-stranded bodies known or expected to constitute the double-stranded body.

[0116] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably in their broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or peptide mimetic compounds. The subunits may be linked by peptide bonds. In other embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein or peptide sequence. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptide mimetic compounds.

[0117] As used herein, the term “gene” broadly includes any nucleic acid sequence transcribed into an RNA molecule, whether the RNA is coding (e.g., mRNA) or non-coding (e.g., ncRNA).

[0118] As used herein, the term “isolated” refers to a molecular, biological, or cellular material that is substantially free of other materials.

[0119] Definitions of other terms used herein are provided below in the context in which they are used.

[0120] Patch graft composition and strategy In another aspect, the present disclosure relates to a patch graft comprising a mixture of epithelial cells and mesenchymal cells and one or more biomaterial layers, wherein the layers are at least

[0121] A first inner layer for contacting solid organs, exhibiting first viscoelastic properties, incorporating a mixture of epithelial cells and mesenchymal cells, supporting the ability of epithelial and mesenchymal cells to produce secretory matrix metalloproteinases (MMPs), and promoting the viability and immaturity of the aforementioned epithelial and mesenchymal cells,

[0122] A backing that selectively provides a barrier to cells migrating in directions other than solid organs, and exhibits a second viscoelastic property, and

[0123] Optionally, a third outer layer of covering or material near the patch graft to minimize adhesion of the patch graft to the inner wall and / or inner surface of the body cavity, including organs. Includes The aforementioned viscoelastic properties are determined by measuring the fluidic properties and expressed in Pascals (Pa). ru.

[0124] As used herein, the term “patch graft” refers to a composition of cells embedded in a suitable biomaterial that enables the transplantation of donor cells into a host. In some embodiments, the term refers to a composition of cells embedded in a suitable biomaterial that enables the transplantation of donor cells into a host. The biomaterial can be prepared under generally defined conditions (e.g., a basic medium consisting of trophic factors, vitamins, amino acids, carbohydrates, minerals, insulin, transferrin / Fe, and / or lipids), solidified into a soft gel (about 100 Pa), and covered with a backing, which consists of chemicals having sufficient tensile strength to enable surgical attachment to host tissues or organs, and further having minimal impact on donor cell differentiation and minimal adverse effects on host tissue.

[0125] As used herein, the term “backing” means a material that (i) is biocompatible with the object in which it is implanted, (ii) exhibits mechanical resilience to withstand compressive and shear forces generated on organs and tissues (especially internal ones), thereby enabling the material to function as a surgical tissue, and (iii) has a neutral or minimal effect on the differentiation state of cells in contact with the material. Suitable materials in this regard include, but are not limited to, Seri-Silk® (or its derivatives), and / or patches consisting of PGA and / or PLLA. Non-limiting examples of suitable patches of synthetic materials include woven patches consisting of 91% PGA-co-9% PLLA, knitted patches consisting of 91% PGA-co-9% PLLA, or nonwoven patches consisting of 100% PGA. Other possibilities include matrix extracts of amniotic membrane or reticular or reticular matrix extracts. In some embodiments, the backing includes silk. In some embodiments, the silk backing includes refined fibroin of silkworm silk woven into the scaffold, including Seri-Silk® or Contour Seri Silk®.

[0126] In some embodiments, the backing is also bioabsorbable. As used herein, “bioabsorbable” means a material that can be broken down by the body of the graft's host or recipient and does not require mechanical removal. In some embodiments, the bioabsorbable backing is bioabsorbable for periods of about 2 to about 10 weeks, about 2 to about 20 weeks, about 2 to about 52 weeks, about 4 to about 16 weeks, about 4 to about 12 weeks, or about 4 to about 8 weeks.

[0127] As used herein, biomaterials in grafts include those capable of forming hydrogels independently of the backing. The term “gel” refers to a solid, jelly-like material that can possess properties ranging from soft and weak to hard and strong. A gel is defined as a substantially dilute crosslinked system that does not flow in a steady state. By weight, gels are generally liquid, but they behave like solids due to a three-dimensional crosslinking network within the liquid. It is the crosslinking in the fluid that gives gel structure (hardness) and contributes to its adhesiveness. Thus, a gel is a dispersion of liquid molecules within a solid, where the solid is a continuous phase and the liquid is a discontinuous phase. “Hydrogels” are a non-limiting example of gels, consisting of high-molecular-weight polymer gels constructed from a network of polymer chains. Hydrogels are synthesized from hydrophilic monomers or hydrophilic dimers (e.g., hyaluronan) by either chain growth or step growth, along with network formation. Along with empty defects, the reticular structure enhances the hydrogel’s ability to absorb large amounts of water via hydrogen bonding. As a result, hydrogels develop characteristic, rigid, yet elastic mechanical properties, where new bonds spontaneously form when old bonds are broken within the material. Along with electrostatic attraction, the structure of the hydrogel promotes the formation of new bonds via non-covalent hydrogen bonds. One successful material for hydrogels is thiol-modified hyaluronan, which can be induced to form a hydrogel when exposed to oxygen and / or poly(ethylene glycol) diacrylate (PEGDA), and can be easily "tuned" by the precise ratio of hyaluronan and PEGDA concentrations (and / or oxygen levels). The viscoelastic properties of the hydrogel are determined by measuring its fluidic properties and can be expressed in Pascals (Pa).

[0128] In some embodiments, the second viscoelastic property (expressed in Pa) is higher than the first viscoelastic property. In some embodiments, one or more biomaterial layers comprise a hydrogel, which further comprises minimally sulfated or non-sulfated glycosaminoglycans. In some embodiments, the non-sulfated glycosaminoglycan comprises hyaluronan. In some embodiments, the hyaluronan comprises thiol-modified hyaluronan, and its gelation by disulfide crosslinking is induced in the presence of polyethylene glycol diacrylate (PEGDA). In some embodiments, the fluidic properties are determined, at least in part, by the initial concentrations and stiffness of the thiol-modified hyaluronan and PEGDA before gelation, and by the final stiffness of the hydrogel after gelation, which is achieved by the precise ratio of the volumes of thiol-modified hyaluronan and PEGDA.

[0129] In some embodiments, the fluid pathway of the patch graft is also determined by the temperature required to achieve the crosslinking process. The temperature for forming the inner layer hydrogel from about 50 Pa to about 150 Pa can be room temperature (RT) or 37°C. The temperature for forming the hydrogel from about 200 to about 300 Pa can be 4°C or room temperature (RT). The temperature for forming the hydrogel from about 600 to about 800 Pa can be room temperature (RT) or 37°C.

[0130] As used herein, the term “hyaluronan” refers to a polymer of disaccharide units composed of glucosamine and glucuronic acid [1-3] linked by β1-4 and β1-3 links, and its salts. Therefore, the term hyaluronan refers to both the natural and synthetic forms of hyaluronan. Naturally occurring hyaluronan (HA) is a water-soluble polysaccharide containing disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), which are alternately linked to form a linear polymer. High molecular weight HA can contain 100 to 10,000 disaccharide units. HA often occurs naturally as sodium hyaluronate, its sodium salt. HA; sodium hyaluronate and preparations of either HA or sodium hyaluronate are often referred to as “hyaluronan.” Non-limiting examples of acceptable hyaluronic acid salts include potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.

[0131] Other glycosaminoglycans (GAGs) can also be used in hydrogels. These include polymers of chondroitin sulfate (CS) and dermatan sulfate (DS), glucuronic acid and galactosamine, and heparan sulfate (HS) and heparin (HP), glucuronic acid and glucosamine. The degree and pattern of sulfation of these GAGs are important because the sulfation pattern determines the formation of complexes with multiple protein families (e.g., coagulation proteins, growth factors, cytokines, neutrophil enzymes). See, for example, Powell AK, Yates EA, Fernig DG, Turnbull JE. Interactions of heparin / heparan sulfate with proteins: appraisal of structural factors and experimental approaches. Glycobiology. April 2004; 14(4):17R-30R. Suitable patch grafts for optimizing engraftment include hyaluronan, non-sulfated GAGs, and minimally sulfated forms such as chondroitin sulfate found in stem cell niches, as shown below: Karumbaiah L, et al. Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells. Bioconjug Chem. 2015 Dec 16;26(12):2336-49 and Hayes AJ, et al. Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells. J Histochem Cytochem. 2008 Feb;56(2):125-38 (incorporated herein by reference).

[0132] The viscoelastic properties of the hydrogel can be set to approximately 10 Pa to approximately 50 Pa, approximately 50 Pa to approximately 100 Pa, approximately 100 Pa to approximately 150 Pa, approximately 150 Pa to approximately 200 Pa, approximately 200 Pa to approximately 250 Pa, approximately 250 Pa to approximately 300 Pa, approximately 300 Pa to approximately 350 Pa, approximately 350 Pa to approximately 400 Pa, approximately 400 Pa to approximately 450 Pa, approximately 450 Pa to approximately 500 Pa, approximately 500 Pa to approximately 550 Pa, approximately 550 Pa to approximately 600 Pa, approximately 600 Pa to approximately 650 Pa, approximately 650 Pa to approximately 700 Pa, approximately 700 Pa to approximately 750 Pa, approximately 750 Pa to approximately 800 Pa, and approximately 800 Pa to approximately 850 Pa. In some embodiments, the viscoelastic properties of the hydrogel are approximately 50 Pa, 100 Pa, 150 Pa, 200 Pa, 250 Pa, 300 Pa, 350 Pa, 400 Pa, 450 Pa, 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, or 800 Pa.

[0133] In some embodiments, the first inner layer exhibits a first viscoelasticity of about 50 Pa to about 150 Pa. In some embodiments, an optional backing contains a hyaluronan hydrogel layer exhibiting a viscoelasticity of about 600 to about 800 Pa. In some embodiments, an optional third outer layer contains a hyaluronan hydrogel layer having viscoelastic properties of about 200 to about 300 Pa. In some embodiments of the patch graft, the viscoelastic properties of the backing are greater than those of the first layer. In some embodiments of the patch graft, the viscosity of the backing is about 1.5 to about 15 times greater than that of the first layer. In some embodiments of the patch graft, the viscoelastic properties of the backing are about twice as great as those of the first layer.

[0134] The applicant has shown that hyaluronan can influence epithelial cells, stem cells, and / or progenitor cells to express factors that regulate key cell adhesion molecules required for cell adhesion and intercellular interactions, and can prevent stem cells and / or progenitor cells from internal migration of these adhesion factors after cell suspension preparation, cryopreservation, or transplantation. Non-limiting examples of such adhesion factors include integrins. Integrins are a large family of heterodimeric transmembrane glycoproteins that function to cause cells to adhere to extracellular matrix proteins of the basement membrane, ligands on other cells, and soluble ligands. Integrins contain large and small subunits called α and β, respectively. These subunits form αβ heterodimers, and in humans, at least 18 α and 8 β subunits are known, generating 24 heterodimers. In some embodiments, stem cells and / or progenitor cells express higher levels of integrin subunits, such as ITGα1, ITGα2, ITGα2B, ITGα3, ITGα4, ITGα5, ITGα6, ITGα7, ITGα8, ITGα9, ITGα10, ITGα11, ITGαD, ITGαE, ITGαL, ITGαM, ITGαV, ITGαX, ITGβ1, ITGβ2, ITGβ3, ITGβ4, ITGβ5, ITGβ6, ITGβ7, and ITGβ8. In one preferred embodiment, stem cells and / or progenitor cells express higher levels of integrin subunit beta 1 (ITGβ1) and / or integrin subunit beta 4 (ITGβ4). Takada Y. et al. (2007) Genome Biol. 8(5):215.

[0135] In some embodiments, the epithelial cells, stem cells, and / or progenitor cells of this disclosure differ from naturally occurring stem cells and / or progenitor cells in that they express integrin subunits in amounts exceeding at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and 200% of the amount of integrin subunits in unmodified stem cells and / or progenitor cells. The increase in integrin subunits is thought to help stem cells and / or progenitor cells adhere and form intercellular interactions.

[0136] Hyaluronan (HA), a major component of the stem cell niche, is a candidate stem cell coating used in cell therapy because it promotes viability, proliferation, and engraftment in damaged liver. The chemical and mechanical properties of HA are related to essential requirements for stem cells. Furthermore, since the liver is a major site for HA clearance, HA coating represents a beneficial strategy for selectively targeting transplanted cells to the liver.

[0137] Stem cells and / or progenitor cells can be coated with hyaluronan (HA) using any method known in the art. For example, stem cells and / or progenitor cells can be incubated with a certain amount of HA and gently mixed for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or longer. The HA-coated stem cells and / or progenitor cells can then be further incubated in a culture medium, for example, Kubota medium.

[0138] In some embodiments, HA comprises salts of HA, such as alkali metal salts. HA occurs naturally, often as sodium hyaluronate, which is a sodium salt. Non-limiting examples of acceptable additional hyaluronic acid salts include potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate. To prepare HA for use in coating stem cells and / or progenitor cells, HA can be resuspended in any pharmaceutically acceptable carrier, such as phosphate-buffered saline (PBS), basal medium, Kubota medium, hormone-regulated medium, etc. In some embodiments, the pharmaceutically acceptable carrier further comprises one or more growth factors, one or more glycosaminoglycan sugars, or a combination thereof.

[0139] In some embodiments, the pharmaceutically acceptable amount of hyaluronan in the carrier is about 0.05% w / v to about 1% w / v. In a preferred embodiment, the pharmaceutically acceptable amount of hyaluronan in the carrier is about 0.1% w / v.

[0140] As used herein, the term “coated” means either continuous or discontinuous; that is, a hyaluronan (HA) coating can completely cover the surface of stem cells and / or progenitor cells, or only partially cover them to form coated areas (e.g., “islands”) and uncoated areas. While the coatings of the present invention contain HA, it is also envisioned that such coatings may contain other substances. In some embodiments, hyaluronan coats at least a portion of the surface of the aforementioned stem cells and / or progenitor cells or aggregates thereof, for example, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% of the surface of the aforementioned stem cells and / or progenitor cells or aggregates. In some embodiments, at least about half of the exposed surface of the aforementioned aggregates, or individual stem cells and / or progenitor cells, are coated with hyaluronan or a pharmaceutically acceptable salt thereof. In some embodiments, stem cells and / or progenitor cells are modified by the presence of externally added hyaluronan.

[0141] The size of transplanted cells can be a crucial factor for transplant success. If the cells are too large (e.g., polyploid hepatocytes) or if they form large aggregates, their transplantation via vascular pathways can result in potentially life-threatening embolisms. If the cells are too small, their engraftment efficiency may be very low, and they tend to distribute to ectopic sites. Both possibilities are important considerations when evaluating cell therapy. Cells used in cell therapy for liver disease are injected into the liver via the spleen in animal models, or into the portal vein or hepatic artery in humans. Cell sizes range from approximately 8–10 μm for stem cells (HpSCs, BTSCs), approximately 12–15 μm for hepatoblasts and differentiated precursors, approximately 17–18 μm for diploid hepatocytes, which are dominant in neonatal livers, and approximately 25–30 μm for mature hepatocytes, which are dominant in adult livers.

[0142] In some embodiments, the majority of stem cell and / or progenitor cell aggregates contain between about 2 and about 10 stem cells and / or progenitor cells per aggregate. Most commonly, they contain 50 to 100 cells per aggregate. In one embodiment, the aggregate contains about 90 to 100 cells. In one embodiment, the aggregate contains about 80 to 90 cells. In one embodiment, the aggregate contains about 70 to 80 cells. In one embodiment, the aggregate contains about 60 to 70 cells. In one embodiment, the aggregate contains about 50 to 60 cells.

[0143] In one embodiment, the aggregate comprises about five stem cells and / or progenitor cells, as well as associated ELSMCs. In some embodiments, the stem cell and / or precursor aggregate has an average diameter of 50 μm or less, 45 μm or less, 40 μm or less, 35 μm or less, 30 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less. In one preferred embodiment, the stem cell and / or progenitor cell aggregate has an average diameter of 30 μm or less.

[0144] As used herein, the term “cell” refers to one or more cells in a graft. The cells of this disclosure are eukaryotic. In some embodiments, the epithelial cells include early lineage stage epithelial cells (ELSEs) and the mesenchymal cells include early lineage stage mesenchymal cells (ELSMCs), or the epithelial cells and mesenchymal cells are at late lineage stages but are equivalent to each other. In some embodiments, the ELSMCs include angioblasts, endothelial precursors, astrocytes, or a combination thereof. In some embodiments, the ELSEs and / or ELSMCs are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS). In some embodiments, the epithelial cells are at late lineage stages, the mesenchymal cells are early lineage stage mesenchymal cells (ELSMCs), or the mesenchymal cells are at late lineage stages and the epithelial cells are early lineage stage epithelial cells (ELSEs). In some embodiments, the cells are of animal origin and may be stem cells, mature cells, progenitor cells, or intermediates in the lineage stages from stem cells to mature cells. The term “population of cells” refers to a group of one or more cells of the same or different cell types having the same or different origins. In some embodiments, this population of cells may originate from a cell line, newly isolated cells, or in some embodiments, it may originate from a part of an organ or tissue.

[0145] The term "stem cell" refers to a population of cells that can self-replicate (produce daughter cells identical to the parent cell) and are pluripotent, that is, capable of giving rise to one or more types of adult cells. As used herein, the term "progenitor cell" or "precursor" is broadly defined to include the offspring of stem cells and their descendants. Precursors are populations of cells that may be pluripotent, dipotent, or unipotent, but have minimal self-renewal capacity (if any). Differentiated precursors are unipotent and capable of differentiating into a specific lineage leading to only one mature cell type. Non-exclusive examples of stem cells include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ layer stem cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells, mesenchymal stem cells (MSCs), and angioblasts. Intermediates between stem cells and differentiated precursors include cell populations such as hepatoblasts and pancreatic duct precursors, as well as other forms of TA cells that may be pluripotent and have broad proliferative capacity but have more limited self-renewal capacity (if any).

[0146] Cells can be any determined stem cells, such as determined endodermal stem cells or precursors derived therefrom. Furthermore, these determined stem cells or precursors may be derived from lineages of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. ES cells are pluripotent stem cells derived from an early embryo and can give rise to adult cells of all three germ layers. iPS cells are postnatal cells that have been reprogrammed by transcription factors or small molecules to have phenotypic traits similar to ES cells and can give rise to adult destinies of all three germ layers. However, these germ layers retain some molecular characteristics in their chromatin, and the chromatin reflects the germ layer from which the somatic cell is induced. Pluripotent or dipotent precursors and TA cells are precursors that have limited (if any) self-renewal ability and can give rise to two or more adult destinies. Differentiated precursors are unipotent, lack self-renewal ability, and give rise to only one adult cell type.

[0147] Cells derived from the stem cell / precursor types listed above, as well as mature cells, can successfully engraft as long as there is a supply of multiple matrix metalloproteinases (MMPs), both secreted and membrane-bound, ideally cellular sources. MMPs are produced by all cell types, both immature and mature, but differ in which isoforms are produced and at what levels of specific MMP expression. Representative secreted MMPs include MMP1, MMP2, MMP7, and MMP9. Representative membrane-bound MMPs include MMP14 and MMP15. Experimentally, the highest production of secreted MMPs has been found in early lineage cells, stem cells, and early precursors. The biomaterial of the graft supports the ability of both epithelial and mesenchymal cells to produce these multiple forms of matrix metalloproteinases (MMPs), which in turn remodel the capsule surrounding an organ or tissue, thereby remodeling the underlying tissue into a capsule and enabling cell migration by dissolving multiple forms of extracellular matrix components.

[0148] The "rule" for engraftment is that there must be a source of MMPs, especially secreted isoforms, which means that success will occur in the following cases: 1) Both epithelial cells and mesenchymal cells are stem cells / precursors (and therefore both are cellular sources of MMPs), 2) Epithelial cells are precursors of stem cells and partner with mature mesenchymal cells (therefore epithelial stem cells / precursors are the cellular source of MMPs), 3) Epithelial cells are mature cells that partner with mesenchymal stem cells / precursors (therefore mesenchymal cells are the cellular source of MMPs), 4) Theoretically, it is reasonable to provide a purified source of MMPs (i.e., cloned forms of MMPs) in the graft.

[0149] Matrix metalloproteinase (MMP) More generally, matrix metalloproteinases (MMPs) are a large family of zinc-dependent proteinases involved in the disruption and regulation of extracellular matrix components and in transplantation, invasion, angiogenesis, and migration in normal or pathological processes. There are at least 24 isoforms, including matrixin, adamaricin, astacin, and ceralicin. Their roles have been elucidated in normal processes such as placental transplantation, as well as in pathological processes such as cancer invasion and metastasis. The studies described herein provide evidence for entirely new roles contributing to the engraftment, migration, and integration of transplanted cells. Stem cells / precursors, both epithelial and mesenchymal, express multiple MMP isoforms that are particularly potent in these roles. As cells mature, the expression of one or more potent stem cell / precursor cell-associated MMPs is attenuated, and therefore the processes of invasion and migration are reduced. Adult cells also express MMPs, primarily membrane-bound (MT-MMPs), which are involved in plasticity processes but not in large-scale engraftment and integration of cells into tissues. This understanding can be summarized as follows: the graft biomaterial, backing, and other conditions must optimize the expression of various MMPs, including secreted MMPs, enabling the processes of transplantation and migration. Therefore, factors that promote differentiation of transplanted cells simultaneously attenuate the complex MMP response. This means that factors to be avoided include serum, soluble signals that promote differentiation (e.g., certain growth factors, cytokines, and hormones); extracellular matrix components that promote differentiation (e.g., collagen, adhesion molecules, highly sulfated glycosaminoglycans / proteoglycans); and mechanical forces that contribute to graft rigidity. In some embodiments, one or more biomaterial layers contain recombinant MMPs. In some embodiments, one or more biomaterial layers contain cells engineered to express MMPs.

[0150] The term "mesenchymal cells" refers to cells derived from mesenchyme, and such include, but are not limited to, mesenchymal stem cells, which are pluripotent stromal cells, as well as various subpopulations of mature and precursor mesenchymal cells, which comprise at least two main categories.

[0151] Mature mesenchymal cells produce and are surrounded by extracellular matrix morphologies containing fibrous collagen (e.g., type I, type III, type V) and associated matrix components, as well as binding signals (e.g., growth factors / cytokines) that form cell-related complexes, typically linear (string-like) cell populations. Non-limiting examples of such cells include astrocytes, tendons, stroma, and myofibroblasts.

[0152] Mature mesenchymal cells that generate and are surrounded by an extracellular matrix morphology containing network collagen (e.g., types IV, VI, VIII, X) and associated matrix molecules, as well as binding signals (e.g., growth factors, cytokines) associated with cells having a more flattened, cubic, or cobblestone morphology. Non-limiting examples of such cells include endothelium and myoepithelium.

[0153] Precursors of these mesenchymal cell types include, but are not limited to, pluripotent angioblasts that can differentiate into endothelial cells (in this later stage, fenestrated endothelial cells) or astrocytes (in this later stage, myofibroblasts (stromal cells). Other precursors include pluripotent mesenchymal stem cells (MSCs) that can differentiate into fibroblasts (stromal cells), osteoblasts (osteocytes), chondrocytes (chondrocytes), myocytes (muscle cells), and adipocytes (adipocytes).

[0154] The term “epithelial cell proliferation” correlates with the growth estimate, which is a composite of the colony diameter and the cell diameter of epithelial cells that typically form colonies with a cuboidal or cobblestone morphology. In contrast, the growth estimate of mesenchymal cell colonies correlates with colony density because mesenchymal cells are more migratory and motile, and colony density reflects the net sum of cells that remain within the colony boundary.

[0155] The term "epithelial cell" refers to cells derived from epithelium, which are specialized cells that provide diverse functions to the needs of the host tissue and / or the entire body. They are recognized by their ability to migrate as precursor or immature cells; that is, as they mature, they become quiescent, forming layers of flattened, cobblestone-like, or columnar polarized cells with apex, base, and lateral vertices, these layers being interconnected by various junctions (connexins, tight junctions, adhesions). Their potential for proliferation is indicated by the diameter of the colony (rather than by density). Mature epithelial cells provide diverse functions, including the secretion of specialized products or contributions to metabolism (hepatocytes, cholangiocytes), detoxification (hepatocytes), enzyme production (acinocytes), endocrine factor production (e.g., pancreatic islets or other endocrine cells), electrical activity (neurons), and absorption (intestinal cells).

[0156] As used herein, the term “supportive” is used to describe a cell that can assist the propagation of cells from another lineage or that can provide support to neighboring cells through the production of “paracrine signals,” which are factors active in influencing neighboring cells with respect to survival, proliferation, migration, differentiation, and maturation. For example, a supportive mesenchymal cell is defined by its ability to selectively influence epithelial cells through the secretion of matrix metalloproteinases (MMPs) and / or one or more other / or factors.

[0157] The term “lineage stage partner” as used herein refers to mesenchymal cells that are at a lineage stage appropriate to a given epithelial lineage stage and capable of supporting the engraftment of its epithelial cells. For liver or biliary stem cells, these consist of angioblasts (CD117+, CD133+, VEGFr+, CD31-negative) and their direct offspring, endothelial precursors (CD133+, VEGFr+, CD31+), and astrocyte precursors (CD146+, ICAM-1+, alpha-smooth muscle actin+ (ASMA), vitamin A-negative). Paracrine signaling between epithelial and mesenchymal cells is also lineage stage dependent, meaning that specific paracrine signals and their levels are distinct in early, mid, and late-stage cells. These can be partially mimicked by using mesenchymal stem cells (MSCs) derived from bone marrow or adipose tissue. The inventors collectively refer to these as early lineage stage mesenchymal cells (ELSMCs).

[0158] In some embodiments, epithelial cells include biliary dendritic stem cells (BTSCs), and mesenchymal cells include early lineage-stage mesenchymal cells (ELSMCs). In some embodiments, ELSMCs include angioblasts and their direct offspring, endothelial precursors, astrocyte precursors, or combinations thereof. In some embodiments, angioblasts express CD117, CD133, and VEGFr, but do not express CD31. In some embodiments, endothelial cell precursors express CD133, VEGFr, CD31, and von Willebrand factor. In some embodiments, astrocyte precursors express CD146, ICAM-1, alpha-smooth muscle actin (ASMA), and are negative for vitamin A.

[0159] The term "biliary tree stem cells" (BTSCs) refers to stem cells found throughout the intrahepatic and extrahepatic biliary trees, within the extramural and intramural periductal glands (PBGs), and within the crypts of the gallbladder villi and Brunner's glands. They possess the ability to differentiate into determined hepatic progenitor cells and / or pancreatic progenitor cells. To date, at least seven subpopulations of stem cell populations have been identified within the intramural network of the biliary tree ducts, ranging from highly primitive BTSCs with overlapping characteristics to stem cell populations that can be defined as hepatic or pancreatic stem cells.

[0160] What is known about these stem cell subpopulations is described below and outlined in Figure 22. The most primitive are found in both extramural periductal glands—those connected to the surface of the bile ducts—and intramural periductal glands—those found within the bile duct wall. Intramural periductal glands (PBGs) near the fibromuscular layer in the center of the bile duct wall are crypts (similar to intestinal crypts) and can be considered the niche where the most primitive stem cell populations are found. The largest number of PBGs in the biliary tree network are found in the common hepatopancreatic duct and in the large intrahepatic bile ducts. PBGs do not develop in the gallbladder, but instead, there is a stem cell niche within the gallbladder at the base of the gallbladder villi, which contains a population of mid-to-late stage stem cells that are precursors to hepatic stem cells.

[0161] In some embodiments, the patch graft contains a BTSC that is positive for at least one marker selected from the group consisting of pluripotency genes such as OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1; at least one marker selected from the group consisting of endodermal transcription factors such as Sox9, Sox17, Pdx1, HNF4 alpha, and ONECUT2; and at least one marker selected from the group consisting of one or more isoforms of EpCAM, LGR5, NCAM, CD44, CXCR4, sodium-iodine cotransporter (NIS), CD49 (integrin A6), CD29 (integrin B1), and integrin B4. BTSCs give rise to two stem cell subpopulations: hepatic stem cells (HpSCs) and pancreatic stem cells (PSCs), both of which express pluripotency genes at lower levels, express stem cell / precursor surface markers at similar levels, and have the following markers that differentiate HpSCs from PSCs: constitutive expression of SOX9, SOX17, HNF4 alpha, ONECUT2, and albumin for HpSCs; constitutive expression of Pdx1, Ngn3, PTF1A, HNF1B, MUC6, PAX6, and insulin for PSCs. BTSCs, HpSCs, and PSCs are negative for markers of mature liver genes or mature pancreatic genes, such as expression of P450, aquaporins, enzymes involved in bile production, digestive enzymes produced by mature acinar cells, and regulated expression of albumin or regulated expression of insulin and islet hormones.

[0162] Generally, all subpopulations of BTSCs express biomarkers including endodermal transcription factors of both liver and pancreas (e.g., SOX9, SOX17, PDX1), pluripotency genes (e.g., OCT4, SOX2, NANOG, SALL4, KLF4 / KLF5, BMI-1); one or more hyaluronan receptor isoforms of CD44 (standard and / or variant isoforms); CXCR4; and cytokeratins 8 and 18. Stem cell subpopulations within the biliary tree may include: 1: Cells found only in the submucosa of the duodenum and not anywhere else in the intestine, specifically within Brunner's gland (possibly another form of BTSC or a distinct cell population – research to definitively identify this is still ongoing). These also express the markers mentioned above, as well as Tra-160, Tra-181, and cytokeratin 7. They are distinguishable from intestinal stem cells by their characteristics. 2: Early-stage biliary dendritic stem cells (BTSCs) that express sodium-iodine cotransporter (NIS) and CXCR4, OCT4, SOX2, and NANOG, but do not express LGR5 or EpCAM; 3. An intermediate stage of BTSC, expressing less NIS but increasing LGR5 expression without increasing EpCAM expression; 4. Late-stage BTSCs (the only BTSCs found in the gallbladder) are found in large intrahepatic bile ducts and the hepatopancreatic common duct. These express both LGR5 and EpCAM. They are precursors to hepatic and pancreatic stem cells. 5. Hepatic stem cells refer to stem cells found in Hering's ducts, PBGs of large intrahepatic bile ducts, PBGs of extrahepatic bile trees, and PBGs of the hepatopancreatic common duct, with the largest number found in the internal hepatic region. Hepatic stem cells retain the ability to self-replicate and become pluripotent. Biomarkers for these cells include SOX9, SOX17, HNF-alpha, ITGB1 (CD29), ONECUT 2, SALL4, LGR5, CD44, epithelial cell adhesion molecules (EpCAM) and neural cell adhesion molecules (NCAM) found in the cytoplasm and cell membrane, constitutively regulated low levels (or no expression) of albumin, complete absence of alpha-fetoprotein (AFP), absence of P450 A7, and absence of secretin receptor (SR). Hepatic stem cells and their progeny, hepatoblasts, express cytokeratins 8, 18, and 19. 6. Pancreatic stem cells are found in small numbers throughout the biliary tree (even in PBGs of large intrahepatic bile ducts), but in large numbers in PBGs of the hepatopancreatic common duct. These differ in that they express pluripotency genes and other genes found in all stem cell populations, but lack SOX17. Subpopulations whose lineage is limited to pancreatic islets express NGN3. These express EpCAM at the cell membrane throughout the cell and express (or not express) insulin with low, constitutive regulation. Their maturation correlates with increased insulin expression and its ability to be regulated by various factors.

[0163] The intermediates within the lineage network are called "TA cells," which can be dipotent (or pluripotent), possess significant proliferative capacity but exhibit little to no true self-renewal (if any), have low to moderate (or even absent) pluripotency gene expression, and express traits that indicate differentiation into liver (e.g., albumin, alpha-fetoprotein) or pancreas (e.g., insulin, MUC6). These include hepatoblasts (the network that gives rise to the liver) and pancreatic duct precursors (the network that gives rise to the pancreas).

[0164] As used herein, the term “pancreatic duct precursor” refers to pluripotent cells found within the pancreatic duct glands (PDGs) of the pancreas that give rise to acinar cells and islets. The inventors have found in their studies that these cells express SOX9, PDX1, HNF1β, EpCAM, LGR5, ICAM-1, and CD44, with subpopulations expressing NGN3 or MUC6.

[0165] As used herein, the term “hepatoblast” refers to pluripotent hepatocytes capable of giving rise to hepatocyte and cholangiocyte lineages, and found in PBGs within or adjacent to Hering’s ducts or in large intrahepatic bile ducts. These cells possess an extraordinary ability to proliferate (i.e., expand) compared to hepatic stem cells, along with a lower self-renewal capacity (if any). These cells are characterized by biomarker profiles that overlap with, but differ from, those of hepatic stem cells. These cells express SOX9, low (or negligible) levels of SOX17, high levels of LGR5, HNF4-alpha, and EpCAM, although these are mainly found on the cell membrane; they also express P450A7, cytokeratin 7, and secretin receptors; all hepatoblasts consistently and regulatoryly express albumin; they express high levels of alf-fetoprotein (AFP) and cell adhesion molecule (ICAM-1) but do not express NCAM; they express pluripotency genes (e.g., SALL4, KL4 / KLF5, OCT4, SOX2, NANOG) at negligible levels or not at all; and they do not express mature liver parenchymal markers (e.g., P450 such as P4503A).

[0166] As used herein, the term “differentiated precursor” refers to unipotent progenitor cells that give rise to a single cell type, such as differentiated hepatocyte progenitor cells. In some embodiments, these do not express pluripotency genes. Differentiated hepatocyte precursors are recognized by the expression of albumin, AFP, glycogen, ICAM-1, various enzymes involved in glycogen synthesis, and the gap junction gene, connexin 28. These give rise to hepatocytes. Differentiated bile duct (or cholangiocyte) precursors give rise to cholangiocytes and are recognized by the expression of EpCAM, cytokeratin 7 and 19, aquaporins, CFTR (cystic fibrosis membrane conductance regulator), and membrane pumps involved in bile production. In some embodiments, differentiated islet precursors express insulin, glucagon, and other islet hormones, albeit at low levels, and as they mature, the expression levels of islet hormones increase, although certain cells preferentially express certain hormones.

[0167] As used herein, the term “aggregate” refers to a group of cells collected together. Aggregates may vary in both size and shape, or they may be substantially uniform in size and / or shape. Cell aggregates used herein can be of various shapes, such as spherical, cylindrical (preferably having equal height and diameter), or rod-shaped in particular. While aggregates of other shapes may be used, in one embodiment of this disclosure, cell aggregates are generally preferred to be spherical or cylindrical. The term “unaggregated” refers to an organism, or a single cell, stem cell, and / or progenitor cell. In some embodiments, compositions provided herein may include substantially aggregated cells, substantially unaggregated cells, or mixtures thereof.

[0168] In this specification, the term “organoid” refers to a specific cell aggregate of donor epithelial cells having mesenchymal cells that self-organize by the simple panning method described herein. Organoids can be obtained by mixing early-stage epithelial cells (ES cells, iPS cells, determined stem cells, TA cells, precursors) with early-stage mesenchymal cells (angioblasts, endothelial precursors, astrocyte precursors).

[0169] Mixtures of adult epithelial cells and mature mesenchymal cells, and chimeric mixtures of mature epithelial cells and early lineage mesenchymal cells (ELSMCs), do not typically produce organoids but can be used as cell mixtures in suspensions of graft biomaterials. When mature epithelial cells (e.g., hepatocytes, cholangiocytes, pancreatic islets, acinar cells, intestinal cells, etc.) are partnered with mature mesenchymal cells (e.g., endothelial cells, astrocytes, stromal cells, myofibroblasts), the mixture does not produce grafts with good results, but rather those that persist on the surface of organs or tissues. This is thought to be because they express membrane-bound MMPs but express secreted MMPs at minimal levels. When chimeric mixtures are used (e.g., mature hepatocytes with angioblasts), engraftment occurs because there is a source of secreted MMPs that enables cell engraftment and migration.

[0170] A protocol for establishing organoids. According to one embodiment disclosed herein, organoids of bile dendritic stem cells (BTSCs) and early lineage-stage mesenchymal cells ("ELMCs") have proven to be the most successful method for incorporating cells into grafts. Disclosed herein is that BTSCs and ELMCs can self-select organoids by panning to remove mature astrocyte / stromal cells, and this has proven to be more efficient and effective in establishing appropriate epithelial-mesenchymal partners at the lineage stage for grafts. In another embodiment, the disclosure provides a method for forming organoids by culturing a first type of cell with a second type of cell that is an appropriate lineage partner for the first type of cell stage, removing mature cells attached to the culture dish by panning, and recovering the self-assembled organoids from the culture suspension. The first type of cell may be epithelial stem cells or differentiated epithelial cells. The second type of cell may be mesenchymal lineage cells, mesenchymal stem cells, or early lineage-stage mesenchymal cells.

[0171] In some embodiments, the mesenchymal cells are supportive mesenchymal cells. In some embodiments, the organoids are formed after culturing aggregated cells in suspension on a low-adhesion dish under serum-free and generally defined conditions that correspond to the lineage stage.

[0172] In some embodiments, a mixture of epithelial and mesenchymal cells is produced by selectively repeating a panning procedure to remove cells adhering to a tissue culture dish or surface within about 15 to 30 minutes at 37°C, thereby depleting a cell suspension of mature mesenchymal cells. As used herein, the terms “produced” and its equivalents (e.g., produce, generate, etc.) are used interchangeably with “generated” or “formed” and their equivalents when referring to method steps leading to the presence of the organoids of this disclosure. By performing such a panning procedure multiple times (e.g., 4 to 5 times), a cell suspension of early lineage stage cells is concentrated. The cell suspension is then transferred again to a low-adhesion dish and serum-free medium designed for early lineage stage cells and left in an incubator at 37°C for several hours or even overnight. In some embodiments, the remaining cell suspension is cultured on the low-adhesion dish and in serum-free medium until multiple organoids are formed by the self-organization of epithelial and mesenchymal cells. In some embodiments, the serum-free medium comprises a basic medium (copper-free, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, fatty acid-free, fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg / ml transferrin / Fe, 5 μg / ml insulin, and a mixture of purified free fatty acids that exist in complex with highly purified, fatty acid-free albumin). In some embodiments, the serum-free medium further comprises 10 μg / mL of high-density lipoprotein. Additional serum-free medium compositions are described elsewhere in this specification.

[0173] In some embodiments, multiple organoids are, At least one marker selected from the pluripotency gene group consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1, At least one marker selected from the endodermal transcription factor group consisting of Sox9, Sox17, Pdx1, HNF4 alpha, HNFB1, and ONECUT2, A BTSC that is positive for at least one marker selected from a group of stem cell / precursor-related surface markers consisting of one or more isoforms of EpCAM, NCAM, LGR5, CD44, CXCR4, sodium-iodine cotransporter (NIS), CD49 (integrin A6), CD29 (integrin B1), and integrin B4; BTSCs are negative for markers of mature hepatocytes or pancreatic cells, including P450, aquaporins, enzymes involved in bile production, amylase, and digestive enzymes.

[0174] In some embodiments, the remaining cell suspension is cultured on a low-adhesion dish in serum-free medium until multiple organoids are formed by the self-organization of cells remaining in the cell suspension. In some embodiments, multiple organoids are formed after about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours.

[0175] Cell culture conditions. The terms "culture" or "cell culture" refer to the maintenance of cells in an artificial in vitro environment. "Cell culture system" is used herein to refer to culture conditions under which a population of cells can grow ex vivo (outside of a living organism).

[0176] The term "basic culture medium" refers to a buffer used in cell culture, consisting of various nutrients in a composition that mimics the chemical components of amino acids, sugars, lipids, vitamins, minerals, salts, trace elements, and the interstitial fluid surrounding cells.

[0177] In this specification, “culture medium” is used to refer to a nutrient solution for culturing, growing, or proliferating cells. Culture media can be characterized by their functional properties, for example, but not limited to, the ability to maintain cells in a particular state (e.g., pluripotent, proliferative, or quiescent state), the ability to mature cells—in some cases specifically, the ability to promote the differentiation of progenitor cells into cells of a particular lineage. A non-limiting example of a culture medium is serum-supplemented medium (SSM), which is any basic medium supplemented with serum, typically at levels of about 10% to about 20%. The serum can be autologous (from the same species as the cells) or, more generally, serum obtained from animals routinely slaughtered for commercial purposes (e.g., chickens, cattle, pigs, etc.).

[0178] For grafting techniques, conditions are used to maintain cells as stem cells or early progenitor cells. That is, serum or any typical supplements that could drive cells into differentiation pathways and lead them toward the fate of mature cells are avoided. In addition to the usual basic medium, various nutritional supplements and lipids (a mixture of free fatty acids that form complexes with carrier molecules such as albumin and high-density lipoprotein) are added. Only two hormones / growth factors are added: insulin, which is necessary for carbohydrate metabolism, and transferrin, which is necessary as an iron carrier for polymerases.

[0179] As used herein, “Kubota medium” refers to any basic medium containing a mixture of copper-free, low-calcium (<0.5 mM), selenium, zinc, insulin, transferrin / Fe, and free fatty acids conjugated to purified albumin and optionally conjugated to high-density lipoprotein (HDL). In some embodiments, Kubota medium is copper-free, low-calcium (e.g., 0.3 mM), and about 10 -9 M selenium, approximately 0.1% bovine serum albumin or human serum albumin (highly purified and fatty acid-free), approximately 4.5 mM nicotinamide, approximately 0.1 nM zinc sulfate heptahydrate, approximately 10 -8The medium comprises any basic culture medium (e.g., RPMI 1640 or DMEM-F12) containing M hydrocortisone (an optional component used for liver precursors but not for pancreatic precursors), approximately 5 μg / ml transferrin / Fe, approximately 5 μg / ml insulin, approximately 10 μg / ml high-density lipoprotein, and a mixture of purified free fatty acids added after being bound to purified serum albumin. The free fatty acid mixture consists of approximately 100 mM palmitic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and stearic acid, respectively. Non-limiting exemplary methods for the preparation of this medium are published elsewhere, e.g., Kubota H, Reid LM, Proc. Nat. Acad. Scien. (USA) 2000;97:12132-12137, the entire disclosure of which is incorporated herein by reference.

[0180] An example of a non-restrictive medium for differentiation is hormone-restricted medium (HDM) used for the differentiation of endodermal stem cells into adult destiny. Supplements can be added to Kubota medium to create serum-free hormone-restricted medium (HDM) that promotes the differentiation of normal liver or biliary stem cells into specific adult destiny. Non-restrictive examples of restrictive media for differentiating endodermal stem cells into adult destiny include calcium reaching concentrations of 0.6 mM or higher, 1 nM triiodothyronine (T3), and 10 -12 Examples include media supplemented with M copper, 10 nM hydrocortisone, and 20 ng / ml basic fibroblast growth factor (bFGF). The following are the additional media conditions necessary to selectively produce hepatocytes (HDM-H), cholangiocarcinomas (HDM-C), and pancreatic islets (HDM-P): Further supplementation with HDM-H: 7 μg / L glucagon, 2 g / L galactose, 10 ng / ml epidermal growth factor (EGF), and 20 ng / ml hepatocyte growth factor (HGF); Further supplementation with HDM-C: 20 ng / ml of vascular endothelial growth factor (VEGF) and 10 ng / ml of HGF; and HDM-P: Prepared without glucocorticoids, supplemented with 1% B27, 0.1 mM ascorbic acid, 0.25 μM cyclopamine, 1 μM retinoic acid, and 20 ng / ml FGF-7 for 4 days, then switched to a supplement with 50 ng / ml exendin-4 and 20 ng / ml HGF for a further 6 days of induction.

[0181] The HDM provided herein may be supplements containing additional growth factors, which may include, but are not limited to, Wnt ligand, R-spongin, epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), neurotrophic factors, various interleukins, leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), colony-stimulating factor (CSF), GM-CSF, erythropoietin, thrombopoietin, heparin-binding growth factor, IGF-binding protein, and / or placental growth factor.

[0182] The HDMs provided herein can supplement cytokines, including, but not limited to, interleukins, lymphokines, monokines, colony-stimulating factors, chemokines, interferons, and tumor necrosis factor (TNF).

[0183] In some embodiments, the medium may be a “seeding medium” used to present or introduce cells into a given environment. In other embodiments, the medium may be a “differentiation medium” used to promote cell differentiation. Such a medium is a hormone-restricted medium (HDM) consisting of a “basic medium” which is a mixture of nutrients, minerals, amino acids, sugars, lipids, and trace elements, supplemented with either serum (serum supplementation medium or SSM) or a restricted mixture of purified hormones, growth factors, and nutrients, and used for the survival, maintenance, or differentiation of cells ex vivo. As used herein, “HDM-H” is an HDM used in combination with a base of type IV collagen and laminin to promote the differentiation of endodermal stem cells / precursors into mature hepatocytes. HDM-C is an HDM used in combination with a base of type I collagen and fibronectin to promote the differentiation of cells into mature bile duct cells.

[0184] When used herein, HDM can also be used in combination with a base of purified extracellular matrix components or concentrated extracts of the extracellular matrix, or with a matrix base that promotes differentiation to a specific fate. One example is the use of "HDM-H" combined with a base of purified type IV collagen and laminin to promote the differentiation of endothelial stem cells / precursors into mature hepatocytes. HDM-C is an HDM that can be used in combination with a base of type I collagen and fibronectin to make cells mature bile duct cells.

[0185] HDM can also be used in combination with extracellular matrix extracts resulting from decellularization processes, such as those used for the isolation of biomatrix scaffolds. Further details regarding HDM can be found in WO2012 / 003463, U.S. Patent No. 9,102,913, U.S. Patent No. 8,802,081, and WO2012003450, which are incorporated herein.

[0186] Basic media are buffers used in cell culture and consist of various nutrients in a composition that mimics the chemical composition of the interstitial fluid surrounding cells, including amino acids, sugars, lipids, vitamins, minerals, salts, trace elements, and other components. Furthermore, cell culture media typically consist of basic media supplemented with a small percentage (typically 2-10%) of serum. For grafting techniques, conditions are used to maintain cells as stem cells or early progenitor cells. Therefore, serum or any typical supplements that might push cells towards differentiation pathways or towards the fate of mature cells are avoided. In addition to the usual basic media, various nutritional supplements and lipids (a mixture of free fatty acids complexed with carrier molecules such as albumin and high-density lipoproteins) are added. Only two hormones / growth factors are added: insulin, necessary for carbohydrate metabolism, and transferrin, necessary as an Fe carrier for polymerase. Kubota medium, a serum-free medium designed for endodermal stem cells / progenitors, consists of basic media supplemented with zinc, selenium, insulin, transferrin, and lipids, but without cytokines or growth factors. Other growth factors and cytokines, and especially serum, should be avoided because they induce differentiation of donor cells, thereby minimizing the production of MMPs necessary for the engraftment and migration processes.

[0187] In some embodiments, the conditions for these patch grafts therefore contradict the conventional use of culture media supplemented with small percentages (typically 2–10%) of serum. While serum has been added for extended periods to provide essential signaling molecules (hormones, growth factors, cytokines) necessary to promote biological processes (e.g., proliferation, differentiation), serum should be avoided in these strategies for patch grafts to allow engraftment to occur. In some embodiments, serum is omitted to avoid cell differentiation and / or to avoid inactivation or attenuation of the secreted form of MMPs.

[0188] In some embodiments, the serum-free medium comprises a basic medium (copper-free, low-calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, fatty acid-free, fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg / ml transferrin / Fe, 5 μg / ml insulin, and a mixture of purified free fatty acids that exist in complex with highly purified, fatty acid-free albumin). In some embodiments, the serum-free medium further comprises 10 μg / mL of high-density lipoprotein.

[0189] Method of using patch graft composition In one embodiment, the present disclosure relates to a method for engrafting cells onto a solid organelle of an object that requires them, the method being This involves bringing the patch graft into contact with a solid organ. The patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a biomaterial having first viscoelastic properties, and the biomaterial facilitates contact between at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both, to promote engraftment between cells of a solid organ. This includes clearly indicating that at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both are engrafted among the cells of a solid organ.

[0190] In some embodiments, demonstrating that at least a portion of the aforementioned epithelial cells are engrafted among the cells of the solid organ is demonstrated by measuring the secretion level from the solid organ or the metabolic effect of the solid organ in a biological sample obtained from the subject.

[0191] In another aspect, the disclosure relates to a method for engrafting cells onto a solid organelle of an object that requires them, the method being This involves bringing the patch graft into contact with a solid organ. The patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a hydrogel layer having first viscoelastic properties, and the hydrogel facilitates the migration of at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both, from the patch across the outer surface of a solid organ, thereby facilitating contact. This includes demonstrating that at least a portion of the aforementioned epithelial cells, mesenchymal cells, or both are migrating along the outer surface of the solid organ.

[0192] As used herein, the term “engraftment” refers to the integration of cells into a tissue or organ. Alternatively, the terms graft, transplant, engraftment, inraftation, or inraftment may be used interchangeably with engraftment.

[0193] The term "migration" refers to the movement of cells from one part of a tissue or organ to another.

[0194] The term "integration" refers to the process where a cell connects with cells in a host organ or tissue, becoming part of that organ or tissue, but without fusing with the host cell.

[0195] In some embodiments, at least a portion of the mixture of epithelial cells and mesenchymal cells migrates over a substantial width of the solid organ and is distributed throughout the solid organ. In some embodiments, the patch graft further includes a backing that facilitates the migration of at least a portion of the mixture of epithelial cells and mesenchymal cells into the solid organ.

[0196] In some embodiments, epithelial cells are early lineage stage epithelial cells (ELSEs), and mesenchymal cells are early lineage stage mesenchymal cells (ELSMCs).

[0197] In some embodiments, ELSMCs include angioblasts, endothelial precursors, astrocytes, or a combination thereof. In some embodiments, ELSE and / or ELSMCs are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS). In some embodiments, epithelial cells are mature and mesenchymal cells are ELSMCs. In some embodiments, at least one of two categories of donor cells may be a stem cell / precursor that enables the production of MMPs. In preferred embodiments, MMPs are secreted isoforms of MMPs.

[0198] The term “tissue” is used herein to mean any tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, injured by trauma, damaged, and / or have genetic variations. The terms “natural tissue” or “living tissue” and their variations, as used herein, mean living tissue as it exists in its natural state or in its unaltered state from when it was obtained from an organism. “Microorganism” refers to a category of “bioengineered tissue” that mimics “natural tissue.”

[0199] Living tissue may include any single tissue (e.g., a collection of interconnected cells) or a group of tissues that constitute an organ, part, or region of the body of an organism. Tissue may consist of homogeneous cellular material or it may be a complex structure, such as that found in a region of the body including the chest, which may include lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, pancreas, gallbladder, lungs, intestines, thyroid gland, thymus, bladder, kidneys, prostate, uterus, breasts, skin, brain, spinal cord, blood vessels (e.g., aorta, iliac veins), heart, muscle, and any combination thereof.

[0200] In some embodiments, a mixture of stem cells / progenitor cells and mesenchymal cells migrates across most of the width of the organ, or at least throughout, and is uniformly distributed throughout the organ. In some embodiments, the solid organ is an endodermal organ. In some embodiments, the solid organ is an endodermal organ that includes the liver, pancreas, intestine, lung, bile duct, thymus, thyroid gland, parathyroid gland, and the urogenital sinus regions of the prostate and vagina. In some embodiments, the endodermal organ includes the liver, and engraftment involves remodeling of the Glisson's sheath.

[0201] As used herein, the term “remodeling” refers to histological changes in tissue initiated by engraftment and partially caused by secreted MMPs. For example, in some embodiments, the engraftment process disclosed herein results in remodeling of the Glisson’s sheath and the host tissue near the graft. Tissue remodeling is transient and returns to normal histological structure after the cells have fully integrated into the host organ / tissue. Remodeling can be visualized by multiple stains, such as trichrome staining, which identifies extracellular matrix components. These are complemented by H&E staining.

[0202] In some embodiments, the disclosure provides a method for further generating (i) engrafted epithelial and mesenchymal cells and (ii) host cells. In some embodiments, the method of the disclosure generates functional hepatocytes. In some embodiments, the parenchymal cells include hepatocytes and cholangiocytes.

[0203] In some embodiments, the patch comprises a backing positioned over a hydrogel containing a mixture of stem cell / precursor cells and mesenchymal cells. In some embodiments, the backing is used to tether the hydrogel layer to a target organ or site. In some embodiments, the endodermal organ includes the pancreas, and engraftment involves remodeling of the pancreatic capsule and pancreatic tissue near the transplantation site. In some embodiments, the method of this disclosure generates functional pancreatic cells. In some embodiments, the functional pancreatic cells include acinar cells and islets.

[0204] In some embodiments, explicit indication includes measuring a parameter or a change in a parameter that indicates the physiological effect on the target brought about by migrating cells.

[0205] In another aspect, the disclosure relates to introducing, restoring, increasing, or improving the functionality of a diseased, impaired, or dysfunctional solid organ, and the method includes contacting the diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells under conditions that promote engraftment of epithelial cells and mesenchymal cells, and demonstrating the introduction, restoration, increasing, or improving the functionality of the diseased, impaired, or dysfunctional solid organ.

[0206] In some embodiments, a portion of the mixture of stem cells / progenitor cells and mesenchymal cells is present in combination with cells of the target organ. In some embodiments, the patch graft used in the method of restoring organ function includes a covering that inhibits the adhesion of the patch graft to nearby organs and tissues.

[0207] In some embodiments, expressing includes measuring the level of secretory or metabolic products or effects in a biological sample obtained from a subject. In some embodiments, the method of the present disclosure further includes expressing that at least a portion of the mixture of epithelial cells and mesenchymal cells is distributed among the cells of the host organ. In some embodiments, the exposed surface of the patch graft includes a covering that inhibits the adhesion of the patch graft to nearby organs and tissues. In some embodiments, the solid organ includes an endodermal organ. In some embodiments, the endodermal organ includes a region derived from the urogenital sinus of the liver, pancreas, intestine, lung, bile duct, thymus, thyroid gland, parathyroid gland, or prostate or vagina. In some embodiments, the solid organ includes the pancreas, and an increase in the secretion level of at least one of insulin, c-peptide glucagon, somatostatin, or pancreatic polypeptide is measured.

[0208] In some embodiments, the solid organ includes the pancreas, and a decrease in blood glucose levels is measured. In some embodiments, the solid organ includes the pancreas, and an increase in glucose tolerance is demonstrated. In some embodiments, the solid organ includes the pancreas, and an increase in the levels of digestive enzymes or bicarbonate fluid is demonstrated. In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. In some embodiments, the solid organ includes the pancreas, and an increase in the levels of products derived from digestive enzymes secreted by the pancreas is measured. In some embodiments, the digestive enzymes include amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. In some embodiments, the solid organ includes the pancreas, and an improvement in digestion is demonstrated.

[0209] In some embodiments, the solid organ includes a liver, and the secretions include fluids rich in urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonates, blood clotting factors, or a combination thereof.

[0210] In some embodiments, the solid organ includes the liver, and the metabolic effect is a decrease in the levels of one or more of the following: cholesterol, blood glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase. In some embodiments, the metabolic effect is a decrease in the levels of tyrosine or alpha-fetoprotein.

[0211] In another aspect, the disclosure relates to a method for treating a subject diagnosed with a condition at least partially resulting from having a disease, disorder, or dysfunction of a solid organ, the method being (i) Contacting a diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells, (ii) Enabling epithelial cells and mesenchymal cells to migrate to and distribute among the cells of the host solid organ, (iii) A method comprising demonstrating that the negative effects of a diseased, impaired, or dysfunctional solid organ are mitigated in the subject of treatment. In some embodiments, explicit representation includes measuring the level of secreted or metabolic products or effects in a biological sample obtained from the subject. In some embodiments, the migration and distribution phases result in the mitigation of disease, impairment, or dysfunction.

[0212] In some embodiments, the solid organ is an endodermal organ. In some embodiments, the endodermal organ includes the liver, pancreas, intestine, lungs, bile ducts, thymus, thyroid gland, parathyroid gland, and urogenital sinus regions of the prostate or vagina. In some embodiments, the endodermal organ is the pancreas, and the subject suffers from diabetes. In some embodiments, an increase in the level of at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide is measured. In some embodiments, a decrease in blood glucose levels is demonstrated. In some embodiments, an increase in glucose tolerance is demonstrated.

[0213] As used herein, the terms “subject” and “patient” are to be used interchangeably and are intended to mean any animal. In some embodiments, the subject may be a mammal. In some embodiments, the mammal is a cow, horse, pig, dog, cat, monkey, mouse, human, or rat. In some embodiments, the subject is a human. In some embodiments, the subject includes mammals. In some embodiments, the mammal is a human.

[0214] As used herein, “treating” a disease or “treatment” of a disease means (1) preventing the onset of symptoms or disease in a subject who is predisposed to the disease or who has not yet shown symptoms of the disease; (2) suppressing the disease or preventing its onset; or (3) reducing or regressing the disease or symptoms of the disease. As understood in the Art, “treatment” is an approach to obtain a beneficial or desired outcome, including clinical outcomes. For the purposes of this Art, beneficial or desired outcomes may include, but are not limited to, one or more of the following, detectable or undetectable: relief or remission of one or more symptoms; reduction of the severity of a condition (including disease); stabilization (i.e., no worsening) of the condition (including disease); delay or slowing of the condition (including disease); progression of the condition (including disease); remission or relief; and improvement (whether partial or whole).

[0215] Also provided herein is a method for treating a subject having a liver disease or disorder, the method comprising, comprising, or essentially comprising contacting the liver of the subject with a patch graft comprising at least a first layer of hydrogel containing epithelial cells and mesenchymal cells, a second layer of hydrogel, and a third layer containing a biocompatible and biodegradable backing, and optionally comprising a fourth layer of hydrogel. In some embodiments of the method, the liver disease or disorder is hepatic fibrosis, cirrhosis, hemochromatosis, liver cancer, biliary atresia, non-alcoholic fatty liver disease, hepatitis, viral hepatitis, autoimmune hepatitis, phylum hepatitis, alcoholic liver disease, alpha-1 antitrypsin deficiency, glycogen storage disease type II, transthyretin-associated hereditary amyloidosis, Gilbert's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Budd-Chiari syndrome, hepatic trauma, or Wilson's disease.

[0216] In other embodiments, provided herein are methods for treating a subject having a disease or disorder of the pancreas, the method comprising, comprising, or essentially comprising contacting the subject's pancreas with a patch graft comprising at least a first layer of hydrogel containing epithelial and mesenchymal cells, a second layer of hydrogel, and a third layer containing a biocompatible and biodegradable backing, and optionally a fourth layer of hydrogel. In some embodiments of the method, the disease or disorder of the pancreas is diabetes mellitus, exocrine pancreatic insufficiency, pancreatitis, pancreatic cancer, sphincter of Oddi dysfunction, cystic fibrosis, pancreatic duct fusion anomaly, annular pancreas, pancreatic trauma, or pancreatic duct hemorrhage (hemosuccus pancreaticus).

[0217] In other embodiments, provided herein are methods for treating subjects having a gastrointestinal disease or disorder, the method comprising, comprising, or essentially comprising contacting the intestine of one or more subjects with a patch graft comprising at least a first layer of hydrogel containing epithelial and mesenchymal cells, a second layer of hydrogel, and a third layer containing a biocompatible and biodegradable backing, and optionally a fourth layer of hydrogel. In some embodiments, the gastrointestinal disease or disorder is gastroenteritis, gastrointestinal cancer, ileitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, peptic ulcer disease, celiac disease, fibrosis, angiogenesis, Hirschsprung's disease, pseudomembranous colitis, or gastrointestinal trauma.

[0218] Method of Disclosure The patch graft compositions provided herein are intended for the direct transplantation of cells into solid organs. This method is safe, avoids embolism and ectopic cell distribution, and optimizes the engraftment and distribution of cell numbers within and throughout the tissue.

[0219] Aspects of this disclosure relate to compositions and methods for engrafting cells within organs. Efforts to transplant cells from solid organs to viscera typically involve either direct injection or delivery of cells via vascular pathways. Lanzoni, G. et al. Stem Cells 31, 2047-2060 (2013). These transplantation methods result in a small number of transplanted cells and carry the risk of potentially life-threatening embolism. Transplantation is improved when cells are delivered by an "injection graft," which involves suspending cells in hyaluronan and then co-injecting a hyaluronan gel-inducing agent (PEGDA) in situ. The injection graft method, while still involving a small number of cells, typically delivers 10 per injection site. 6 ~10 7 This provides a strategy for localizing individual cells to specific sites. This strategy eliminates or minimizes ectopic cell distribution and optimizes cell integration at the site. However, when mature functional cells are used, they are highly immunogenic and may require prolonged immunosuppression.

[0220] To address these obstacles and concerns, the challenges are overcome by the “patch grafting” strategies described herein. In some embodiments, the “band-aid-like” grafts are surgically attached to the surface of an organ or tissue. The condition for this graft is that the cells fully engraft within the site, migrate throughout the organ / tissue, and then mature into the relevant adult cell type. A large number of cells (e.g., >10) 8 The potential for transplanting individual cells is entirely determined by the size of the patch, the number of cells in the graft, and the source of multiple forms of MMPs, ideally a cellular source of MMPs. Furthermore, considering the ease with which organoids can be cryopreserved under generally defined serum-free conditions in some embodiments, the use of organoids facilitates the ability to stockpile donor cells.

[0221] The patch graft compositions provided herein are intended for the transplantation of cells into solid organs. This method is safe, avoids embolism and ectopic cell distribution, and optimizes the engraftment and distribution of cell numbers within and throughout the tissue.

[0222] This disclosure provides a novel method for transplanting cells into solid organs or tissues, including viscera, as demonstrated herein by studies on the liver. The method is safe, avoids embolism and ectopic cell distribution, and optimizes the rapid engraftment and distribution of a large number of cells throughout the host tissue. Within one week, all of the donor cells (≥10 in pigs) are transplanted into a substantial portion of the liver. 8 , for mice ≥10 6 Grafting occurred, followed by cell migration and integration at a considerable distance from the graft site. Maturation into mature cell types occurred over two weeks throughout the entire region of the engrafted cells, and subsequently, the histological structure and composition of the Glisson's sheath and tissue recovered over three weeks. Importantly, patch grafts were able to save animals from the disease, as demonstrated here using patch grafts in a mouse model of type I tyrosinemia.

[0223] The cell transplantation strategies for solid organs disclosed herein are fundamentally different from those known in the art. In some embodiments, the cell transplantation methods for solid organs disclosed herein may involve directly placing grafts on the surface of a target site / organ and using graft biomaterials designed to allow donor cells to engraft and migrate within the tissue. While this corresponds to cell therapy strategies for skin, for internal tissues, such as internal organs of the abdominal cavity, modifications are needed to address mechanical effects, i.e., wear or compression of organs close to each other, and to recognize the unique fluid microenvironment surrounding specific organs.

[0224] The inventors of this disclosure as expressed herein are several exemplary embodiments of a strategy using grafts of biliary dendritic stem cells (BTSCs), which are determined endodermal stem cells / precursors that are precursors to both liver and pancreas. In some embodiments, BTSCs were implanted on the surface of the liver by patch grafting. In some embodiments, BTSCs were implanted on the surface of the pancreas by patch grafting.

[0225] The animal models used in some embodiments of this disclosure include mice (Mus musculus) and pigs (Sus scrofa domestic) transplanted with donor cells containing a transgene, the transgene being conjugated to green fluorescent protein (GFP), a fluorescent probe linked to the histone (H2-B) locus, and thus providing a nuclear biomarker. In some embodiments, the methods of this disclosure used the mouse model NOD-Rag1- / -IL2R gamma C-null (NRG), which is immunodeficient and has been genetically constructed (using Crisp / Cas9 technology) to be deficient in fumarylacetoacetate hydrolase (FAH), a key enzyme in the tyrosine metabolic pathway. Its loss results in type I tyrosinemia. Mice and pigs are major animal species in translational research and are increasingly being used as alternatives to non-human primates in preclinical studies.

[0226] The inventors of this disclosure have previously reported that engraftment requires co-transplantation of epithelial cells with mesenchymal cell partners appropriate to their lineage stage. Turner, R., et al. Successful Transplantation of Human Hepatic Stem Cells With Restricted Localization to Liver Using Hyaluronan Grafts. Hepatology 57, 775-784 (2013). For liver and biliary stem cells, these mesenchymal cells consist of angioblasts (CD117+, CD133+, VEGFr+, CD31-negative) and their direct offspring, endothelial precursors (CD133+, VEGFr+, CD31+, von Willebrand factor), and astrocyte precursors (CD146+, ICAM-1+, alpha-smooth muscle actin+ (ASMA), vitamin A-negative). The inventors collectively refer to these as early lineage mesenchymal cells (ELSMCs).

[0227] In some embodiments, matching epithelial and mesenchymal cell partners may be isolated by determining the ratio of lineage stage partners of epithelial and mesenchymal cells in the cell suspension using multiparametric flow cytometry, and then those ratios may be used in grafts with immunoselective cells. In some preferred embodiments, it may be more efficient to deplete the cell suspension of mature mesenchymal cells by repeating the panning procedure (see Methods) and then culturing the remaining cell suspension on a low-adhesion dish in serum-free Kubota medium for 6–8 hours. Self-organized organoids consisting of each aggregate containing approximately 50–100 cells. Marker analysis showed partnering between BTSCs and ELSMCs (Figures 1A–1D). As summarized in Figure 1A, BTSCs / ELMCs were used immediately or cryopreserved under specified conditions and thawed when grafting was needed. BTSC / ELSMC organoids were characterized using immunofluorescence (IF), qRT-PCR, and RNA-seq, and were shown to express the classical traits of BTSC (Figures 1A-1D) and ELSMC (data not shown). BTSCs in the organoids expressed low levels of pluripotency genes (e.g., OCT4, SOX2) and endodermal stem cell genes (e.g., EpCAM, SOX9, SOX17, PDX1, LGR5, CXCR4, MAFA, NGN3, and NIS), but did not express genes of mature liver or pancreas. These findings were confirmed in pre-transplant cells using representative qRT-PCR assays (Figure 1D). Immunohistochemical (IHC) assays showed that more primitive cells (e.g., those expressing the highest levels of pluripotency genes) were distributed within the organoid, while those in later mature lineage stages were distributed in the periphery (Figure 1C). The results shown in Figures 1A to 1D are exemplary embodiments for the formation of porcine organoids. In some embodiments, organoids may be similarly formed from any mammal. In some embodiments, organoids are formed from mice, or preferably from human cells.

[0228] The patch grafts were fixed to the liver surface by sutures or surgical glue, as shown in Figures 2A–2E. To achieve a specified level of stiffness, which is determined fluidically and expressed as the dynamic shear coefficient (G*), the graft composition included the use of thiol-modified hyaluronan (HA) hydrogel prepared with precise concentrations of HA and PEGDA (Figure 2C). Donor cells were embedded in a soft HA layer (less than 100 Pa), positioned against the liver surface, covered with a backing impregnated with a more rigid HA layer (approximately 700 Pa), and the graft was sutured or adhered to the liver surface at the corner of a silk patch. The soft hydrogel in which the donor cells were positioned maintained stem cell characteristics essential for the production of matrix metalloproteinases (MMPs) necessary for engraftment. The HA-impregnated silk backing acted as a barrier against migration in directions other than the target tissue. During surgery, an HA hydrogel with a rigidity of approximately 200-300 Pa was used, allowing the outer surface of the graft to be painted or covered. The hydrogel played a role in minimizing adhesion to the surrounding tissue.

[0229] The only variant of patch grafting that was attempted and abandoned involved surgically removing the capsule with a sharp edge. Excessive bleeding and the adverse effects of serum on the donor cells rendered its future use unnecessary, both in hosts with altered blood flow due to liver failure and even in normal hosts. Aside from such attempts to alter organ capsules, patch grafting proved to be a readily available surgical procedure.

[0230] As shown in Tables 1 and 2 below, several buckings were tested, focusing on those already clinically used in abdominal surgery. Only SERI Silk Surgical Scaffolds (Sofregen, Medford, Massachusetts) did not cause problems. Problems with other buckings (Table 1, Figures 12A–12E) included fragility (e.g., Seprafilm, Retroglyde), induction of necrosis or fibrosis and significant levels of adhesion (e.g., Surgisis, Vetrix), as well as severe adhesion formation between the abdomen and either the fibrous sponge version made from reconstituted silk protein or the bucking supplemented with carboxymethylcellulose ("berry jelly") (Figures 12A–12E). [Table 1]

[0231] Two forms of SERI Silk (Sofregen, Medford, Massachusetts) provided the best combination of mechanical support and minimal adhesion (Table 2), and the effect was further enhanced by applying 2×HA to the outer (free) surface of the silk backing after attachment to the target site. The product is purified fibroin from silkworm silk, which is woven into the scaffold to provide soft tissue support. Due to the stiffness of the original version of Seri-Silk, application to sites with significant curvature was difficult. In later studies, the inventors used "Contour Seri Silk" (Sofregen, Medford, Massachusetts), which has very high flexibility, allowing for graft application to target sites with any degree of curvature. At 3 weeks, the SERI-Silk graft was encased in a collagen band, suggesting mild fibrosis. [Table 2]

[0232] Evidence of engraftment one week postoperatively was verified by trichrome staining (Figures 3A and 3B) and hematoxylin / eosin (H&E) staining (Figures 3A2 and 3B2), revealing a remarkably extensive area of ​​remodeling in the parenchymal tissue beneath the graft site, in addition to Glisson's sheath remodeling (see also Figures 9A–9B). In this area, histological structure was either completely lost or in the process of lysis (see Figures 6E–6H). Reorganization of Glisson's sheath and lobular structure occurred over three weeks following HA reabsorption, which led to donor cell maturation and reduced MMP expression (Figure 3B). Engraftment in the liver of NRG / FAH mice was completed within 3–4 weeks postoperatively, with cells becoming uniform throughout the tissue (Figure 3C, and Figures 5A–5P).

[0233] In several embodiments, donor organoids of BTSC / ELSMCs derived from transgenic GFP+ pigs were transplanted into wild-type pigs (Figures 3A and 3B, Figure 4A) or NRG / FAH mice (Figure 3C, Figures 5L-5P) and identified by GFP expression (Figure 3C, and Figure 8A). In the liver, autofluorescence may originate from many different molecules (e.g., aromatic amino acids, flavins, vitamin A, lipofuscin). The autofluorescence of lipofuscin peaks at wavelengths overlapping with GFP (Figure 8A). Therefore, donor cell identification in the liver may be performed by binding an antibody against GFP (rabbit anti-GFP antibody, Novus, NB600-308), a secondary antibody with a red fluorescent probe that causes donor cells to have pink nuclei (donkey anti-rabbit 555, Invitrogen) (Figures 4A and 10), merging the red fluorescent probe with a blue color derived from DAPI in diploid cells, and by punctate pink entities associated with blue nuclei in cells with larger nuclei (presumably polyploid cells). In some embodiments, host cells were observed to have blue nuclei but no GFP expression (Figures 3C, 4A, and 10). Numerous donor GFP+BTSCS / ELSMCs were observed in the host liver near the graft (Figure 4A). In some embodiments, donor GFP+BTSC / ELSMCs were also observed near host lobules on the transverse side of the lobe, approximately 1.5 cm from the graft site (Figures 4B and 10). Therefore, in some embodiments, cells can migrate across the entire width of a lobule in one week.

[0234] Mature hepatocyte lobules contain lipofuscin in the cytosol, which is autofluorescent green. However, in young animals, the amount of lipofuscin is minimal and easily distinguishable from nuclear GFP-labeled cells (Figure 4B, Figure 4Cii, and Figure 8A). In some embodiments, mature donor GFP+ cells were observed as aggregates of hepatocytes with pink nuclei (Figure 4C, Figure 4Ci).

[0235] In some embodiments, transplantation of BTSC / ELSMC patch grafts resulted in organoid-mediated remodeling of Glisson's sheath and surrounding tissues, followed by merging of host and donor cells within one week (Figures 3A-3C-5A-5P, 6E-6H). Hematoxylin / eosin (H&E) staining of the remodeling regions suggested an inflammatory response involving both donor and host cells (Figures 6E-6H, 9A-9B). GFP labeling was found only in the nuclei of GFP+ BGTSC / ELSMCs in the grafts and in the nuclei of GFP+ adult cells (Figures 4A-4E). However, some GFP cytoplasmic staining was also observed in mature cells during the first week, and then completely or predominantly migrated to the nucleus within one or two weeks (Figures 4A-4E, 5A-5P).

[0236] In some embodiments, integration of donor cells within large areas of the liver was completed within two weeks, by which time HA was almost completely reabsorbed, and some donor cells had lineages limited to the fate of adult hepatocytes, including cholangiocarcinoma (pancytic keratin, pCK) and hepatocytes (albumin) (Figures 4A–4E). In some embodiments, donor GFP+ cells were present throughout the organ, acquiring a classic sinusoidal plate- or tubular morphology (Figure 4E), and expressing the functions of intermediates (e.g., SOX9, alpha-fetoprotein, HNF4a) and adult cells (albumin, pCK) (Figure 4E).

[0237] Due to the high engraftment efficiency of the patch transplant, essentially all donor cells remained viable after being transplanted into the host liver. They were not found on the liver surface or in the remainder of the graft, and there was no evidence of ectopic cell distribution to other organs (e.g., lungs). The migration rate of donor cells in the BTSC / ELSMC graft through the liver resulted in donor cells being delivered to most areas of the organ (liver) by the end of the first week, and by two to three weeks, uniform cell distribution occurred throughout the tissue (liver) (Figures 3A-3C-5A-5P).

[0238] In some embodiments, the present disclosure provides a method for rescuing a host from a pathological condition by cell engraftment. In one embodiment of the present disclosure, a method for rescuing a mouse model of type I tyrosinemia due to deficiency of fumarylacetoacetate hydrolase (NRG / FAH) in mice was achieved by cell engraftment. NRG / FAH mice were obtained from Dr. Lishan Su (UNC, Department of Microbiology and Immunology). NRG / FAH mice were established in immunodeficient mice using CRISPR / Cas9 technology to obtain a mouse model of type I tyrosinemia due to fumarylacetoacetate hydrolase (FAH) deficiency that also tolerates xenografts. The mice were maintained under conventional conditions of an immunodeficient host and sustained with normal liver and kidney by supplying their water with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and nitisinone (20 ug / ml). Niticinone has been shown to block the tyrosine pathway before FAH enzyme deficiency occurs, thus preventing the accumulation of toxic intermediates that affect both the liver and kidneys. NRG / FAH mice were treated with a patch graft of porcine BTSC / ELSMC organoids, followed by deactivation of niticinone. Controls were given a cell-free patch graft, and niticinone was deactivated.

[0239] All animals with porcine BTSC / ELSMC patch grafts remained healthy for more than four weeks after discontinuation of nitisinone and gained weight (approximately 1 mg / every 2-3 days), whereas controls (given cell-free patch grafts and withheld nitisinone) began losing weight (approximately 1 mg / day) by day 17 post-transplantation (Figure 11). Control mice with cell-free patch grafts had to be euthanized by day 17. Mice with BTSC / ELSMC-containing patch grafts were euthanized on day 30. Histological assays showed that mice with BTSC / ELSMC-containing patch grafts had livers and kidneys similar to those of animals maintained on nitisinone (Figures 5A, 5B, and 5N). In contrast, the livers and kidneys of animals with cell-free control grafts showed significant damage (Figures 5C, 5D, and 5N). These effects on the liver and kidneys are known to be due to toxins derived from tyrosine metabolism in FAH-deficient hosts. Histological assays showed that there were no or very few cells remaining in the patch graft (Figure 5E). GFP signaling from porcine donor cells was observed throughout the grafted liver (Figures 3C, 5G, 5H, 5I), with some cytoplasmic staining of GFP, but mostly expressed in the nucleus. Furthermore, independent assays for H2B histone showed co-expression with GFP both in the nucleus and cytoplasm (Figure 5P). In addition to histological examination of normal liver and kidney in animals with patch grafts containing BTSC / ELSMCs, donor cells in the host liver were shown to express porcine FAH (Figure 5O).

[0240] In some embodiments, the patch grafts of this disclosure maintain stem cells in an immature state that preserves the expression of matrix-metallic proteinases (MMPs) in donor stem cells. Remodeling of Glisson's sheath and adjacent hepatic lobules correlates with increased expression of multiple MMPs, enzymes known to lyse extracellular matrix components and be involved in cell migration. Figures 6A–6H summarize RNA sequencing and IHC assay data of MMPs expressed in comparison between stem cells / precursors and adult cells. BTSCs expressed high levels of multiple MMPs, consisting of both secretory forms (e.g., MMP2, MMP7) and membrane-bound forms (e.g., MMP14, MMP15). ELSMCs, precursors of endothelial and astrocyte cells, also contributed to the expression of multiple MMPs.

[0241] Findings from RNA-seq data were confirmed by IHC assays for proteins (enzymes) encoded by MMP genes (Figures 6E-6H). The IHC assays confirmed the presence of secreted forms of MMPs, such as MMP1, MMP2, MMP7, and MMP9, particularly in the remodeling regions. MMP1 protein expression was found in BTSC / ELSMC organoids and also in the remodeling regions of grafts. However, existing RNA-seq databases do not include MMP1 because there are no annotated species of porcine MMP1 used for analysis. Therefore, recognition of its presence in the remodeling zone is based on the IHC assays.

[0242] Depending on the factors that induce the differentiation of donor cells, the expression of secreted MMPs was attenuated, and accordingly, the potential for engraftment and migration was lost (data not shown). These include serum, various soluble regulatory signals known to affect the differentiation of donor cells (such as growth factors, cytokines, hormones), extracellular matrix components in the hydrogel or backing (especially type I collagen), and the stiffness of the HA hydrogel (e.g., greater than about 200 - 300 at the Pascal level). When the differentiation of ELSMCs preferentially proceeded into the stroma (e.g., in the presence of serum), the grafts became fibrotic; when directed towards the endothelium (in the presence of factors that promote angiogenesis), the grafts contained viable cells, but remained superficial when directed towards the organ (data not shown).

[0243] The present disclosure provides a novel method for the transplantation of cells into solid organs or tissues, including viscera, as demonstrated herein by studies on the liver. The method is safe, avoids embolization and ectopic cell distribution, and optimizes the rapid engraftment and distribution of numerous cells throughout the host tissue. Within one week, there was engraftment of all donor cells (≥10 8 in pigs, ≥10 6 ) over a substantial portion of the liver, and subsequently, there was cell migration and incorporation at a significant distance from the graft site. Maturation into mature cell types occurred over a two-week period throughout the area of engrafted cells, and subsequently, the histological structure and composition of the Glisson capsule and tissue were restored over a three-week period. Importantly, patch grafts were able to rescue animals from the disease state, as demonstrated herein using patch grafts in mice with type I tyrosinemia.

[0244] The method of the present disclosure is superior to methods of transplanting cells into solid organs by direct injection or by delivering cells via the vascular route. These past transplant methods have resulted in a small number of transplanted cells, a risk of life-threatening embolisms, and a significant level of potentially clinically significant but unknown ectopic cell distribution. These problems have led to cell therapy for internal solid organs being minimally used or not used at all.

[0245] In the present disclosure, it has been discovered that organoids provide the most successful placement for the cells used in the graft to ensure appropriate lineage-stage-specific epithelial-mesenchymal cell partnering. Thus, in some preferred embodiments, the donor cell mixture is formed by self-selecting them into the organoid after removal by mature mesenchymal panning. In some embodiments, relevant cells are immunologically selected by flow cytometry using characteristic surface antigens from the cell suspension, and then the donor cell mixture may be formed by co-transplanting epithelial-mesenchymal partners by mixing BTSCs and ELSMCs according to the ratios found in the cell suspension from fresh isolated tissue. Without being bound by theory, it is the hypothesis of the present disclosure that establishing lineage-stage-appropriate epithelial-mesenchymal partners provides relevant paracrine signaling to the graft, produces organoids under defined (serum-free) conditions, and that they are easily cultured or cryopreserved.

[0246] In one aspect, the primary design of the graft consists of a mixture of cells and a suitable biomaterial that can form a hydrogel capable of keeping the cells localized at the target site. The cells in the soft hydrogel are protected by a backing that is neutral with respect to the effects on donor cells. Preferred embodiments of the graft biomaterial can be non-sulfated or minimally sulfated glycosaminoglycans (GAGs) such as hyaluronic acid (HA), although HA is found in every stem cell niche together with receptors for HA, and HA receptors are classical stem cell traits. Without being bound by theory, it is the hypothesis of the present disclosure that HA supports maintenance as an immature form (i.e., as a stem cell / precursor) and optimizes the expression of secreted MMPs essential for engraftment and migration and integration into host tissue.

[0247] In some embodiments, the present disclosure has demonstrated that the methods of the present disclosure induced an engraftment process that resulted in remodeling of the Glisson's capsule and remodeling of the host tissue in the vicinity of the graft (FIGS. 3A-3C, FIGS. 5A-5P, and FIGS. 9A-9B). To verify the findings of remodeling, trichrome staining was used, and in addition to that staining of extracellular matrix components (FIGS. 3A-3C), staining with H&E (FIGS. 3A-3C and FIGS. 9A-9B) confirmed the remodeling phenomenon associated with inflammation. During the 3 weeks following surgery and subsequent clearance of HA, there was reorganization of the tissue structure of the Glisson's capsule and normal tissue. The remodeling zone (see FIGS. 3A-3C-5A-5P) contains multiple forms of MMPs (FIGS. 6A-H) and was shown to transiently return to a normal histological structure within 3 weeks.

[0248] While multiple types of HA exist, thiol-modified HA can be crosslinked with PEGDA to form hydrogels with precise biochemical and mechanical properties. These HA hydrogels are clinically useful for in vivo cell and molecular delivery. These properties of HA are reproducible, stable, and elastic, allowing access to all soluble signals from blood, lymph, or interstitial fluid within the graft, and minimizing donor cell maturation until engraftment and migration occur. The ability to alter fluidity factors with simple changes in HA and PEGDA concentrations allows for the "tuning" of HA, providing additional advantages in guiding cell migration direction and minimizing adhesion. Soft HA hydrogels mimic the properties in the stem cell niche and have been tolerant of the stem cell / progenitor cell-associated repertoire of MMPs, particularly secreted MMPs. Therefore, the mechanical properties of HA, which have been studied for many years in terms of skeletal tissue function, are also important in managing graft strategies.

[0249] In patch grafts containing stem cells / precursors, a remarkable phenomenon occurred: the graft "thawed" within the tissue within a few days, followed by fusion of donor and host cells, and distribution of cells over a significant portion of the organ within a week (see Figures 3A-3C and 5A-5P). During the remodeling phase, primarily in the first week after transplantation, GFP-labeled cytoplasmic expression was frequently observed in donor cells, especially if they had lineages limited to adult fate (Figure 4C). This was problematic because the GFP label was tagged at the H-2B histone locus, meaning the label should only be found in the nucleus. While not bound by theory, the hypothesis of this disclosure is that, given the presence of histones in the cytoplasm during inflammatory processes, there was abundant evidence of such inflammatory processes in the remodeling zone (Figures 9A-9B). These inflammatory processes decreased over time after transplantation, resulting in the reconstruction of Glisson's sheath, stabilization of histological structure, and donor cells exhibiting predominantly or completely nuclear GFP staining by 3-4 weeks (Figures 4D, 4E, 5G). In control studies, antibodies against histones produced a similar pattern to those against GFP (Figure 5P).

[0250] The discovery of this invention is that the engraftment and integration processes correlated with the expression of multiple MMPs, a family of calcium-dependent zinc-containing endopeptidases that degrade extracellular matrix components.

[0251] Immature cells express high levels of both secretory forms (e.g., MMP2, MMP7) and membrane-bound forms (e.g., MMP14, MMP15). IHC assays have shown that secretory MMPs (e.g., MMP1, MMP2, MMP7) are abundantly expressed in the remodeling region (Figure 7).

[0252] The biomaterials used in grafts, particularly HA, have been shown to maintain stem cell-like characteristics within cells both ex vivo and in vivo. Since grafts lack known signals that can trigger fate determination, the findings of donor cells that matured to different adult destinies suggest that the local microenvironment of the host tissue serves as a logical source of fate-determining factors during maturation.

[0253] Patch grafting is a safe and effective method for transplanting a large number of cells into solid organs or tissues, resulting in the replacement of missing functions or the alleviation of disease conditions. The number of cells that can engraft per patch is considerable (10 in pigs). 8 10 for mice 7 These findings are determined by the number of cells (e.g., ≤10) that can be achieved by vascular delivery or injection grafts, the dimensions of the patch graft, and the number of organoids. 6 This is in contrast to the individual.

[0254] The conditions for differentiating donor cells (soluble growth factors, cytokines, serum, matrix components, mechanical forces) resulted in a decrease in secreted MMPs and inhibited the processes of engraftment and migration. Complementing these findings was a control study using patch grafts of mature hepatocytes partnered with endothelium, where donor cells survived and were functional but did not engraft (data not shown). Therefore, engraftment requires a source of secreted MMPs, and ideally, this source is a cellular source capable of dynamically interacting to produce multiple forms of secreted MMPs as well as membrane-bound MMPs. Mature cells cannot do this because they express only or primarily the membrane-bound form (Figures 6A-6H).

[0255] This approach offers an alternative to cell therapy. The biomaterials and backings used have been proven safe insofar as they are neutral with respect to the host tissue, collectively support the maintenance of donor cells as immature cells, and thus produce the relevant repertoire of MMPs necessary for engraftment, migration, and integration.

[0256] Abbreviation ADHEP, adult hepatocytes; AFP, alpha-fetoprotein; ALB, albumin; BTSC, biliary dendritic stem cells; CD, common determinant; CD44, hyaluronan receptor; CD133, prominin; Cdx2, tail-type homeobox 2; CFTR, cystic fibrosis transmembrane permeability regulator; CK, cytokeratin protein; CXCR4, CXC-chemokine receptor 4 (also called fucin or CD184; also called platelet factor 4); EGF, epidermal growth factor; ELSMC, early lineage mesenchymal cells consisting of angioblasts and their progeny, endothelium and astrocyte precursors; EpCAM, epithelial cell adhesion molecule; FAH, fumaryl acetoacetate hydrolase, an enzyme crucial for tyrosine metabolism (its absence leads to type I tyrosinemia); FGF, fibroblast growth factor; HB, hepatoblasts; HGF, hepatocyte growth factor; HpSC , hepatic stem cells; KM, Kubota medium, a serum-free medium designed for endodermal stem cells; KRT, cytokeratin gene; LGR5, R-spongin-binding leucine-rich repeat-containing G protein-coupled receptor 5; Mafa, V-Maf myofascial fibrosarcoma oncogene homolog A; MMP, matrix metalloproteinase, a large family of proteinases involved in extracellular matrix lysis, cell migration, and regeneration responses; NANOG, transcription factor critically involved in self-regeneration; NCAM, neuronal cell adhesion molecule; NGN, neurogenin; NRG, NOD-Rag1- / -IL2R gamma C-null; NIS, sodium / iodine cotransporter; OCT4, (octamer-binding transmutation Transcription factor 4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a gene expressed by stem cells; PDX1, homeobox 1 of the pancreas and duodenum and a transcription factor crucial for pancreatic development; PBG, periductal gland, a stem cell niche for biliary stem cells; SALL4, Sal-like protein 4, found to be important for stem cell self-renewal; SOX, Sry-related HMG box; SOX2, a transcription factor essential for maintaining pluripotency in self-renewing or embryonic stem cells and determined stem cells; SOX9, a transcription factor associated with endodermal tissues (liver, intestine, and pancreas); SOX17, a transcription factor essential for liver differentiation; VEGF, vascular endothelial growth factor. [Examples]

[0257] Example 1: Preparation and Characterization of Patch Grafts This embodiment describes an exemplary method for preparing and characterizing patch grafts.

[0258] material Companies providing instruments, reagents, and / or supplies: Abcam, Cambridge, Massachusetts; ACD Labs, Toronto, Canada; Acris Antibodies, San Diego, California; Advanced Bioscience Resources (ABR), Rockville, Maryland; Agilent Technologies, Santa Clara, California; Alpco Diagnostics, Salem, New Hampshire; Applied Biosystems, Foster City, California; BD Pharmingen, San Jose, California; Becton Dickenson, Franklin Lakes, New Jersey; Bethyl Laboratories, Montgomery, Texas; BioAssay Systems, Hayward, California; Cambridge Isotope Laboratories, Tucksbury, Massachusetts; Biotime, Alameda, California; Carl Zeiss Microscopy, Thornwood, New York; Carolina Liquid Chemistries, Winston-Salem, North Carolina; Charles River Laboratories International, Wilmington, Massachusetts; Chenomx, Alberta, Canada; Cole-Parmer, Court Vernon Hills, Illinois; DiaPharma, West Chester Township, Ohio; Fisher Scientific, Pittsburgh, Pennsylvania; Gatan, Pleasanton, California; Illumina, San Diego, California; Ingenuity, Redwood, California; Life Technologies, Grand Island, New York; Leica, Washington, DC; LifeSpan Biosciences, Seattle, Washington; Molecular Devices, Sunnyvale, California; Olympus Scientific Solutions Americas, Waltham, Massachusetts;PhoenixSongs Biologicals (PSB), Branford, Connecticut; Polyscience, Warrington, Pennsylvania; Quiagen, Germantown, Maryland; R&D Systems, Minneapolis, Minnesota; RayBiotech, Norcross, Georgia; Roche Diagnostics, Mannheim, Germany; Santa Cruz Biotechnology, Dallas, Texas; Sigma-Aldrich, St. Louis, Missouri; Sofregen, Medford, Massachusetts; Takara, Otsu, Japan; Tousimis Research, Rockville, Maryland; Triangle Research Labs (TRL), Research Triangle Park, North Carolina; Umetrics, Umeå, Sweden; Varian Medical Systems, Palo Alto, California; Vector Laboratories, Burlingame, California; VWR Scientific, Radnoll, Pennsylvania.

[0259] animal Location of the facility. Animals used as hosts or cell donors were maintained at facilities within the College of Veterinary Medicine at NCSU (Raleigh, North Carolina). Surgery, autopsies, and collection of all bodily fluids and tissues were performed at these facilities. All procedures were approved by the NCSU IACUC Committee.

[0260] The pig hosts used for the grafts were a six-way cross consisting of a mix of six different breeds: Yorkshire, Large White, Landrace (from sows), Duroc, Spot, and Pietran (from boars). This highly heterogeneous genetic background is desirable in that it is analogous to the heterogeneous genetic composition of human populations. All host animals were female, approximately 6 weeks old, and weighed about 15 kg.

[0261] Pig donors for cell transplantation. There were two classifications: a) transgenic donor animals carrying the GFP transgene (all studies in this report), and b) male pigs weighing approximately 15 kg and about 6 weeks old used as donors for cell transplantation into females (parallel studies whose findings are reported in subsequent reports). GFP+ donor animals were obtained by breeding transgenic H2B-GFP male pigs with wild-type nulliparous pigs using standard artificial insemination. This model was developed via CRISPR-Cas9-mediated homologous recombination repair (HDR) of IRES-pH2B-eGFP to the endogenous β-actin (ACTB) locus. Transgenic animals showed ubiquitous expression of pH2B-eGFP in all tissues.

[0262] By fusing GFP to H2B, the GFP marker is localized to nucleosomes, enabling clear visualization of the nucleus and study of chromosome dynamics. Extensive analysis of established lines confirmed ubiquitous and nuclear-localized expression. Furthermore, breeding demonstrated the transmission of H2B-GFP to the next generation. All animals were healthy, multiple pregnancies were established, and the offspring showed the expected Mendelian ratio for H2B-eGFP transmission. Genotypes were identified at birth for male offspring, and those positive for the transgene were humanely euthanized for tissue collection and isolation of donor cells.

[0263] Animal genotyping analysis. For each donor and recipient animal, the porcine leukocyte antigen class I (SLA-I) and class II (SLA-II) loci were PCR-amplified using primers designed to amplify known alleles in these regions, based on a PCR sequence-specific primer strategy. The system consists of a set of 47 distinguishive SLA-I primers that amplify 63 loci of SLA-1, SLA-2, and SLA-3, and a set of 47 distinguishive SLA-II primers that amplify the loci of DRB1, DQB1, and DQA. These primer sets were developed to distinguish alleles by groups sharing similar sequence motifs and are clearly and simply demonstrated for the detection of known SLA-I and SLA-II alleles. When used together, these primers effectively yield the haplotype of each animal tested, thus providing an assay for easily confirming matching or mismatched haplotypes between donor and recipient animals.

[0264] Mating pairs of NRG / FAH mice were obtained from Dr. Lishan Su (Department of Microbiology and Immunology, UNC, Chapel Hill, North Carolina) and maintained in the animal facility at UNC (Chapel Hill, North Carolina) under appropriate conditions for immunodeficient hosts. These FAH mice were established by CRISPR / Cas9 technology using NRG mice, which means that these mice are immunodeficient and secondarily deficient in fumarylacetoacetate hydrolase (FAH). The FAH gene encodes fumarylacetoacetate hydrolase, the final enzyme in the tyrosine and phenylalanine catabolic pathways. FAH is highly expressed in liver and kidney cells and less so in endocrine tissues. NRG (NOD-Rag1- / -IL2RgammaC-null) / FAH (fumarylacetoacetate hydrolase) knockout mice exhibit a phenotype of progressive liver (and kidney) injury that mimics the major features of hereditary tyrosinemia type 1 (HT1) in humans, such as tyrosinemia, the appearance of succinylacetone in blood and urine, and liver and kidney injury. Complications of FAH deficiency in mice were managed by adding 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; also called nitisinone) (20 μg / mL) to the animal's water supplementation. NTBC interrupts the production of toxic metabolites in the tyrosine catabolic pathway that results from the absence of FAH. The livers and kidneys of these mice exhibit normal histological architecture unless the animals are provided with nitisinone-free normal water. When regular water is provided, evidence of severe tyrosinemia occurs within two weeks, resulting in the need to euthanize the mice within three weeks.

[0265] Culture medium and solution All media were filter sterilized (0.22 μm filter) and kept at 4 °C in the dark before use. Basal media and fetal bovine serum (FBS) were purchased from GIBCO / Invitrogen. All growth factors were purchased from R&D Systems. All other reagents were obtained from Sigma, except as otherwise described.

[0266] Cell washing solution. 599 ml of basic medium (e.g., RPMI1640; Gibco #11875-093) was supplemented with 0.5 g of serum albumin (Sigma, #A8896-5G, fatty acid-free), 10⁻⁹ M selenium, and 5 ml of antibiotic (Gibco #35240-062, AAS). This was used to wash tissues and cells during processing.

[0267] Collagenase buffer. It consists of 100 mL of cell wash solution supplemented with collagenase (Sigma # C5138) at a final concentration of 600 U / mL (25 mg R1451) for bile duct tissue and 300 U / mL (12.5 mg) for organs (e.g., liver).

[0268] Cell suspensions, organoids, and HA hydrogels were prepared using Kubota medium, a globally defined, serum-free medium designed for endodermal stem cells / precursors. This medium consists of any basic medium (here RPMI 1640) containing copper-free, low-calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, fatty acid-free, fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg / ml transferrin / Fe, 5 μg / ml insulin, 10 μg / ml high-density lipoprotein, and a mixture of purified free fatty acids that exist in complex with highly purified, fatty acid-free albumin. Its preparation is described in detail in Methods Review 67. It is also available from PhoenixSongs Biologicals (Branford, Connecticut).

[0269] Hyaluronan (HA). Soluble, long-chain HA (Sigma catalog number 52747) was used for stabilizing and cryopreserving organoid cultures. The thiol-modified HA used in hydrogel production was obtained from Glycosan Biosciences, a subsidiary of Biotime. These thiol-modified HA components were produced by a proprietary bacterial fermentation process using Bacillus subtilis as the host, following the ISO 9001:2000 process (www.bioppolymers.novozyme.com / ).

[0270] This component is produced by Novozymes under the trademark HyaCare® and is 100% free of animal-derived raw materials and organic solvent residues. No animal-derived components are used in production, protein levels are very low, and it is endotoxin-free. (Production volume conforms to the standards set forth in the European Pharmacopoeia). The HA hydrogel was prepared using Glycosil (HyStem® HA, ESI BIO-CG313), a thiol-modified HA capable of inducing disulfide crosslinking in the presence of oxygen, or by forming thioether links using polyethylene glycol diacrylate (PEGDA). Glycosil® is reconstituted as a 1% solution of thiolated HA in 1% phosphate-buffered saline (PBS) using degassed water, or in the inventors' case, in serum-free Kubota medium. Once reconstituted, it remains liquid for several hours but can gel somewhat upon exposure to oxygen. Treatment of Glycosil with a crosslinking agent such as PEGDA, which induces gelation within minutes, results in more precise gelation without changing temperature or pH.

[0271] The level of crosslinking is the primary contributor to the level of stiffness or rigidity, and can be controlled by adjusting the ratio of thiol-modified HA to PEGDA. In preliminary studies, stem cell populations were tested in HA hydrogels of varying levels of stiffness, and it was found that stem cells remained viable in terms of both antigenicity and functionality (e.g., with respect to migratory ability) only when the stiffness level was less than 100-200 Pa. Using this finding, the inventors designed grafts with a hyaluronane hydrogel backing consisting of a very soft layer and a more rigid layer, forming a barrier against migration in directions other than the target tissue and minimizing cell adhesion from nearby tissues. Three versions of the hydrogel with different levels of stiffness are characterized in Figures 2-2E, and the characteristics include direct measurements of fluidic properties. The barrier of the most rigid barrier, 10×HA hydrogel (stiffness = 760 Pa), could be prepared in advance on the backing and cryopreserved as desired. During surgery, donor cells were prepared on a soft 1×HA hydrogel (rigidity = 60 Pa), placed on a more rigid 10× hydrogel (already on the backing), and the patch was attached to the target site. After attachment, the outside of the graft was coated or painted with 2×HA hydrogel (rigidity = 106 Pa) using a NORM-JECT 4010.200V0 plastic syringe fitted with a BD Micro-Fine® IV permanent implantable needle.

[0272] The macroscopic fluid properties of the hydrogel were determined using a stress-controlled conical plate rheometer (TA Instruments, AR-G2, cone diameter 40 mm, angle 1°). The gel was actively polymerized on the rheometer while oscillating at a frequency of 1 rad / s and a stress amplitude of 0.6 Pa, while the coefficients were continuously monitored to indicate the complete completion of the crosslinking reaction.

[0273] After equilibration, the hydrogels were subjected to vibrational frequency sweeps (stress amplitude: 0.6 Pa, frequency range: 0.01–100 Hz). The fluid properties of the three versions of hyaluronane hydrogel used are summarized in Figures 2A–2E, including a soft hydrogel (approximately 100 Pa), a more rigid one (approximately 700 Pa), and an intermediate level (approximately 200–300 Pa).

[0274] donor cells Donor cells were obtained from transgenic H2B-GFP pigs, as described above. These offer significant advantages to cell transplantation research in that all cells are tagged with H2B-GFP. By using a fluorescent protein as a molecular tag, it became possible to track the migration and engraftment of donor cells after transplantation. This fusion protein targets nucleosomes by fusing GFP to the nucleosomal H2B protein, resulting in a GFP signal in the nucleus / chromatin.

[0275] In the characterization of the grafts, the presence of autofluorescence for both silk backing (light green) and lipofuscin (dark green) in mature hepatocytes presented a challenge, considering that their wavelengths overlap with the wavelength of GFP. Therefore, the inventors used an antibody against GFP, and secondarily an antibody with a red fluorescent probe, to shift the GFP+ signal to pink or rose (Figures 4A-4E, Figure 10). As a result, stem cells were recognized as small cells with pink nuclei (a combination of blue DAPI staining of the nucleus and a rose-colored GFP+ label tagged by the antibody). Any mature hepatocyte donor cells were found to have pink nuclei and lipofuscin autofluorescence in the cytoplasm (Figures 4A-4E). In patch grafts on mouse liver, some cells had completely pink nuclei, while others had large blue nuclei with mottled pink bodies (Figures 5A-5P). The inventors hypothesize that this is due to a mixture of donor cells (diploid) with a single pink nucleus and some donor cells that had matured into polyploid cells with a larger nucleus, and therefore the GFP label may have been localized to a single region of the nucleus. Alternatively, the localization of individual GFP labels may indicate fusion of donor and host cells with young mouse hepatocytes, which are known to have ploidy levels ranging from 4N to 32N.

[0276] Cell preparation. Extrahepatic bile dens (gallbladder, common duct, hepatic duct) from transgenic pigs was obtained. Parenchymal cells were removed by striking the tissue with a sterile stainless steel mallet, carefully maintaining the connection between the intrahepatic and extrahepatic bile ducts. The bile dens were then washed with a "cell washing" buffer consisting of sterile serum-free basal medium supplemented with antibiotics, 0.1% serum albumin, and 1 nM selenium (10⁻⁹ M). The aggregates were then mechanically dissected with a crossed scalpel and enzymatically dispersed in a cell suspension in RPMI-1640 supplemented with 0.1% bovine serum albumin (BSA), 1 nM selenium, 300 U / ml type IV collagenase, 0.3 mg / ml deoxyribonuclease (DNAse), and antibiotics. Digestion was carried out at 32°C for 30–60 minutes with frequent stirring. Most tissues required two rounds of digestion, followed by centrifugation at 4°C and 1100 rpm. The cell pellets were combined and resuspended in cell wash. The cell suspension was centrifuged at 30G for 5 minutes at 4°C to remove red blood cells. The cell pellets were again resuspended in cell wash, filtered through a 40 μm nylon cell strainer (Becton Dickenson Falcon #352340), and fresh cell wash was added. Cell counts were determined, and viability was assessed using trypan blue. Cell viability exceeding 90–95% was routinely observed.

[0277] Mesenchymal stem cells / precursors required as partners. In previous studies, the inventors defined the antigenic profile of a population of mesenchymal cells that shows the correspondence between important paracrine signals required for hepatic and biliary dendritic stem cells and other signals required for mature parenchymal cells. The mesenchymal cells required as partners for BTSCs are subpopulations that do not contain MHC antigens and exhibit low lateral scattering, and can be identified as angioblasts (CD117+, CD133+, VEGF receptor+, and CD31-negative), endothelial precursors (CD133+, VEGF receptor+, and CD31+), and astrocyte precursors (CD146+, ICAM1+, VCAM+, alpha smooth muscle actin (ASMA)+, and vitamin A-negative). The inventors collectively refer to these three subpopulations as early lineage stage mesenchymal cells (ELSMCs).

[0278] In contrast, adult hepatocytes are associated with mature sinusoidal endothelial cells (CD31+++, type IV collagen+, VEGF receptor+, and CD117-negative) and adult bile duct cells associated with mature astrocytes and stromal cells (ICAM-1+, ASMA+, vitamin A++, type I collagen+).

[0279] Organoid formation. The cell suspension was added to a multi-well flat-bottom cell culture plate (Corning #353043) in serum-free Kubota medium and incubated at 37°C for approximately 1 hour to promote the adhesion of mature mesenchymal cells. Even in serum-free medium, mature mesenchymal cells adhered to the dish within 10-15 minutes. The cells remaining in the suspension were transferred to another dish and incubated again for up to 1 hour. This repetition resulted in the depletion of a significant portion of the mature mesenchymal cells. After the depletion of the mature mesenchymal cells, the remaining suspension cells were transferred to Corning's ultra-low adhesion dish (Corning #3471) in serum-free Kubota medium at a rate of approximately 2 × 10⁶ cells per well. 5 Cells were seeded individually and incubated overnight at 37°C in a CO2 incubator. Organoids consisting of biliary dendritic stem cells (BTSCs) and ELMSCs were formed overnight (Figures 1A-1D). These organoid cultures survived for several weeks in Kubota medium, especially when supplemented with soluble form of HA(Sigma) (0.1%). They could also be cryopreserved as described below. From each gram of neonatal piglet biliary dendritic tissue, the inventors obtained approximately 1.5 × 10⁶ cells. 7 The inventors obtained approximately 3–6 × 10 cells per well in a 6-well ultra-low adhesion plate incubated in serum-free Kubota medium. 5 Cells were used. The cells produced an average of 6,000 to 20,000 small organoids (approximately 50 to 100 cells / organoid / well). For grafts, the inventors used at least 100,000 organoids (>10 7Cells were used. Depending on the size of the backing, the inventors embedded the number of organoids in the graft in approximately 1 ml of soft hyaluronan hydrogel on a 3 cm × 4.5 cm backing. 8 More than one (i.e., about 10) 9 We were able to increase the number of organoids (individual cells) to that level.

[0280] Cryopreservation of stem cell organoids. Isolated stem cell organoids were cryopreserved in Cryostor10 (Bioliife, Seattle, Washington; https: / / www.stemcell.com / products / cryostor-cs10.html), an isotonic cryopreservation buffer containing antifreeze factors, dextran, and DMSO. Cell viability was further improved by supplementing with 0.1% HA (Sigma #52747). Cryopreservation was performed using a CryoMed® Controlled-Rate Freezer. Since the viability after thawing was over 90%, the cells were able to form or attach organoids, proliferate and expand ex vivo and in vivo, and produce expected mature cells in vitro and in vivo.

[0281] Graft composition (Figures 1A-1D and 2A-2E). Cell isolation and graft assembly are depicted in schematic diagrams in Figures 1A-1D and 7, and detailed summaries are provided in Figures 2A-2E. Grafts were formed using backing (Table 1, above), and stem cell organoids embedded in soft hyaluronan hydrogel were placed on top. These were briefly prepared in advance and maintained overnight in culture dishes in an incubator. The grafts were found to be stable at the target site throughout the experimental period. Cryopreservation of organoids was easily achieved, however, cryopreservation of organoids in soft hydrogel was not achieved. This means that organoid embedding in soft hydrogel needs to be performed immediately before surgery.

[0282] surgery Surgical procedure. Anesthesia was induced by intravenous administration of ketamine / xylazine (2-3 mg / kg body weight each) or intramuscular administration of 20 mg / kg ketamine and 2 mg / kg xylazine, and maintained with isoflurane in oxygen administered via a closed-circuit gas anesthesia unit. The animals were placed in a supine position, and the abdomen was shaved from the xiphoid process to the pubic bone. The skin was prepared aseptically using alternating iodine scrubs and alcohol solutions. After entering the operating room, the skin was repeatedly prepared using sterile techniques, the area was covered with topical iodine solution, and then a sterile surgical drape was applied. The surgeon used appropriate aseptic techniques. A central abdominal incision was made through the skin, subcutaneous tissue, and linea alba, starting from the xiphoid process and extending caudally for 8-12 cm. The left hepatic region was exposed, and a 3 × 4.5 cm patch graft was applied to the ventral surface of the liver. This patch graft contained a 1 × HA (approximately 60 Pa) layer with embedded organoids on a 10 × HA (approximately 760 Pa) backing, and the patch was placed in direct contact with the surface of the liver capsule. The patch graft was sutured to the liver using 4-0 polypropylene single-knot sutures with 4-6 needles. The exposed surface of the graft was then treated with 2 ml of 2 × HA hydrogel (approximately 106 Pa). The stiffness level of this gel was sufficiently fluid to allow application or covering of the outside of the graft and to minimize adhesion from adjacent tissues. After surgical graft placement, the rib alba was closed with simple continuous sutures using 0-PDS. The rib alba was blocked by intramuscular injection of 2 mg / kg of 0.5% bupivacaine. The subcutaneous tissue and skin were closed with 2-0 PDS sutures and 3-0 Monocryl sutures, respectively. Tissue adhesive was placed on the skin surface.

[0283] Immunosuppression. Graft transplantation from transgenic pigs to wild-type recipients was allogeneic, and therefore immunosuppression was necessary. The immunosuppression protocol used was established by a third party. All pigs were orally administered the immunosuppressants tacrolimus (0.5 mg / kg) and mycophenolic acid (500 mg) twice daily, starting 24 hours prior to surgery. The drugs were administered continuously throughout the entire experimental period. These drugs could be easily administered to the animals by mixing them with palatable foods.

[0284] Autopsy Procedure: All animals were humanely euthanized at the designated time by intravenous injection of a lethal dose of pentobarbital sodium after sedation with ketamine / xylazine and isofluorane anesthesia. Upon confirmation of death, the carcasses were carefully dissected, target organs were removed and placed in chilled Kubota medium for transport to the laboratory. In addition to the liver, the lungs, heart, kidneys, and spleen were recovered and fixed in 10% neutral formalin.

[0285] Patch grafts for NRG / FAH mice. Using neonatal piglet hepatocytes (10⁶ H₂B-GFP cell mixture), organoids were formed embedded in 100 μl of 1×HA hydrogel and transferred to a 0.7 cm × 1.2 cm Seri-silk contour backing impregnated with 10×HA hydrogel to form patch grafts. The patch grafts were then transplanted onto NRG / FAH mouse livers by sliding them between the medial lobe and the left lateral lobe. Using a micropipette, surgical glue was applied to the edge of the patch to fix it to the graft site. Subsequently, 200 μl of 2×HA hydrogel was applied as an adhesion barrier.

[0286] The muscle layer and skin were closed using sutures or clips, and mice were given 400 μL of saline for hydration and 100 μL of buprenorphine as postoperative treatment. Controls received a cell-free patch. All animals were allowed to recover overnight. On day 1 post-transplant, a 7-day gradual withdrawal process was performed to remove NTBCs (reduced to 25% on day 1, 12% from days 2 to 3, 6% from days 4 to 6, and 0% on day 7). In later studies, the inventors found that this gradual withdrawal process was unnecessary; the drug could simply be removed within approximately 24 hours after the patch implantation surgery. Body weight measurements were used to check for abnormalities in the animals.

[0287] Graft Characterization Histological Examination: More than 48 hours after fixation, tissue samples were placed in a labeled cassette in 70% ethanol and processed in a Leica ASP300S tissue processor at 60°C for approximately 10 hours. After overnight processing, the samples were embedded using a Leica EG1160 embedded station. The mold was filled with wax, and the sample was placed in the correct orientation to allow for the collection of desired sections. The cassette was cooled until the block and tissue sample could be removed from the mold as a single unit. This block was cut into 5-micron sections using a Leica RM2235 microtome, and the sections were suspended in a water bath and placed on slides. The slides were air-dried overnight before staining. Sections were stained with hematoxylin and eosin (H&E; reagents #7211 and #7111) or Masson's trichrome (Masson's trichrome staining: Blue Collagen Kit #87019) using Richard Allan Scientific Histology Products, according to the manufacturer's recommended protocol. The above protocol is programmed into Leica Autostainer XL.

[0288] Immunofluorescence (IF) of unstained frozen liver sections. Autofluorescent liver sections (Figure 8) from wild-type and transgenic pigs were prepared from tissues embedded in OCT and frozen, and rapidly frozen at -200°C for frozen section preparation. Frozen sections were imaged to observe donor cells containing GFP linked to H2B (Figure 8). High autofluorescence in the cytoplasm of hepatocytes (lipofuscin) and autofluorescence of the Seri-Silk backing presented problems in visualizing GFP+ cells. In some embodiments, paraffin sections were prepared and stained for GFP using a rabbit polyclonal antibody against GFP (Novus Biologcoils, NE600-308), i.e., a rabbit anti-GFP antibody was used in combination with a secondary antibody, donkey anti-rabbit IgG H&L (Alexa Fluor 568; ab175470, Invitrogen), and a donkey anti-goat IgG Alexa Fluor 488 antibody was used to exclude nonspecific staining of liver autofluorescence (Figures 3C, 4, and 10). For immunofluorescence of GFP following antibody treatment against GFP, frozen sections were thawed at room temperature for 1 hour and then fixed in 10% buffered formaldehyde, acetone, or methanol. After fixation, sections were washed three times in 1% phosphate-buffered saline (PBS) and then blocked with 2.5% horse serum in PBS at room temperature for 1 hour. Primary antibodies diluted in 10% goat serum in PBS were added and incubated overnight at 4°C. The following morning, the sections were rinsed three times with PBS and incubated with secondary antibodies diluted in 2.5% horse serum in PBS at room temperature for 2 hours. Images were acquired using a Zeiss CLSM 710 spectral confocal laser scanning microscope (Carl Zeiss Microscopy). The antibodies are listed in Table 3 or Table 4. Autofluorescence was reduced by quenching with a dye containing trypan blue. Trypan blue was used in tissues / cells at 0.4% in PBS. This significantly reduced background.

[0289] Images in Figures 4D and 4E were taken in Sapienza, Rome, Italy. Sections (3 μm) were stained with hematoxylin-eosin and Sirius Red according to standard protocols. For immunohistochemistry, endogenous peroxidase activity was blocked by incubation in methanol hydrogen peroxide (2.5%) for 30 minutes. Antigens were retrieved by applying proteinase K (code S3020, Dako, Grosstrup, Denmark) at room temperature for 10 minutes, as indicated by the supplier. Sections were then incubated overnight at 4°C with primary antibodies (pancytic keratin, Dako, code: Z0622, dilution: 1:100; Sox9, Millipore, code: AB5535, dilution: 1:200). [Table 3] JPEG0007854806000004.jpg60162

[0290] The samples were rinsed twice with PBS for 5 minutes each and incubated with biotinylated secondary antibody (LSAB+System-HRP, code K0690, Dako, Grostorup, Denmark) and then with streptavidin-HRP (LSAB+System-HRP, code K0690, Dako, Grostorup, Denmark) at room temperature for 20 minutes. Diaminobenzidine (Dako, Grostorup, Denmark) was used as the substrate, and sections were counterstained with hematoxylin (PMID: 29248458). Nonspecific protein binding was blocked with 5% normal goat serum for immunofluorescence. The specimens were incubated overnight at 4°C with primary antibodies (chicken anti-GFP, Abcam, code: ab13970, dilution = 1:200; rabbit anti-HNF4 alpha, Abcam, code: 92378, dilution = 1:50; rabbit anti-albumin, ab2406, dilution = 1:500). The specimens were washed and incubated for 1 hour with labeled isotype-specific secondary antibodies (anti-chicken AlexaFluor-546, anti-mouse AlexaFluor-488, anti-rabbit AlexaFluor-488, Invitrogen, Life Technologies, Paisley, UK), and counterstained with 4,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei (PMID: 26610370). Negative controls (primary antibody replaced with preimmune serum) were included for all immunoreactions. Sections were examined using a coded method with a Leica Microsystems DM 4500 B optical and fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Jenoptik Prog Res C10 Plus Videocam (Jena, Germany). Immunofluorescence staining was also analyzed using a confocal microscope (Leica TCS-SP2). Slides were further processed using an image analysis system (IAS-Delta Sistemi, Rome, Italy) and independently evaluated by two researchers in a blinded manner. Immunofluorescence staining was scanned using a digital scanner (Aperio Scanscope FL System, Aperio Technologies, Oxford, UK) and processed with ImageScope.

[0291] Quantitative reverse transcription and polymerase chain reaction (qRT-PCR) were performed. Total RNA was extracted from organoids or grafts using Trizole (Invitrogen). First-strand cDNA synthesized using the Primescript 1st strand cDNA synthesis kit (Takara) was used as a template for PCR amplification. Quantitative mRNA-level analysis was performed using a Faststart Universal Probe Master (Roche Diagnostics) equipped with an ABI PRISM 7900HT sequence detection system (Applied Biosystems). Primers were designed by the Universal Probe Library Assay Design Center (Roche Applied Science). The primer sequences are listed in Table 5 below. Primers were annealed at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles at 95°C (15 seconds) and 60°C (1 minute). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was generally used as a control and standard. [Table 4] JPEG0007854806000006.jpg96161

[0292] RNA sequencing and gene expression analysis. RNA was purified from cells using the Qiagen Rneasy kit. RNA integrity (RIN) analysis was performed using the Agilent 2000 Bioanalyzer. cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA preparation kit and sequenced on the Illumina HiSeq 2500 platform. Two samples were sequenced per lane, occupying a total of eight lanes (one flow cell) for all samples. Quality control analysis was completed using FastQ. Sequence reads were mapped to the human genome (hg19) using MapSplice2 with default parameters. Transcript quantification was performed by RSEM analysis, gene expression was normalized using DESeq, and differentially expressed genes were identified. Candidate fusion transcripts were also detected using MapSplice2. Fusion calls were based on the depth and complexity of reads extending across candidate fusion junctions. Gene expression profiles were compared using Pearson correlation analysis, and hierarchical clustering was performed in R. Hierarchical clustering was performed according to the variance stabilization transformation provided by the DESeq package. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) software. Differential gene expression analysis was performed only on genes with a minimum mean normalization number > 50 in at least one category.

[0293] Statistical analysis. Statistical significance between samples was calculated using Student's two-tailed t-test, and the results were expressed as mean ± standard deviation (SD). A p-value less than 0.05 was considered statistically significant.

[0294] Example 2: Administration of patch grafts to pig livers Representative findings from a study in which porcine GFP+BTSC / ELSMC organoids were transplanted into the livers of wild-type pigs. (Findings are shown in Figures 3-4, 6, 8-10, and 14). Cells were able to engraft within one week, and the engraftment process induced remodeling of the Glisson's sheath and the underlying tissue (Figures 3-4, 6, 8-10). The remodeling process showed similarities to the inflammatory process (Figures 9A-9B). By weeks 2-3, the engrafted cells showed maturation to adult cell types, including hepatocytes and cholangiocytes (Figures 4A-4E). The engraftment process was demonstrated to be mediated by multiple matrix metalloproteinases (Figures 6A-6H), which caused dramatic remodeling of the host tissue. With clearance of the grafted biomaterial, paracrine signaling domains (matrix and soluble signaling) resulted in attenuation of MMPs, and subsequently, the donor cells matured to adult cell types. Within three weeks, the host tissue stabilized and exhibited a complete integration of donor and host cells. PCR analysis of GFP-encoding DNA confirmed the distribution of donor cells throughout the host liver (Figure 14). This patch implantation method proved safe for both the host and tissue. The only drawback was the exacerbation of skin infection at the surgical site due to immunosuppression in the pigs (Figures 12A-12E).

[0295] Example 3: Administration of patch graft to mouse liver Patch grafts containing porcine GFP+BTSC / ELSMC organoids were surgically implanted into the livers of FAH / NRG mice. These livers were evaluated one month after transplantation. Donor cells were found throughout the host liver (Figure 3C, Figures 5A-5P, Figure 11, Figures 13A-13C). Similar to patch grafts on porcine liver, patch grafts on mouse liver resulted in rapid engraftment and integration throughout the host liver (Figures 13A-13C). In fact, donor cells largely survived the host liver up to one month. Ongoing research is evaluating the degree of dominance between donor and host cells at various time points after surgical graft implantation.

[0296] Example 4: Administration of patch graft to the pancreas This example describes an exemplary method for administering a patch graft to an organ such as the pancreas. Specifically, this detailed protocol describes the administration of a patch graft to the pancreas of a mutant mouse (Akita / NRG) that is immunodeficient and has a genetic condition that predisposes it to diabetes.

[0297] Preparation of the work area. Surgical instruments are autoclaved before surgery. The entire surgical procedure is performed in a laminar flow cabinet to minimize environmental contamination. Necessary supplies are assembled in the preparation area, surgical area, and recovery area using appropriate aseptic techniques. A circulating water heating pad is prepared. The circulating water heating pad has a temperature of 38°C and is used for temperature stabilization during surgery. The heating pad is covered with a sterile waterproof pad. A surgical microscope, such as the UB56 provided by the UNC animal facility, is used during surgical procedures. Instrument sterilizers, such as a hot bead sterilizer, are used to sterilize instruments between surgical procedures. Finally, a recovery area is prepared, consisting of a clean cage covered with a flat paper bed.

[0298] Preparation of animals for surgery. Eight-week-old male mice were used for patch graft surgery. Eight-week-old male mice were housed in standard cages, maintained on a 12-hour / 12-hour light-dark cycle, and fed a standard rodent diet as needed. Mice were anesthetized by peritoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine. Adequate anesthesia was assessed by observing the gradual loss of voluntary movement and muscle relaxation. Loss of reflexes was examined by pinching the toes. Eye ointment was applied to prevent dryness of the eyes under anesthesia.

[0299] Preparation of the surgical site. The rib cage and abdomen were disinfected with an antiseptic chlorhexidine solution. Fur was removed from an abdominal area (approximately 2.5 cm x 1.5 cm) by shaving. The shaved area was disinfected with gauze soaked in chlorhexidine solution, then disinfected with an alcohol solution, and finally applied with chlorhexidine solution. The animal was positioned in the surgical area so that the prepared surgical site faced upwards to the surgeon. To create a sterile working area, a waterproof surgical drape was placed over the mouse, exposing the disinfected abdominal area while covering the rest of the body. The mouse was monitored for anesthetic depth before the procedure.

[0300] The patch is fixed to the surface of the pancreas. An upper midline incision is made using a sterile surgical blade, extending from the xiphoid process to the navel and into the skin. To expose the four compartments of the upper abdomen, the underlying glandular leucoma and peritoneum are separated using sterile scissors. To prevent the internal organs from drying out, the organs are periodically sprinkled with a sterile 0.9% sodium chloride solution. The stomach is lifted using sterile forceps or a swab, exposing the spleen and the splenic lobe (tail region) of the pancreas. The patch graft (see below for preparation) immersed in fresh serum-free Kubota medium is placed on the surface of the pancreas in a culture dish. The corners of this patch are fixed with surgical adhesive (Patterson Veterinary, Devens, MA).

[0301] Patch graft preparation. The backing for the patch graft is thoroughly prepared before surgery. The patch graft, containing cells in a soft hydrogel on top of the backing, is prepared several hours before surgery. The cells and the hyaluronan mixture that produces the hydrogel are all prepared in serum-free Kubota medium. There are three different hyaluronan hydrogels used with varying levels of stiffness at 1×, 2×, and 10× viscoelastic (rigidity) levels, which are achieved by varying the ratio of thiol-modified hyaluronan concentration to polyethylene glycol diacrylate (PEGDA) concentration. The stiffest of the three hydrogels (10×) (approximately 600-800 Pa) is used to impregnate the silk backing and is thoroughly prepared before the surgical procedure (e.g., the day before). During surgery, a soft hydrogel is prepared by mixing hyaluronan with PEGDA in a 4:1 ratio (20% of 50 μl of hyaluronan is PEGDA), and allowed to gel for approximately 5 minutes. Then, the organoid (1 × 10) is prepared. 5 The cells were mixed into this soft hydrogel, the mixture was layered onto the backing, and the gelling process of the hydrogel / cell mixture was allowed to continue for a further 30 minutes to 2 hours. This allowed the patch graft to be attached to the target site. For most tissues and organs, this could be done by suturing the corners of the graft. For delicate tissues such as the pancreas, surgical glue could be used, and if the patch graft was placed partially over the duodenum and partially over the pancreas, sutures were used for the duodenum and surgical glue for the pancreas. After graft attachment, the patch graft was covered with 200 μL of HA hydrogel (viscoelasticity = 200-300 Pa) to minimize adhesion by nearby tissues and organs.

[0302] The organs are returned to the abdominal cavity. The incision is closed using 3.0 polyglycol filamentous sutures, with a continuous suture pattern for the muscle / peritoneal layer and a discontinuous suture pattern for the skin.

[0303] Postoperative care and monitoring. After the surgical procedure is completed, mice are placed in a recovery area consisting of a cage placed on a heating pad and covered with a flat paper bed to maintain a normal body temperature. To avoid postoperative hypoglycemia, nutritional supplements (DietGel Recovery, ordered from ClearH2O) are administered by placing moistened food at the bottom of the cage. Moistened food is prepared by soaking standard rodent food pellets in water until softened. Fluid support is provided by moistened food and optional water administration. Buprenorphine is administered twice daily as an analgesic (0.05-0.1 mg / kg) for three days postoperatively. Throughout the entire experiment, mice are observed for signs of possible infection, including fluid or pus discharge from the wound, or for physical deterioration characterized by decreased grooming behavior and activity levels, decreased appetite, and weight loss.

[0304] Example 5: In diabetic NRG / Akita mice, engrafted BTSC / ELSMC rapidly reverses hyperglycemia. In this example, we considered the use of patch grafts for the treatment of diabetes.

[0305] Mouse models. In this example, two mouse models were used: Akita mice and NRG / Akita mice. DBA-background Akita mice (DBA-Akita Ins2 Akita) are a recently described mouse mutant model of type 1 diabetes. Diabetes is a result of selective pancreatic β-cell toxicity and depletion due to insulin 2 misfolding. Akita spontaneous mutant (Ins2A AkitaHeterozygous mice are viable and reproductive mice. They develop hyperglycemia, hypoinsulinemia, polydipsia, and polyuria around 3-4 weeks of age. Studies conducted by the Diabetic Complications Consortium Animal Model (AMDCC) have shown that the DBA / 2J strain is particularly prone to developing diabetic nephropathy. Therefore, the advantages of this model of type 1 diabetes are the presence of early-onset, progressive functional and structural glomerular damage and the fact that diabetes is spontaneous.

[0306] NRG / Akita mice are immunodeficient mice genetically modified to have a gene defect in the Akita mouse. Breeding pairs of these two mouse models were ordered from Jackson Laboratory (Bar Harbor, Maine) and used to establish colonies at UNC. Eight-week-old mice were used in patch graft tests and maintained for approximately one month before being euthanized.

[0307] Patch graft administration and results. Patch grafts were prepared as described in Example 1. In patch graft surgery, patch grafts containing cells were administered to the pancreas of mice as described in Example 4. In the control group, surgery was performed using graft biomaterial but without cells (e.g., mice treated with graft biomaterial only).

[0308] Postoperatively, blood glucose levels were obtained from mice treated with patch grafts, mice treated with graft biomaterial only, and untreated mice (e.g., mice that received neither patch grafts nor graft biomaterial). Mice treated with patch grafts showed a gradual decrease in blood glucose levels compared to mice treated with biomaterial only and untreated mice. This decrease was significant up to one week after patch implantation, and the effect lasted for up to 3-4 weeks, after which the mice were euthanized. Blood glucose levels were dynamically monitored every two days using a glucose meter starting three days after surgery in the animals, each time after a 5-hour fast. The results showed that hyperglycemia was reduced in diabetic mice treated with patch grafts within approximately 7-10 days, and several mice were corrected to normal blood glucose within 3 weeks (Figure 15A, triangle). Such a reduction in hyperglycemia was not observed in untreated diabetic mice (Figure 15A, circle) or diabetic mice treated with biomaterial only (Figure 15A, square). Furthermore, a gradual decrease in blood glucose levels was found to correlate with a significant increase in serum levels of C-peptide, which indicates insulin secretion (see Figure 15B, third column for days 7, 14, and 21, which correspond to diabetic mice treated with patch grafts). Mouse serum C-peptide levels were lowest in untreated diabetic mice (Figure 15B, first column for days 0, 7, 14, and 21), diabetic mice treated with biomaterials alone (Figure 15B, second column for days 0, 7, 14, and 21), and diabetic mice treated with patch grafts on day 0 (Figure 15B, third column for day 0).

[0309] Serum insulin levels also gradually increased in diabetic mice treated with patch grafts (Figure 15C, third column on days 21 and 28). Serum insulin levels did not increase in untreated diabetic mice (Figure 15C, first bar on days 21 and 28) or in diabetic mice treated with biomaterials alone (Figure 15C, second column on days 21 and 28).

[0310] An intraperitoneal glucose tolerance test (IPGTT) was also performed to evaluate hyperphysiological glucose-stimulated insulin release. The glucose spike measurements were then evaluated over a period of time to determine the insulin response to increased plasma glucose. Compared to non-diabetic mice (normal mouse controls, Figure 15D, triangle), diabetic mice treated with biomaterials alone (Figure 15D, circle) showed high basal blood glucose levels and changes in the glucose tolerance test with persistent hyperglycemia. In contrast, diabetic mice treated with cell-containing patch grafts (Figure 15D, square) showed normal (or near-normal) basal blood glucose levels and responses to the IPGTT compared to non-diabetic control mice (Figure 15D, triangle). This data provides additional evidence of how the patch grafts enabled these conventionally diabetic mice to tolerate and manage glucose spikes. The complete return to euglycemia in organoid-grafted mice within 240 minutes is remarkable evidence of the BTSC cells' impressive ability to respond to hyperphysiological glucose spikes. However, the correction for hyperglycemia was significantly slower than the correction observed in non-diabetic control mice.

[0311] Figures 16A-16B-18A-18B show images of pancreatic sections obtained from Akita / NRG mice with patch grafts derived from DS-red mice. These sections were stained for insulin (Figures 16A-16B) or NGN3 (Figures 17A-17C and 18A-18B). Donor cells are shown in red in Figures 18A-18B, when visualized using DS-red staining.

[0312] Example 6. Patch graft containing porcine GFP+BTSC / ELSMC organoids surgically attached to the pancreas of a wild-type piglet. In this example, a patch graft containing GFP+BTSC / ELSMC organoids derived from transgenic pigs was transplanted onto the pancreas of a wild-type pig.

[0313] Patch grafts containing porcine GFP+ BTSC / ELSMC organoids were ligated to the pancreases of wild-type piglets and evaluated after one week. GFP+ donor cells engrafted within one week and were found to be widely dispersed throughout the pancreas (Figures 19A-19B-21A-21C). At the first week, the engrafted cells had matured into both islet and acinar cells. However, while the nuclear biomarker GFP was observed in the nuclei of the islets, it was observed in the cytoplasm of the acinar cells. Studies are ongoing to evaluate whether GFP stabilizes in the nucleus when it engrafts in the liver at a later stage.

[0314] In Figures 19A, 19B, 21A, and 21C, the green cells, whether islets or acinar cells, are donor cells. Sections were also stained for insulin (red) to ensure that red cells with blue nuclei represent host pancreatic islets, while those with red / purple nuclei and yellowish cytoplasm represent donor cell-derived beta cells in the islets. The antibodies used to detect insulin, glucagon, GFP, and amylase are shown in the table below. [Table 5]

[0315] The immunofluorescence staining of porcine pancreases grafted with transgenic pig-derived GFP+BTSC / ELSMCs is shown in Figures 19A and 19B. Immunofluorescence staining for insulin is shown as red, and GFP is shown as green. Graft regions were collected and analyzed on day 7 post-transplant. The nuclei were stained with DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) and appeared blue. GFP+ cells were originally green due to the transgene linked to H2B histone, but the intensity of the green color was enhanced by staining with an antibody against GFP bound to a green fluorescent probe. Numerous GFP+ donor-derived cells are visible near the region where the patch graft was placed. Insulin expression, a characteristic of pancreatic islet beta cells, was identified using an anti-insulin antibody bound to a red fluorescent probe. Endogenous (host) islet beta cells appeared red and were clearly visible in the upper part of the pancreas. Donor-derived beta cells are visible with yellowish nuclei resulting from the merging of blue (DAPI) and green (GFP), and red / orange cytoplasm resulting from insulin staining in the lower pancreas proximal to the patch graft placement site. This low-magnification image clearly shows the degree of engraftment in the pancreas, as well as engraftment in the submucosa of the duodenum and the location of Brunner's glands (presumably the starting point of the cell network contributing to organogenesis of the liver and pancreas).

[0316] Immunofluorescence staining for amylase and GFP in serial sections from the same tissue block is shown in Figure 19B. Amylase (green) is detected primarily in pancreatic acinar tissue, as well as in the mucosal layer and duodenal lumen. Insulin (red) does not overlap with amylase (green). This staining, when combined with the staining in Figure 19A, suggests that the majority of GFP+ donor-derived cells are differentiated to a pancreatic acinar-like fate.

[0317] Figures 20A–20C show immunofluorescence staining for insulin and GFP in pancreatic tissue sections obtained from three recipients of GFP+BTSC / ELSMC patch graft transplantation (7 days post-transplant). GFP+ cells were observed in the pancreatic parenchyma and near the patch graft site in all recipients. In particular, GFP+ cells appeared to emerge at a distance of a few millimeters from the patch material and were well integrated into the recipient pancreatic parenchyma. The patch material (SERI silk) exhibited some fluorescence in different channels and was still visible 7 days post-transplant.

[0318] Figures 21A–20C clearly show the coexistence of endogenous (host) islet beta cells (insulin+ / GFP-: red cytoplasm) and donor-derived islet beta cells (insulin+ / GFP+: yellow to orange cytoplasm) in the pancreas 7 days after transplantation of a GFP+ BTSC / ELSMC patch graft. Both donor-derived and endogenous (host) islet beta cells were observed in all cases. The majority of GFP+ cells exhibited a phenotype consistent with that of acinar cells. GFP+ cells organized to form tubular structures can be seen in the lower part of Figure 21A.

[0319] Example 7. Use of BTSC / ELSMC patch grafts to treat streptozocin (STZ)-induced diabetes in animals. This embodiment describes a detailed protocol of an exemplary method for treating diabetes in mice that have been induced with STZ by administering a patch graft.

[0320] Animal care and rearing. 8-14 week old C57BL / 6 (Jax Stock 000664), BALB / c (Jax Stock 000651), and DsRed.MST B6 (Jax Stock 006051) are housed in standard cages, maintained on a 12-hour / 12-hour light-dark cycle, and fed a standard rodent diet as needed.

[0321] Immunodeficient NSG mice (Jax Stock 005557) aged 8-14 weeks were housed in a sterile environment with a 12-hour / 12-hour light-dark cycle and fed autoclaved food as needed.

[0322] DSRed.MST B6 mice will be used as donors for islets and BTSC / ELMC.

[0323] C57BL / 6, BALB / c, and NSG mice were used as recipients.

[0324] Induction of diabetes over 14 days. Diabetes is induced in the target recipient via streptozotocin treatment at a dose of 250 mg / kg. After the first injection, blood glucose levels are monitored, and if blood glucose is found to be >350 mg / dL for three consecutive days, the animal is considered to have diabetes. In rare cases, the initial dose of streptozotocin may only be partially effective (e.g., blood glucose does not reach >350 mg / dL for three consecutive days), in which case the animal receives additional doses to complete beta-cell depletion and induction of persistent hyperglycemia. Streptozotocin treatment may be repeated up to three times, with each injection spaced three days apart. 95-100% of animals treated with streptozotocin are expected to develop diabetes.

[0325] LP insulin pellets are placed subcutaneously to maintain diabetic animals. The insulin pellets are removed after patch graft implantation on day 15.

[0326] Day 1: Hyaluronic acid (HA) hydrogel was combined with a 20% polyethylene glycol diacrylate (PEGDA) linker solution. After mixing the soft HA gel components, it was allowed to gel for about 5 minutes, and then BTSC / ELMC organoids (1 × 10⁻¹⁶) were added. 5Add individual (or island) (2000 IEQ) particles and allow gelation to continue for a further 30 minutes to 3 hours. Add this to a pre-prepared backing containing a more rigid hyaluronan layer. Prepare this one day in advance and complete the gelatin overnight by culturing it in a 5% CO2 incubator at 37°C at the air-liquid interface.

[0327] Preparation of the work area. Autoclave surgical instruments before surgery. Wear appropriate protective covers during surgery. Make multiple sets of sterile gloves available for use in the surgical work area. Perform all surgical procedures in a laminar flow cabinet to minimize environmental contamination. Assemble necessary supplies in the preparation area, surgical area, and recovery area using appropriate aseptic techniques. Prepare a 38°C heating pad for temperature stabilization during surgery. Perform surgery using a surgical microscope. Use instrument sterilizers such as hot bead sterilizers to sterilize instruments between surgical procedures. Prepare a recovery area consisting of a clean cage covered with a flat paper bed.

[0328] Preparation of animals for surgery. The series of transplants will be sex-mismatched; that is, cells from male donors will be transplanted into female recipients to allow for Y chromosome tracking and fluorescence tracking to be confirmed.

[0329] For patch graft transplantation of DsRed.MST B6 cells in a syngeneic background, 8-14 week old C57BL / 6 recipient mice will be used.

[0330] For patch graft transplantation of DsRed.MST B6 cells in an allogeneic background, 8-14 week old BALB / c recipient mice will be used.

[0331] For human cell patch graft transplantation, immunodeficient NSG recipient mice aged 8-14 weeks are used.

[0332] Weigh the mice and calculate the required doses of ketamine (100 mg / kg) and xylazine (10 mg / kg). Anesthetize the mice by intraperitoneal injection of 100 mg / kg of ketamine and 10 mg / kg of xylazine. Assess appropriate anesthesia by observing the gradual loss of voluntary movement and muscle relaxation. Examine the loss of reflexes by pinching the toenails. Apply eye ointment to prevent dryness of the eyes under anesthesia. Alternatively, mice may be anesthetized using 2% isoflurane via inhalation.

[0333] Preparation of the surgical site. Fur was removed from the abdomen (approximately 2.5 cm x 1.5 cm area) by shaving. The rib cage and abdomen were disinfected with an antiseptic chlorhexidine solution. The shaved area was disinfected with gauze soaked in chlorhexidine solution, wiped with alcohol solution, and then chlorhexidine solution was applied again. The animal was positioned and secured within the surgical area by placing it on its back so that the prepared surgical site in the abdominal area faced upward to the surgeon. The mouse was covered with a waterproof surgical drape that had an opening to expose the disinfected abdominal area and covered the rest of the body, creating a sterile working area. The mouse was monitored for anesthetic depth before the procedure.

[0334] Secure the patch to the surface of the pancreas. An upper midline incision is made in the skin using a sterile surgical blade, extending from the xiphoid process to the navel. Extend the skin incision 1 cm in each direction from the xiphoid process toward the upper limb. Extend this incision 1 cm in each direction from the navel toward the lower limb. Separate the underlying glandular leucoma and peritoneum using sterile scissors or a scalpel to expose the four compartments of the upper abdomen. Separate and widen the incision with retaining clips. To prevent the internal organs from drying out, sprinkle them periodically with sterile 0.9% sodium chloride. Using sterile forceps or a swab, lift the stomach and pull down the intestines to expose the spleen and the splenic lobe (caudal region) of the pancreas. Immerse the patch filled with cells embedded in HA gel in fresh Kubota medium in a culture dish. Orient the gel side facing the pancreas (this patch resembles a band-aid, with the gel on only one side). Place the patch on the surface of the pancreas so that the gel side is in contact with the pancreas. Secure the corners of the two patches with surgical adhesive (Patterson Veterinary, Devens, MA). To minimize adhesion between organs, cover the patches with 200 μl of 2×HA hydrogel. Return the organ to the abdominal cavity. Close the incision with 3.0 polyglycol filamentous sutures, using a continuous suture pattern for the muscle / peritoneal layer and a discontinuous suture pattern for the skin.

[0335] Postoperative care and monitoring. After completing the surgical procedure, the mice are placed in a recovery area. The recovery area consists of a cage placed on a heating pad and covered with a flat paper bed to maintain a normal body temperature. To avoid postoperative hypoglycemia, nutritional supplements (DietGel Recovery, ordered from ClearH2O) are to be administered by soaking standard rodent food pellets in water until soft and placing the moistened food at the bottom of the cage. Fluid support is provided by moistened food and voluntary water supply. Buprenorphine is to be used as an analgesic (0.05-0.1 mg / kg) twice daily for 3 days postoperatively, or as an SR version that acts for 72 hours with a single injection. Throughout the entire experiment, the mice are observed for the development of signs of possible infection, including fluid or pus discharge from the wound, or for physical deterioration characterized by decreased grooming behavior and activity levels, decreased appetite, and weight loss. Administer prophylactic antibiotics such as Clavamox or Baytril in the drinking water for up to 14 days after surgery. (Note) [Note 1] A method for engrafting cells into a solid organelle that requires them, Attaching the patch graft to a solid organ; Here, the patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a biomaterial having first viscoelastic properties, wherein the biomaterial facilitates the engraftment of at least a portion of the epithelial cells, mesenchymal cells, or both, between the cells of the solid organ. A method comprising making it clear that at least a portion of the epithelial cells, mesenchymal cells, or both are engrafted among the cells of the solid organ. [Note 2] The method according to Appendix 1, wherein the explicit indication includes measuring the level of secretion from the solid organ or the metabolic effect of the solid organ in a biological sample obtained from the subject, thereby indicating that at least a portion of the epithelial cells are engrafted among the cells of the solid organ. [Note 3] A method for engrafting cells into a solid organelle that requires them, Attaching the patch graft to a solid organ; Here, the patch comprises a mixture of epithelial cells and mesenchymal cells incorporated into a hydrogel layer having first viscoelastic properties, wherein the hydrogel facilitates the migration of at least a portion of the epithelial cells, mesenchymal cells, or both, from the patch across the outer surface of the solid organ. A method comprising demonstrating that at least a portion of the epithelial cells, mesenchymal cells, or both are migrating across the outer surface of the solid organ. [Note 4] The method described above, as described in Appendix 3, includes measuring a parameter or a change in a parameter that indicates the physiological effect on the subject brought about by the migrating cells. [Note 5] The method according to Appendix 1, further comprising a backing that facilitates the migration of at least a portion of the mixture of epithelial cells and mesenchymal cells to the solid organ. [Note 6] The method according to Appendix 5, wherein at least a portion of the mixture of epithelial cells and mesenchymal cells migrates over the substantial width of the solid organ and is distributed throughout the solid organ. [Note 7] The method according to Appendix 1, wherein the solid organ is an endoderm organ. [Note 8] The method according to Appendix 1, wherein the solid organ is an endoderm organ including the liver, pancreas, intestine, lung, bile duct, thymus, thyroid gland, parathyroid gland, and the urogenital sinus region of the prostate and vagina. [Note 9] The method according to Appendix 8, wherein the endodermal organ includes the liver and engraftment is accompanied by remodeling of the Glisson's sheath. [Note 10] The method described in Appendix 8, further comprising (i) creating combinations of engrafted epithelial cells and mesenchymal cells with (ii) host cells. [Note 11] The method described in Appendix 8 for producing functional hepatocytes. [Note 12] The method according to Appendix 11, wherein the parenchymal cells include hepatocytes and bile duct cells. [Note 13] The method according to Appendix 8, wherein the endodermal organ includes the pancreas, and engraftment is accompanied by remodeling of the pancreatic capsule and pancreatic tissue near the transplantation site. [Note 14] The method described in Appendix 13 for generating functional pancreatic cells. [Note 15] The method according to Appendix 14, wherein the functional pancreatic cells include acinar cells and islets. [Note 16] The method according to Appendix 14, wherein the pancreas secretes at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide at an increased level. [Note 17] The method described in Appendix 14, wherein the pancreas exhibits a metabolic effect of reducing blood glucose levels. [Note 18] The method described in Appendix 14, wherein the pancreas exhibits a metabolic effect of increased glucose tolerance. [Note 19] The method according to Appendix 14, wherein the pancreas secretes digestive enzymes or bicarbonate fluid at increased levels. [Note 20] The method according to Appendix 19, wherein the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or a combination thereof. [Note 21] The method described in Appendix 14, which results in an increase in the level of metabolites derived from digestive enzymes secreted by the pancreas. [Note 22] The method according to Appendix 21, wherein the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or a combination thereof. [Note 23] The method described in Appendix 21, which improves digestion. [Note 24] The method according to Appendix 11, wherein the liver secretes a fluid rich in urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonates, or blood coagulation factors. [Note 25] The method according to Appendix 4, wherein the biological sample obtained from the subject shows a decrease in at least one level of cholesterol, blood glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase. [Note 26] The method according to Appendix 1, wherein the patch comprises a backing positioned over the hydrogel containing the mixture of epithelial cells and mesenchymal cells. [Note 27] The method according to Appendix 26, wherein the backing is used to connect the hydrogel layer to the host organ. [Note 28] The method according to Appendix 1, wherein at least one or both of the epithelial cells and mesenchymal cells are early lineage stage cells. [Note 29] The method according to Appendix 28, wherein the early lineage mesenchymal cells (ELSMCs) include angioblasts, endothelial precursors, astrocytes, or a combination thereof. [Note 30] The method according to Appendix 28, wherein the early lineage stage epithelial cells (ELSE), ELSMC, or both are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS). [Note 31] The method according to Appendix 1, wherein the epithelial cells are mature and the mesenchymal cells are ELSMCs. [Note 32] A method for introducing, restoring, increasing, or improving the functionality of a target diseased, impaired, or dysfunctional solid organ, comprising: contacting the diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells under conditions that promote engraftment of epithelial cells and mesenchymal cells; and demonstrating the introduction, restoration, increase, or improvement of the functionality of the diseased, impaired, or dysfunctional solid organ. [Note 33] The method according to Appendix 32, wherein the explicit statement includes measuring the level of secreted or metabolized products or effects in a biological sample obtained from the subject. [Note 34] The method according to Appendix 32, further comprising specifying that at least a portion of the mixture of epithelial cells and mesenchymal cells is distributed among the cells of the host organ. [Note 35] The method according to Appendix 32, wherein the exposed surface of the patch graft includes a covering that inhibits adhesion of the patch graft to nearby organs and tissues. [Note 36] The method according to Appendix 32, wherein the solid organ includes an endoderm organ. [Note 37] The method according to Appendix 36, wherein the endoderm organ includes a region derived from the liver, pancreas, intestine, lung, bile duct, thymus, thyroid gland, parathyroid gland, or the urogenital sinus of the prostate or vagina. [Note 38] The method according to Appendix 33, wherein the solid organ includes the pancreas, and an increase in the secretion level of at least one of insulin, c-peptide glucagon, somatostatin, or pancreatic polypeptide is measured. [Note 39] The method according to Appendix 32, wherein the solid organ includes the pancreas and the reduction in blood glucose levels is measured. [Note 40] The method according to Appendix 32, wherein the solid organ includes the pancreas and an increase in glucose tolerance is explicitly demonstrated. [Note 41] The method according to Appendix 32, wherein the solid organ includes the pancreas and an increase in the level of digestive enzymes or bicarbonate fluid is explicitly indicated. [Note 42] The method according to Appendix 41, wherein the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. [Note 43] The method according to Appendix 33, wherein the solid organ includes the pancreas, and an increase in the level of products derived from digestive enzymes secreted by the pancreas is measured. [Note 44] The method according to Appendix 43, wherein the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. [Note 45] The method according to Appendix 32, wherein the solid organ includes the pancreas, and improvement in digestion is explicitly demonstrated. [Note 46] The method according to Appendix 33, wherein the solid organ includes a liver and secretes fluids rich in urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonates, blood coagulation factors, or combinations thereof. [Note 47] The method according to Appendix 33, wherein the solid organ includes a liver, and the metabolic effect is a reduction in the levels of one or more of the following: cholesterol, blood glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase. [Note 48] The method according to Appendix 32, wherein the solid organ includes a liver and the subject suffers from type 1 tyrosinemia. [Note 49] The method according to Appendix 33, wherein the metabolic effect is a decrease in the levels of tyrosine or alpha-fetoprotein. [Note 50] A method for treating a subject diagnosed with a condition at least partially attributable to having a disease, disorder, or dysfunction of a solid organ, (i) Contacting the diseased, impaired, or dysfunctional solid organ with a patch graft containing a mixture of epithelial cells and mesenchymal cells, (ii) enabling the epithelial cells and mesenchymal cells to migrate to the cells of the host solid organ and to be distributed among the cells, (iii) A method comprising demonstrating that the negative effects of the disease, disorder, or dysfunction of a solid organ are mitigated in the subject of treatment. [Note 51] The method according to Appendix 50, wherein the explicit indication includes measuring the level of secreted or metabolized products or effects in a biological sample obtained from the subject. [Note 52] The method according to Appendix 50, wherein the migration and distribution stages result in the alleviation of the disease, disorder, or dysfunction. [Note 53] The method according to Appendix 50, wherein the solid organ is an endoderm organ. [Note 54] The method according to Appendix 53, wherein the endoderm organs include the liver, pancreas, intestine, lung, bile duct, thymus, thyroid gland, parathyroid gland, and urogenital sinus region of the prostate or vagina. [Note 55] The method according to Appendix 53, wherein the endoderm organ is the pancreas and the subject suffers from diabetes. [Note 56] The method according to Appendix 55, wherein an increase in the level of at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide is measured. [Note 57] The method described in Appendix 55, which clearly demonstrates a decrease in blood glucose levels. [Note 58] The method described in Appendix 55 demonstrates increased glucose tolerance. [Note 59] The method described in Appendix 50, wherein the subject includes mammals. [Note 60] The method described in Appendix 59, wherein the mammal is a human. [Note 61] A patch graft comprising a mixture of epithelial cells and mesenchymal cells, and one or more layers of biomaterial, wherein the layers are at least a) A first inner layer for contacting solid organs, which exhibits first viscoelastic properties, incorporates a mixture of epithelial cells and mesenchymal cells, supports the ability of the epithelial cells and mesenchymal cells to produce secretory matrix metalloproteinases (MMPs), and promotes the viability and immaturity of the epithelial cells and mesenchymal cells; b) A backing that optionally provides a barrier to cells migrating in directions other than the solid organ, and exhibits a second viscoelastic property, c) Optionally, a third outer layer of covering or material in the vicinity of the patch graft to minimize adhesion of the patch graft to the inner wall and / or inner surface of the body cavity, including organs. and Includes, before The viscoelastic properties are determined by measuring the fluidic properties and expressed in Pascals (Pa). ru, Patch graft. [Note 62] The patch graft as described in Appendix 61, wherein the epithelial cells include early lineage stage epithelial cells (ELSE) and the mesenchymal cells include early lineage stage mesenchymal cells (ELSMC), or the epithelial cells and mesenchymal cells are at a later lineage stage but are at equivalent lineage stages. [Note 63] The patch graft described in Appendix 62, wherein the ELSMC comprises angioblasts, endothelial precursors, astrocytes, or a combination thereof. [Note 64] The patch graft described in Appendix 62, wherein the ELSE and / or ELSMC are derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS). [Note 65] The patch graft described in Appendix 61, wherein the epithelial cells are in the late lineage stage and the mesenchymal cells are early lineage stage mesenchymal cells (ELSMC), or the mesenchymal cells are in the late lineage stage and the epithelial cells are early lineage stage epithelial cells (ELSE). [Note 66] The patch graft described in Appendix 61, wherein the second viscoelastic property (expressed in Pa) is higher than the first viscoelastic property. [Note 67] The patch graft according to Appendix 61, wherein the one or more biomaterial layers include a hydrogel, and the hydrogel further includes minimally sulfated or non-sulfated glycosaminoglycans. [Note 68] The patch graft according to Appendix 67, wherein the non-sulfate glycosaminoglycan contains hyaluronan. [Note 69] The patch graft according to Appendix 68, wherein the hyaluronane comprises thiol-modified hyaluronane, and its gelation by disulfide crosslinking is induced in the presence of polyethylene glycol diacrylate (PEGDA). [Note 70] The patch graft according to Appendix 69, wherein the fluidity trait is determined at least in part by the initial concentration and stiffness of the thiol-modified hyaluronan and PEGDA before gelation and the final stiffness of the hydrogel after gelation, which is achieved by the precise ratio of the volumes of the thiol-modified hyaluronan and PEGDA. [Note 71] The patch graft described in Appendix 61, wherein the first inner layer exhibits a first viscoelasticity ranging from approximately 50 Pa to approximately 150 Pa. [Note 72] The patch graft according to Appendix 61, wherein the optional backing contains a hyaluronan hydrogel layer exhibiting viscoelasticity from about 600 to about 800 Pa. [Note 73] The patch graft according to Appendix 61, wherein the optional third outer layer comprises a hyaluronane hydrogel layer having viscoelastic properties from about 200 to about 300 Pa. [Note 74] The patch graft described in Appendix 61, wherein the backing includes silk. [Note 75] The patch graft as described in Appendix 74, wherein the silk backing includes refined fibroin of silkworm silk woven into the scaffold, including Seri-Silk (trademark) or Contour Seri Silk (trademark). [Note 76] The patch graft according to Appendix 61, wherein the epithelial cells include bile dendritic stem cells (BTSCs) and the mesenchymal cells include early lineage mesenchymal cells (ELSMCs). [Note 77] The patch graft according to Appendix 76, wherein the ELSMC comprises hemangioblasts and their direct offspring, endothelial cell precursors, astrocyte precursors, or a combination thereof. [Note 78] The patch graft described in Appendix 77, wherein the angioblasts express CD117, CD133, and VEGFr, but do not express CD31. [Note 79] The patch graft described in Appendix 77, wherein the endothelial cell precursor expresses CD133, VEGFr, CD31, and von Willebrand factor. [Note 80] The patch graft described in Appendix 77, wherein the astrocyte precursor expresses CD146, ICAM-1, and alpha-smooth muscle actin (ASMA), and is negative for vitamin A. [Note 81] The patch graft described in Appendix 61 is produced by repeating a panning procedure to selectively remove cells that adhere to the mixture of epithelial cells and mesenchymal cells within about 15 to 30 minutes at 37°C, by depleting the cell suspension of mature mesenchymal cells. [Note 82] The patch graft described in Appendix 81 is cultured on a low-adhesion dish in serum-free medium until multiple organoids are formed by the self-organization of epithelial and mesenchymal cells. [Note 83] The patch graft as described in Appendix 82, wherein the serum-free medium comprises a basic medium (copper-free, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, fatty acid-free, fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg / ml transferrin / Fe, 5 μg / ml insulin, and a mixture of purified free fatty acids that exist in complex with highly purified albumin free of fatty acids). [Note 84] The patch graft described in Appendix 83, wherein the serum-free medium further contains 10 μg / ml of high-density lipoprotein. [Note 85] The patch graft described in Appendix 82, wherein the plurality of organoids are formed after approximately 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. [Note 86] The aforementioned plurality of organoids a) At least one marker selected from the pluripotent gene group consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1, b) At least one marker selected from the endodermal transcription factor group consisting of Sox9, Sox17, Pdx1, HNF4 alpha, HNFB1, and ONECUT2, c) A BTSC that is positive for at least one marker selected from the group of stem cell / precursor-related surface markers consisting of one or more isoforms of EpCAM, NCAM, LGR5, CD44, CXCR4, sodium-iodine cotransporter (NIS), CD49 (integrin A6), CD29 (integrin B1), and integrin B4; The BTSC is negative for markers of mature hepatocytes or pancreatic cells, including P450, aquaporins, enzymes involved in bile production, amylase, and digestive enzymes, as described in Appendix 82. [Note 87] The patch graft according to Appendix 61, wherein one or more biomaterial layers contain recombinant MMP. [Note 88] The patch graft according to Appendix 61, wherein one or more biomaterial layers contain cells engineered to express MMPs.

Claims

1. A patch graft comprising a mixture of epithelial cells and mesenchymal cells for use in a method to induce, restore, increase, or improve the function of a target disease, disorder, or dysfunctional liver suffering from type 1 tyrosinemia, The method includes contacting the diseased, impaired, or dysfunctional liver with a patch graft containing a mixture of epithelial cells and mesenchymal cells, such that the introduction, restoration, increase, or improvement of the functionality of the diseased, impaired, or dysfunctional liver is manifested by measuring a decrease in the levels of tyrosine or alpha-fetoprotein measured in a biological sample obtained from the subject, under conditions that promote the engraftment of epithelial cells and mesenchymal cells. A patch graft containing epithelial cells, including bile dendritic stem cells (BTSCs), and mesenchymal cells, including early lineage mesenchymal cells (ELSMCs).

2. The patch graft according to claim 1, wherein the exposed surface of the patch graft includes a covering that inhibits adhesion of the patch graft to nearby organs and tissues.

3. A patch graft according to any one of claims 1 to 2, comprising one or more biomaterial layers, wherein the biomaterial layers are at least a) A first inner layer for contacting solid organs, exhibiting first viscoelastic properties, incorporating a mixture of epithelial cells and mesenchymal cells, supporting the ability of epithelial and mesenchymal cells to produce secretory matrix metalloproteinases (MMPs), and promoting the viability and immaturity of the aforementioned epithelial and mesenchymal cells. Includes, Depending on the circumstances, b) A backing that provides a barrier to cells migrating in directions other than solid organs, and which exhibits a second viscoelastic property, and / or c) A third outer layer of covering or material that exhibits third viscoelastic properties, which minimizes adhesion of the patch graft to the inner wall and / or inner surface of the body cavity, including organs, in the vicinity of the patch graft. It may include, A patch graft in which the aforementioned viscoelastic properties are determined by measuring the fluidic traits and expressed in Pascals (Pa).

4. The patch graft according to claim 3, wherein the first inner layer contains a hyaluronane hydrogel layer exhibiting a first viscoelasticity of about 50 Pa to about 150 Pa.

5. The patch graft according to claim 3, wherein the backing contains a hyaluronane hydrogel layer exhibiting viscoelasticity from about 600 to about 800 Pa.

6. The patch graft according to claim 3, wherein the third outer layer comprises a hyaluronane hydrogel layer and the third viscoelastic property is about 200 to about 300 Pa.

7. The patch graft according to claim 3, wherein the backing comprises silk.

8. The patch graft according to any one of claims 1 to 7, wherein the mixture of BTSC and ELSMC is depleted of mature mesenchymal cells.

9. The patch graft according to claim 8, wherein the mixture of BTSC and ELSMC comprises a plurality of organoids formed by the self-assembly of BTSC and ELSMC.

10. Multiple organoids, a) At least one marker selected from the pluripotent gene group consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1, b) At least one marker selected from the endodermal transcription factor group consisting of Sox9, Sox17, Pdx1, HNF4 alpha, HNFB1, and ONECUT2, c) A BTSC that is positive for at least one marker selected from the group of stem cell / precursor-related surface markers consisting of one or more isoforms of EpCAM, NCAM, LGR5, CD44, CXCR4, sodium-iodine cotransporter (NIS), CD49 (integrin A6), CD29 (integrin B1), and integrin B4; BTSCs are negative for markers of mature hepatocytes or pancreatic cells, including P450, aquaporins, enzymes involved in bile production, amylase, and digestive enzymes. The patch graft according to claim 9.

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