Progenitor cells, methods and uses related thereto

Abstract

The present invention relates to a substantially pure population of viable pancreatic progenitor cells, and methods for isolating such cells. The present invention further concerns certain therapeutic uses for such progenitor cells, and their progeny.

1 - 30 . (canceled)  
     
     
         31 . A method of isolating progenitor cells comprising: 
 i) obtaining pancreatic ductal cells;    ii) culturing said pancreatic cells in a suitable nutrient medium;    iii) isolating a population of progenitor cells from said culture.    
     
     
         32 . A method of  claim 31 , comprising obtaining pancreatic intralobular ductal epithelial cells.  
     
     
         33 . A method of  claim 31 , wherein said pancreatic ductal epithelial cells are obtained by explant or by enzymatic digestion.  
     
     
         34 . A method of  claim 31 , wherein said pancreatic ductal cells are grown to confluence.  
     
     
         35 . A method of  claim 31 , wherein said progenitor cells are isolated by mechanical separation.  
     
     
         36 . A method of  claim 34 , wherein after growing said culture to confluence non-adherent cells are isolated and further treated with an agent.  
     
     
         37 . A method of  claim 36 , wherein said agent induces differentiation and is selected from the group consisting of Forskolin, Di-butyrl cAMP, Na-Butyrate, dexamethasone and cholera toxin.  
     
     
         38 . A method of  claim 36 , wherein said agent is a growth factor.  
     
     
         39 . A method of  claim 38 , wherein said growth factor is selected from a group consisting of IGF, TGF, FGF, EGF, HGF, hedgehog and VEGF.  
     
     
         40 . A method of  claim 38 , wherein said growth factor is selected from a group consisting of the TGFβ superfamily, BMP2 and BMP7.  
     
     
         41 . (canceled)  
     
     
         42 . A method for stimulating the ex vivo proliferation of mammalian pancreatic β-islet cells, comprising the steps of: 
 (a) preparing a primary culture of mammalian pancreatic cells; and,    (b) contacting said primary culture cells with an effective concentration of a cAMP agonist, wherein the effective concentration is an amount sufficient to induce the primary culture to differenitate to β-islet cells.    
     
     
         43 . The method of  claim 42 , wherein the primary culture cells are human pancreatic cells.  
     
     
         44 . The method of  claim 41 , wherein said cell differentiation comprises an increase in average cellular insulin production.  
     
     
         45 . The method of  claim 41 , further comprising growing said cultured cells in monolayer on an extracellular matrix in the presence of a growth factor.  
     
     
         46 . The method of  claim 41 , further comprising contacting said cells with an agent that upregulates the insulin gene, e.g., a poly (ADP-ribose) synthetase inhibitor such as nicotinamide or a benzamide.  
     
     
         47 . A method for stimulating the ex vivo proliferation of human adult pancreatic beta-cells, comprising the steps of: 
 (a) preparing a monolayer culture of primary human adult pancreatic cells; and    (b) culturing said cells with an effective concentration of a growth factor and a cAMP agonist, wherein the effective concentration is an amount sufficient to induce the primary culture to produce insulin-producing cells.    
     
     
         48 - 54 . (canceled)  
     
     
         55 . A method for preparing a substantially pure non-adherent population of progenitor cells comprising: 
 obtaining a cell suspension from an animal tissue, wherein said cell suspension comprises at least one progenitor cell;    treating the cell suspension with a growth factor preparation; and    allowing proliferation of said at least one progenitor cell such that a substantially pure non-adherent progenitor cell population is obtained, thereby obtaining a substantially pure non-adherent progenitor cell population.    
     
     
         56 . The method of  claim 55 , wherein said non-adherent population of progenitor cells is at least about 50% pure.  
     
     
         57 . The method of  claim 55 , wherein said non-adherent population of progenitor cells is at least about 60% pure.  
     
     
         58 . The method of  claim 55 , wherein said non-adherent population of progenitor cells is at least about 70% pure.  
     
     
         59 . The method of  claim 55 , wherein said non-adherent population of progenitor cells is at least about 80% pure.  
     
     
         60 . The method of  claim 55 , wherein said non-adherent population of progenitor cells is at least about 90% pure.  
     
     
         61 . The method of  claim 55 , wherein said animal tissue is obtained from a mammalian organ.  
     
     
         62 . The method of  claim 55 , wherein said animal tissue is selected from the group consisting of: pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, mesentery tissue, fetal tissue and umbilical tissue.  
     
     
         63 . The method of  claim 55 , wherein said cell suspension is obtained by mechanical disruption of said animal tissue.  
     
     
         64 . The method of  claim 55 , wherein said cell suspension is obtained by enzymatic disruption of said animal tissue.  
     
     
         65 . The method of  claim 55 , wherein said growth factor preparation comprises at least one of: epidermal growth factor, transforming growth factor, hepatocyte growth factor, fibroblast growth factor, leukemia inhibitory factor, insulin-like growth factor and platelet-derived growth factor.  
     
     
         66 . The method of  claim 55 , wherein said substantially pure non-adherent progenitor cells are floating cells.  
     
     
         67 . The method of  claim 55 , wherein said substantially pure non-adherent progenitor cells are non-adherent cells.  
     
     
         68 . The method of  claim 55 , wherein said substantially pure non-adherent progenitor cells forms a homotypic cell sphere.  
     
     
         69 . A method for preparing a substantially pure non-adherent population of progenitor cells comprising: 
 providing an animal tissue;    disrupting said animal tissue so as to obtain a cell suspension comprising at least one progenitor cell; and    allowing proliferation of said at least one progenitor cell such that a substantially pure non-adherent progenitor cell population is obtained, thereby obtaining a substantially pure non-adherent progenitor cell population.    
     
     
         70 . The method of  claim 69 , wherein said animal tissue is selected from the group consisting of: pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, mesentery tissue, fetal tissue and umbilical tissue  
     
     
         71 . The method of  claim 55 , wherein said non-adherent progenitor cell population expresses Nestin.  
     
     
         72 . The method of  claim 55 , wherein said non-adherent progenitor cell population expresses at least one: c-kit and Sca.  
     
     
         73 . The method of  claim 55 , wherein said non-adherent progenitor cell population under proper conditions can give rise to cells that express a marker selected from the group comprising: Pdx-1, glucagon, and insulin.  
     
     
         74 . A composition comprising the substantially-pure nonadherent progenitor cell population obtained by the method of  claim 55 .  
     
     
         75 . The composition of  claim 74 , wherein the substantially-pure nonadherent progenitor cell population expresses a marker selected from the group consisting of: Nestin, c-kit and Sca.  
     
     
         76 . The composition of  claim 74 , wherein the substantially-pure nonadherent progenitor cell population under proper conditions can give rise to cells that express a marker selected from the group comprising: Pdx-1, glucagon, and insulin.  
     
     
         77 . The method of  claim 55 , wherein said substantially pure non-adherent population of progenitor cells is at least about one thousand-fold enriched from said animal cell suspension.  
     
     
         78 . The method of  claim 55 , wherein said substantially pure non-adherent population of progenitor cells is at least about one hundred-fold enriched from said animal cell suspension.