Patch graft compositions for cell engraftment

Abstract

Compositions and methods of transplanting cells by grafting strategies into solid organs (especially internal organs) are provided. These methods and compositions can be used to repair diseased organs or to establish models of disease states in experimental hosts. The method involves attachment onto the surface of a tissue or organ, a patch graft, containing the donor cells. The donor cells may be a mixture of stem cells/progenitors with supporting early lineage stage mesenchymal cells. The patch graft promotes migration of the donor cells into the host organ and supports the successful integration of donor cells with host cells to repair the diseased organ.

What is claimed is: 
     
         1 . A method of engrafting cells into a solid organ of a subject in need thereof, comprising:
 contacting a patch graft onto a solid organ,
 the patch comprising a mixture of epithelial cells and mesenchymal cells incorporated into a biomaterial having a first viscoelasticity property, in which the biomaterial promotes an engraftment of at least a portion of said epithelial cells, mesenchymal cells, or both among the cells of the solid organ; 
   demonstrating that at least a portion of said epithelial cells, mesenchymal cells, or both have engrafted among the cells of the solid organ.   
     
     
         2 . The method of  claim 1  in which said demonstrating comprises measuring a level of a secretion from the solid organ, or a metabolic effect of the solid organ, in a biological sample obtained from the subject to demonstrate that at least a portion of said epithelial cells have engrafted among the cells of the solid organ. 
     
     
         3 . A method of engrafting cells into a solid organ of a subject in need thereof, comprising:
 contacting a patch graft onto a solid organ,
 the patch comprising a mixture of epithelial cells and mesenchymal cells incorporated into a hydrogel layer having a first viscoelasticity property, in which the hydrogel promotes a migration of at least a portion of said epithelial cells, mesenchymal cells, or both from the patch through an outer surface of the solid organ, 
   demonstrating that at least a portion of said epithelial cells, mesenchymal cells, or both have migrated through an outer surface of the solid organ.   
     
     
         4 . The method of  claim 3  in which said demonstrating comprises measuring a parameter or a change in same, which indicates a physiological effect in the subject resulting from the migrated cells. 
     
     
         5 . The method of  claim 1  in which the patch graft further comprises a backing that promotes a migration of at least a portion of the mixture of epithelial cells and mesenchymal cells towards the solid organ. 
     
     
         6 . The method of  claim 5  in which at least a portion of the mixture of epithelial cells and mesenchymal cells migrates over the substantial width of the solid organ and distributes throughout the solid organ. 
     
     
         7 . The method of  claim 1  in which the solid organ is an endodermal organ. 
     
     
         8 . The method of  claim 1  in which the solid organ is an endodermal organ comprising liver, pancreas, intestine, lung, bile duct, thymus, thyroid, parathyroid and the urogenital sinus region of the prostate and vagina. 
     
     
         9 . The method of  claim 8  in which the endodermal organ comprises liver, and engraftment involves a remodeling of Glisson's Capsules. 
     
     
         10 . The method of  claim 8 , which further gives rise to a combination of (i) engrafted epithelial cells and mesenchymal cells and (ii) host cells. 
     
     
         11 . The method of  claim 8 , which gives rise to functional hepatic parenchymal cells. 
     
     
         12 . The method of  claim 11 , in which the parenchymal cells comprise hepatocytes and cholangiocytes. 
     
     
         13 . The method of  claim 8 , in which the endodermal organ comprises pancreas, and engraftment involves a remodeling of pancreatic capsules and pancreatic tissue near to the graft site. 
     
     
         14 . The method of  claim 13 , which gives rise to functional pancreatic cells. 
     
     
         15 . The method of  claim 14 , in which the functional pancreatic cells comprise acinar cells and islets. 
     
     
         16 . The method of  claim 14 , in which the pancreas secretes increased levels of at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide. 
     
     
         17 . The method of  claim 14 , in which the pancreas exhibits a metabolic effect of reduced blood sugar levels. 
     
     
         18 . The method of  claim 14 , in which the pancreas exhibits a metabolic effect of increased glucose tolerance. 
     
     
         19 . The method of  claim 14 , in which the pancreas secretes increased levels of a digestive enzyme or bicarbonate fluid. 
     
     
         20 . The method of  claim 19 , in which the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or combinations thereof. 
     
     
         21 . The method of  claim 14 , which results in increased levels of a metabolic product derived from a digestive enzyme secreted by the pancreas. 
     
     
         22 . The method of  claim 21 , in which the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, or combinations thereof. 
     
     
         23 . The method of  claim 21 , which results in improved digestion. 
     
     
         24 . The method of  claim 11 , in which the liver secretes urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonate-rich fluids, or blood-clotting factors. 
     
     
         25 . The method of  claim 4 , in which a biological sample obtained from the subject indicates reduced levels of at least one of cholesterol, blood sugar, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase. 
     
     
         26 . The method of  claim 1  in which the patch includes a backing positioned over the hydrogel containing of the mixture of epithelial cells and mesenchymal cells. 
     
     
         27 . The method of  claim 26 , in which the backing is used to tether the hydrogel layer to the host organ. 
     
     
         28 . The method of  claim 1 , in which at least one of the epithelial cells, mesenchymal cells, or both are early lineage stage cells. 
     
     
         29 . The method of  claim 28 , in which the early lineage stage mesenchymal cells (ELSMCs) comprise angioblasts, precursors of endothelia, stellate cells, or combinations thereof. 
     
     
         30 . The method of  claim 28 , in which the early lineage stage epithelial cells (ELSEs), ELSMCs, or both are derived from embryonic stem (ES) cells or from induced pluripotent stem cells (iPS). 
     
     
         31 . The method of  claim 1 , in which the epithelial cells are mature and the mesenchymal cells are ELSMCs. 
     
     
         32 . A method of introducing, restoring, increasing, or improving functionality of a diseased, impaired, or malfunctioning solid organ of a subject, comprising contacting the diseased, impaired, or malfunctioning solid organ with a patch graft comprising a mixture of epithelial cells and mesenchymal cells under conditions that promote engraftment of the epithelial cells and mesenchymal cells; demonstrating an introduction, restoration, increase, or improvement of a functionality in the diseased, impaired, or malfunctioning solid organ. 
     
     
         33 . The method of  claim 32 , in which said demonstrating comprises measuring in a biological sample obtained from the subject a level of a secretion or metabolic product or effect. 
     
     
         34 . The method of  claim 32 , which further comprises demonstrating that a least a portion of the mixture of epithelial cells and mesenchymal cells has distributed among the cells of the host organ. 
     
     
         35 . The method of  claim 32 , in which an exposed surface of the patch graft includes a coating that inhibits adhesion of the patch graft to organs and tissues in the vicinity of the patch graft. 
     
     
         36 . The method of  claim 32 , in which the solid organ comprises an endodermal organ. 
     
     
         37 . The method of  claim 36 , in which the endodermal organ comprises liver, pancreas, intestine, lung, bile duct, thymus, thyroid, parathyroid or the regions from the urogenital sinus of the prostate or vagina. 
     
     
         38 . The method of  claim 33 , in which the solid organ comprises a pancreas and in which an increased level of the secretion of at least one of insulin, c-peptide glucagon, somatostatin, or pancreatic polypeptide is measured. 
     
     
         39 . The method of  claim 32 , in which the solid organ comprises a pancreas and in which a reduced blood sugar level is measured. 
     
     
         40 . The method of  claim 32 , in which the solid organ comprises a pancreas and in which increased glucose tolerance is demonstrated. 
     
     
         41 . The method of  claim 32 , in which the solid organ comprises a pancreas and in which increased levels of a digestive enzyme or bicarbonate fluid is demonstrated. 
     
     
         42 . The method of  claim 41 , in which the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. 
     
     
         43 . The method of  claim 33 , in which the solid organ comprises a pancreas and in which increased levels of a product from a digestive enzyme secreted by the pancreas is measured. 
     
     
         44 . The method of  claim 43 , in which the digestive enzyme comprises amylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase. 
     
     
         45 . The method of  claim 32 , in which the solid organ comprises a pancreas and in which improved digestion is demonstrated. 
     
     
         46 . The method of  claim 33 , in which the solid organ comprises liver, and in which a secretion comprises urea, bile acids, phospholipids, lipoproteins, bilirubin, bicarbonate-rich fluids, blood-clotting factors, or combinations thereof. 
     
     
         47 . The method of  claim 33 , in which the solid organ comprises liver, and in which a metabolic effect is a reduced level of one or more of cholesterol, blood sugar, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactate dehydrogenase. 
     
     
         48 . The method of  claim 32 , in which the solid organ comprises a liver, and the subject suffers from type 1 tyrosinemia. 
     
     
         49 . The method of  claim 33 , in which a metabolic effect is a decrease in levels of tyrosine or alpha-fetoprotein. 
     
     
         50 . A method of treating a subject diagnosed with a pathological condition attributable at least in part to having a diseased, impaired, or malfunctioning solid organ, comprising
 (i) contacting the diseased, impaired, or malfunctioning solid organ with a patch graft comprising a mixture of epithelial cells and mesenchymal cells,   (ii) allowing the epithelial cells and mesenchymal cells to migrate into and distribute among the cells of the host solid organ, and   (iii) demonstrating that a negative effect of said diseased, impaired, or malfunctioning solid organ has been alleviated in the treated subject.   
     
     
         51 . The method of  claim 50 , in which said demonstrating comprises measuring in a biological sample obtained from the subject a level of a secretion or a metabolic product or effect. 
     
     
         52 . The method of  claim 50 , in which the migration and distribution steps lead to an alleviation of the disease, impairment, or malfunction. 
     
     
         53 . The method of  claim 50 , in which the solid organ is an endodermal organ.888888 
     
     
         54 . The method of  claim 53 , in which the endodermal organ comprises liver, pancreas, intestine, lung, bile duct, thymus, thyroid, parathyroid, and the urogenital sinus regions of the prostate or vagina. 
     
     
         55 . The method of  claim 53 , in which the endodermal organ is pancreas and in which the subject suffers from diabetes. 
     
     
         56 . The method of  claim 55 , in which increased levels of at least one of insulin, c-peptide, glucagon, somatostatin, or pancreatic polypeptide is measured. 
     
     
         57 . The method of  claim 55 , in which reduced blood sugar levels are demonstrated. 
     
     
         58 . The method of  claim 55 , in which increased glucose tolerance is demonstrated. 
     
     
         59 . The method of  claim 50 , in which the subject comprises a mammal. 
     
     
         60 . The method of  claim 59 , in which the mammal is human. 
     
     
         61 . A patch graft comprising a mixture of epithelial cells and mesenchymal cells and one or more biomaterial layers including, at least:
 a) a first, inner layer for contacting a solid organ, the first inner layer exhibiting a first viscoelastic property, incorporating a mixture of epithelial cells and mesenchymal cells, supporting an ability of the epithelial cells and the mesenchymal cells to produce secreted matrix metallo-proteinases (MMPs) and promoting viability and immaturity of said epithelial cells and mesenchymal cells;   b) optionally, a backing that confers a barrier to the cells migrating in a direction other than towards the solid organ, the backing exhibiting a second viscoelastic property; and   c) optionally, a third, outer layer of a coating or material that minimizes is adhesions of the patch graft to internal surfaces of a body cavity, including internal walls and/or organs, in proximity to the patch graft; wherein said viscoelastic properties are determined by measuring rheological traits and expressed in Pascals (Pa).   
     
     
         62 . The patch graft of  claim 61 , in which the epithelial cells comprise early lineage stage epithelia cells (ELSEs) and the mesenchymal cells comprise early lineage stage mesenchymal cells (ELSMCs), or in which the epithelial and mesenchymal cells are of later lineage stages but are of comparable lineage stages as each other. 
     
     
         63 . The patch graft of  claim 62 , in which the ELSMCs comprises angioblasts, precursors of endothelia, stellate cells, or combinations thereof. 
     
     
         64 . The patch graft of  claim 62 , in which the ELSEs and/or the ELSMCs are derived from embryonic stem (ES) cells or from induced pluripotent stem cells (iPS). 
     
     
         65 . The patch graft of  claim 61 , in which the epithelial cells are of a later lineage stage and the mesenchymal cells are early lineage stage mesenchymal cells (ELSMCs), or the mesenchymal cells are of a later lineage stage and the epithelial cells are early lineage stage epithelial cells (ELSEs). 
     
     
         66 . The patch graft of  claim 61 , in which the second viscoelastic property (expressed in Pa) has a greater value than the first viscoelastic property. 
     
     
         67 . The patch graft of  claim 61 , in which the one or more biomaterial layers comprise a hydrogel, which further comprises minimally sulfated or non-sulfated glycosaminoglycans. 
     
     
         68 . The patch graft of  claim 67 , in which the non-sulfated glycosaminoglycans comprise hyaluronans. 
     
     
         69 . The patch graft of  claim 68 , in which the hyaluronans comprise a thiol-modified hyaluronan, whose gelation by disulfide bridge formation is triggered in the presence of polyethylene glycol diacrylate (PEGDA). 
     
     
         70 . The patch graft of  claim 69 , in which the rheological traits are determined, at least in part, by a starting concentration and rigidity of thiol-modified hyaluronan and PEGDA prior to gelation, a final rigidity of hydrogel post-gelation achieved by the precise ratios of the volumes of thiol-modified hyaluronan and PEGDA. 
     
     
         71 . The patch graft of  claim 61 , in which the first, inner layer exhibits a first viscoelasticity of from about 50 Pa to about 150 Pa. 
     
     
         72 . The patch graft of  claim 61 , in which the optional backing contains a hyaluronan hydrogel layer that exhibits a viscoelasticity from about 600 to about 800 Pa. 
     
     
         73 . The patch graft of  claim 61 , in which the optional third, outer layer comprises a hyaluronan hydrogel layer with viscoelasticity properties of from about 200 to about 300 Pa. 
     
     
         74 . The patch graft of  claim 61 , in which the backing comprises silk. 
     
     
         75 . The patch graft of  claim 74 , in which the silk backing comprises a purified fibroin of Bombyx™ moth silk knitted into a scaffold, including Seri-Silk™ or Contour Seri Silk™. 
     
     
         76 . The patch graft of  claim 61 , in which the epithelial cells comprise biliary tree stem cells (BTSCs) and the mesenchymal cells comprise early-lineage-stage mesenchymal cells (ELSMCs). 
     
     
         77 . The patch graft of  claim 76 , in which the ELSMCs comprise angioblasts and their immediate descendants, precursors to endothelia cells, precursors to stellate cells, or combinations thereof. 
     
     
         78 . The patch graft of  claim 77 , in which the angioblasts express CD117, CD133, VEGFr, but do not express CD31. 
     
     
         79 . The patch graft of  claim 77 , in which the precursors to endothelia cells express CD133, VEGFr, CD31 and Van Willebrand Factor. 
     
     
         80 . The patch graft of  claim 77 , in which the precursors to stellate cells express CD146, ICAM-1, alpha-smooth muscle actin (ASMA) and are negative for vitamin A. 
     
     
         81 . The patch graft of  claim 61 , in which the mixture of epithelial cells and mesenchymal cells is produced by depleting cell suspensions of mature mesenchymal cells, optionally, by repeated panning procedures to remove cells that attach within from about 15 minutes to about 30 minutes on tissue culture dishes or surfaces at 37° C. 
     
     
         82 . The patch graft of  claim 81 , in which a culturing of the remaining cell suspensions is performed on low-attachment dishes and in a serum-free medium until a plurality of organoids is formed by self-assembly of epithelial cells and mesenchymal cells. 
     
     
         83 . The patch graft of  claim 82 , in which the serum-free medium comprises a basal medium (with no copper, 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 are presented complexed with fatty acid free highly purified albumin. 
     
     
         84 . The patch graft of  claim 83 , in which the serum-free medium further comprises 10 μg/ml high density lipoprotein. 
     
     
         85 . The patch graft of  claim 82 , in which the plurality of organoids is formed after about 2 hours, after about 4 hours, after about 6 hours, after about 8 hours, after about 10 hours, after about 12 hours, after about 14 hours, after about 16 hours, after about 18 hours, after about 20 hours, after about 22 hours, or after about 24 so hours. 
     
     
         86 . The patch graft of  claim 82 , in which the plurality of organoids comprises BTSCs positive for:
 a) at least one marker selected from the group of pluripotency genes consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2 and Bmi1,   b) at least one marker selected from the group of endodermal transcription factors consisting of Sox9, Sox17, Pdx1, HNF4alpha, HNFB1 and ONECUT2,   c) at least one marker selected from the group of surface markers associated with stem/progenitors consisting of EpCAM, NCAM, LGR5, one or more isoforms of CD44, CXCR4, sodium iodide symporter (NIS), CD49 (integrin A6), CD29 (integrin B1) and integrin B4;   wherein the BTSCs are negative for markers of mature hepatic or pancreatic cells, including P450s, aquaporin, enzymes involved in bile production, amylase and digestive enzymes.   
     
     
         87 . The patch graft of  claim 61 , in which the one or more biomaterial layers comprise recombinant MMPs. 
     
     
         88 . The patch graft of  claim 61 , in which the one or more biomaterial layers comprise cells engineered to express MMPs.