Methods of Inducing Tissue Regeneration

Abstract

Methods are provided for producing cells within a lineage (lineage restricted cells) from post-mitotic differentiated cells of the same lineage ex vivo and in vivo, and for treating a subject in need of tissue regeneration therapy by employing these lineage-restricted cells. In addition, the production of lineage restricted cells from postmitotic tissues derived from patients with diseases allows for a characterization of pathways that have gone awry in these diseases and for screening of drugs that will ameliorate or correct the defects as a means of novel drug discovery. Also provided are kits for performing these methods.

1 . A method of producing dedifferentiated regenerative cell comprising:
 contacting a differentiated primary cell with one or more agents that downregulate the expression or activity of one or more genes or gene products of the differentiated primary cell to produce a dedifferentiated regenerative cell that is restricted to the lineage of the differentiated primary cell.   
     
     
         2 . A method of producing a dedifferentiated regenerative cell comprising:
 contacting a post-mitotic differentiated cell with one or more agents that downregulate the expression or activity of at least two genes or gene products of the post-mitotic differentiated cell to produce a dedifferentiated regenerative cell that is restricted to the lineage of the post-mitotic differentiated cell.   
     
     
         3 . The method of any of  claims 1 - 2 , wherein the one or more agents downregulate the expression of the gene. 
     
     
         4 . The method of any of  claims 1 - 2 , wherein the one or more agents inhibit the activity of the gene product. 
     
     
         5 . The method of any of  claims 1 - 2 , wherein the gene product is a protein. 
     
     
         6 . The method of any of  claims 1 - 2 , wherein the agent is an siRNA. 
     
     
         7 . The method of any of  claims 1 - 2 , wherein the agent is a small molecule, organic molecule, biomolecule, polypeptide, antisense oligonucleotide, shRNA or any combination thereof. 
     
     
         8 . The method of any of  claims 1 - 2 , wherein the agent is in a vector. 
     
     
         9 . The method of  claim 8 , wherein the vector is a plasmid vector or viral vector. 
     
     
         10 . The method of  claim 9 , wherein the viral vector is derived from a cytomegalovirus, adenovirus, MMLV, HIV-1, or ALV. 
     
     
         11 . The method of any of  claims 1 - 2 , wherein the agent is introduced by electroporation, calcium chloride transfection, lipofection, fusion, or infection. 
     
     
         12 . The method of  claim 1 , wherein the one or more agents inhibit the activity of at least two genes or gene products of the differentiated primary cell. 
     
     
         13 . The method of any of  claims 1 - 2 , wherein the one or more agents transiently inhibit genes or gene products of the differentiated cell. 
     
     
         14 . The method of any of  claims 1 - 2 , wherein the genes or gene products is a member of an RB family gene or gene product thereof. 
     
     
         15 . The method of claim of any of  claims 1 - 2 , wherein the genes or gene products is selected from a group consisting of ARF, RB, and p16INK4A. 
     
     
         16 . The method of any of  claims 2  and  12 , wherein at least two of the genes or gene products are selected from a group consisting of ARF, RB, and p16INK4A. 
     
     
         17 . The method of claim of any of  claims 1 - 2 , wherein the genes or gene products is selected from a member of an ARF family, RB family, and p16INK4A family. 
     
     
         18 . The method of any of  claims 2  and  12 , wherein at least two of the genes or gene products are selected from a member of an ARF family, RB family, and p16INK4A family. 
     
     
         19 . The method of  claim 1 , wherein the differentiated primary cell is a post-mitotic cell. 
     
     
         20 . The method of  claim 2 , wherein the post-mitotic differentiated cell is a primary cell. 
     
     
         21 . The method of any of  claims 1 - 2 , wherein the differentiated cell is of a pancreatic islet cell lineage. 
     
     
         22 . The method of any of  claims 1 - 2 , wherein the differentiated cell is a cardiomyocyte, neuron or hepatocyte. 
     
     
         23 . The method of any of  claims 1 - 2 , wherein the differentiated cell is from a tissue. 
     
     
         24 . The method of  claim 23 , wherein the tissue is a muscle, brain, skin, pancreas, or liver. 
     
     
         25 . The method of any of  claims 1 - 2 , wherein the differentiated cell is not a stem cell. 
     
     
         26 . The method of any of  claims 1 - 2 , wherein the differentiated cell is not a pluripotent cell. 
     
     
         27 . The method of any of  claims 1 - 2 , wherein the dedifferentiated regenerative cell is a mitotic cell. 
     
     
         28 . The method of any of  claims 1 - 2 , further comprising differentiating the dedifferentiated regenerative cell to produce a differentiated cell. 
     
     
         29 . A method comprising:
 administering a dedifferentiated regenerative cell to a subject in need thereof, wherein the dedifferentiated regenerative cell is produced by the method of any of  claims 1 - 28 .   
     
     
         30 . The method of  claim 29 , wherein administering the dedifferentiated regenerative cell comprises injecting the dedifferentiated regenerative cell into the subject. 
     
     
         31 . The method of  claim 29 , wherein administering the dedifferentiated regenerative cell comprises transplanting the dedifferentiated regenerative cell into the subject. 
     
     
         32 . The method of  claim 29 , wherein administering the dedifferentiated regenerative cell comprises administering the dedifferentiated regenerative cell by a catheter. 
     
     
         33 . The method of  claim 29 , wherein administering the dedifferentiated regenerative cell comprises providing the dedifferentiated regenerative cell on a solid support. 
     
     
         34 . The method of  claim 33 , the solid support comprises a bead, filter, or membrane. 
     
     
         35 . The method of  claim 29 , wherein the subject is suffering from a skeletal disease, skeletal muscle disorder, neuronal disease, cardiac disease, pancreatic disease, or hepatic disease. 
     
     
         36 . The method of  claim 29 , wherein the subject is suffering from diabetes. 
     
     
         37 . The method of  claim 29 , wherein the subject is suffering from diabetes mellitus type 1. 
     
     
         38 . The method of  claim 29 , wherein the subject is suffering from a cardiac disease. 
     
     
         39 . The method of  claim 29 , wherein the subject is suffering from a disease of the central nervous system or peripheral nervous system. 
     
     
         40 . The method of  claim 39 , wherein the disease of the central nervous system or peripheral nervous system is selected from Parkinson's disease, Alzheimer's disease, ALS, disorder of olfactory neurons, disorder of spinal cord neurons, or a disorder of peripheral neurons. 
     
     
         41 . The method of  claim 29 , wherein the subject is suffering from a disease selected from a group consisting of ophthalmoplegia, pigmentary retinopathy, cataracts, premature balding, and deafness. 
     
     
         42 . An isolated dedifferentiated regenerative cell, wherein the isolated dedifferentiated regenerative cell is produced the method of any of  claims 1 - 28 . 
     
     
         43 . A pharmaceutical composition comprising the dedifferentiated regenerative cell of  claim 42  and a pharmaceutically acceptable excipient 
     
     
         44 . A kit for producing a dedifferentiated regenerative cell comprising:
 (a) an agent that inhibits a gene or gene product of an ARF family member; and   (b) an agent that inhibits a gene or gene product of an RB family member.   
     
     
         45 . The kit of  claim 44 , wherein the agent is an siRNA. 
     
     
         46 . The kit of  claim 44 , wherein the agent that inhibits a gene or gene product of an ARF family member also inhibits a gene or gene product of a p16INK4A family member. 
     
     
         47 . The kit of  claim 44 , wherein the agent is a small molecule, organic molecule, biomolecule, polypeptide, antisense oligonucleotide, shRNA or any combination thereof.