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.

That which is claimed is: 
     
         1 . A method of producing lineage-restricted cells (LRCs) from a post-mitotic differentiated cell (PMD) of the same lineage, comprising:
 contacting a PMD with an effective amount of an agent that transiently inhibits the activity of a pocket protein and an effective amount of an agent that transiently inhibits the activity of the cyclin-dependent kinase inhibitor 2A alternate reading frame protein (ARF) under conditions sufficient to permit the PMD to transiently divide to produce progeny,   wherein said progeny are LRCs of the same lineage as the PMD.   
     
     
         2 . The method of  claim 1 , wherein said PMD dedifferentiates prior to dividing. 
     
     
         3 . The method of  claim 1 , wherein said PMD is a myocyte. 
     
     
         4 . The method of  claim 3 , wherein said myocyte is selected from the group consisting of a cardiomyoctyte, a smooth muscle myocyte, a skeletal myocyte, and myofiber. 
     
     
         5 . The method of  claim 1 , wherein said pocket protein is retinoblastoma protein (RB). 
     
     
         6 . The method of  claim 5 , wherein said agent that transiently inhibits RB activity is a nucleic acid, a polypeptide, or a small molecule. 
     
     
         7 . The method of  claim 1 , wherein said agent that transiently inhibits ARF activity is a nucleic acid, a polypeptide, or a small molecule. 
     
     
         8 . The method of  claim 1 , wherein about 10% of PMDs of a population that are contacted are induced to transiently divide. 
     
     
         9 . The method of  claim 1 , wherein the PMD are from an individual with a disease. 
     
     
         10 . The method of  claim 1 , wherein the individual is alive. 
     
     
         11 . The method of  claim 1 , wherein the individual is a cadaver. 
     
     
         12 . The method of  claim 1 , wherein the method is effected in vivo. 
     
     
         13 . The method of  claim 1 , wherein the method is effected ex vivo. 
     
     
         14 . The method of  claim 1 , wherein said method further comprises transferring said progeny LRCs to conditions that promote differentiation,
 wherein the population that is produced is a population of PMDs of the same lineage as the PMD that was contacted in said contacting step.   
     
     
         15 . The method of  claim 14 , wherein said transferring is effected by transplanting said progeny into a subject. 
     
     
         16 . The method of  claim 15 , wherein said subject is in need of tissue regeneration therapy. 
     
     
         17 . A method of screening a candidate agent for an effect on a disease condition, comprising:
 producing lineage-restricted cells (LRCs) from a post-mitotic differentiated cell (PMD) from an individual with said disease condition by the method of  claim 1 ,   transferring said LRCs to conditions that promote differentiation to produce a differentiated population of cells,   contacting said differentiated population of cells with a candidate agent, and   comparing the viability and/or function of the cells in said differentiated population to the viability and/or function of differentiated cells not contacted with said candidate agent;   wherein enhanced viability and/or function of the cells in the differentiated population contacted with said candidate agent as compared to a differentiated population not contacted with said candidate agent indicates that the candidate agent will have an effect on said disease condition.   
     
     
         18 . The method of  claim 17 , wherein said disease condition is a muscle disorder. 
     
     
         19 . The method of  claim 18 , wherein the muscle is smooth muscle, skeletal muscle, or cardiac muscle. 
     
     
         20 . The method of  claim 17 , wherein said disease condition is a nervous system disorder. 
     
     
         21 . The method of  claim 20 , wherein the nervous system disorder is Parkinson's Disease, Alzheimer's Disease, ALS, a disorder of olfactory neurons, a disorder of spinal cord neurons, or a disorder of peripheral neurons. 
     
     
         22 . The method of  claim 17 , wherein said individual is alive. 
     
     
         23 . The method of  claim 17 , wherein said individual is a cadaver. 
     
     
         24 . A method of screening a candidate agent for toxicity to a human comprising:
 producing lineage-restricted cells (LRCs) from a post-mitotic differentiated cell (PMD) from a healthy individual by the method of  claim 1 ,   transferring said LRCs to conditions that promote differentiation to produce a differentiated population of cells,   contacting said differentiated population of cells with a candidate agent, and   comparing the viability and/or function of the cells in said differentiated population to the viability and/or function of differentiated cells not contacted with said candidate agent;   wherein a decrease in viability and/or function of the cells in the differentiated population contacted with said candidate agent as compared to a differentiated population not contacted with said candidate agent indicates that the candidate agent is toxic to a human.   
     
     
         25 . The method of  claim 24 , wherein the PMD is a hepatocyte. 
     
     
         26 . The method of  claim 24 , wherein the function of the cells is assess by assessing a cytochrome P450 panel. 
     
     
         27 . A method of producing lineage-restricted cells (LRCs) from post-mitotic differentiated cells (PMDs) in a tissue in a subject, comprising:
 contacting PMDs of said tissue in vivo with an effective amount of an agent that transiently inhibits the activity of a pocket protein and an effective amount of an agent that transiently inhibits the activity of the cyclin-dependent kinase inhibitor 2A alternate reading frame protein (ARF),   wherein the contacted cells are induced to transiently divide in situ so as to produce LRCs of the lineage of the PMD's lineage.   
     
     
         28 . The method of  claim 27 , wherein said PMD dedifferentiates prior to dividing. 
     
     
         29 . The method of  claim 27 , wherein said PMDs are myocytes. 
     
     
         30 . The method of  claim 29 , wherein said myocytes are selected from the group consisting of cardiomyoctytes, smooth muscle myocytes, skeletal myocytes, and myofibers. 
     
     
         31 . The method of  claim 27 , wherein said pocket protein is retinoblastoma protein (RB). 
     
     
         32 . The method of  claim 27 , wherein said agent that transiently inhibits RB activity is a nucleic acid, a polypeptide, or a small molecule. 
     
     
         33 . The method of  claim 27 , wherein said agent that transiently inhibits ARF activity is a nucleic acid, a polypeptide, or a small molecule. 
     
     
         34 . The method of  claim 27 , wherein said agent that transiently inhibits RB activity and said agent that transiently inhibits ARF activity are administered locally to the tissue. 
     
     
         35 . The method of  claim 27 , wherein said subject is a subject in need of tissue regeneration therapy. 
     
     
         36 . A kit for use in transiently inducing post-mitotic differentiated cells to transiently divide, comprising an agent that transiently inhibits the activity of a pocket protein and an agent that transiently inhibits the activity of ARF.