Methods of inducing tissue regeneration
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 lineage-restricted mammalian cells (LRCs) from a post-mitotic differentiated mammalian cell (PMD) of the same lineage, comprising:
contacting a primary culture mammalian myocyte or hepatocyte PMD with an effective amount of a combination of agents comprising a first agent that transiently inhibits the activity of a pocket protein and an effective amount of a second 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 cardiomyocyte, 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 effective to transiently inhibit said activity, a polypeptide effective to transiently inhibit said activity, or a small molecule effective to transiently inhibit said activity.
7. The method of claim 1 , wherein said agent that transiently inhibits ARF activity is a nucleic acid effective to transiently inhibit said activity, a polypeptide effective to transiently inhibit said activity, or a small molecule effective to transiently inhibit said activity.
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 a human individual with a disease.
10. The method of claim 9 , wherein the individual is alive.
11. The method of claim 9 , wherein the individual is a cadaver.
12. 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.
13. The method of claim 12 , wherein said transferring is effected by transplanting said progeny into a subject.
14. The method of claim 13 , wherein said subject is in need of tissue regeneration therapy.
15. The method of claim 1 , wherein the PMD is a hepatocyte.