Production of reprogrammed cells with restored potential
A method for treating cells and/or nuclear transfer units and/or stem cells in culture with such compounds, individually or in combinations, is described. The method results in a globally hypomethylated genome and a restoration of cell differentiation and/or developmental potential, or potentiality. In addition, a method for the in vitro production of reprogrammed cells which have had differentiation potential (totipotential, pluripotential, or multipotential) restored by demethylating the genome is described.
1. An in vitro method for reprogramming comprising:
(a) decreasing S-adenosylmethione-to-S-adenosylhomocysteine ratio (SAM-to-SAH ratio) in a eukaryotic cell having a first phenotype
(b) comparing the first phenotype to a phenotype of the cell obtained after step (a); and
(c) selecting the cell from step (b), wherein differentiation potential has been restored to the cell.
2. The method of claim 1 , further comprising: reducing levels of 5-methylcytosine in DNA within the eukaryotic cell prior to step (b).
3. The method of claim 2 , wherein decreasing S-adenosylmethione-to-S-adenosylhomocysteine ratio and reducing levels of 5-methylcytosine in DNA are performed simultaneously.
4. The method of claim 2 , wherein reducing levels of 5-methylcytosine in DNA comprises specifically suppressing expression, activity, or expression and activity of DNA methyltransferases within the eukaryotic cell.
5. The method of claim 4 , wherein reducing levels of 5-methylcytosine in DNA comprises contacting the cell with a DNA methyltransferase inhibitor.
6. The method of claim 4 , wherein reducing levels of 5-methylcytosine in DNA comprises contacting the cell with a suppression-effective amount of a small-interfering ribonucleic acid (siRNA) dimensioned and configured to suppress expression of DNA methyltransferases.
7. The method of claim 1 , wherein step (a) comprises contacting the cell with a siRNA comprising the nucleic acid sequence of SEQ. ID. No. 14.
8. The method of claim 1 , wherein step (a) comprises increasing expression, activity, or both expression and activity of glycine-N-methyl transferase within the eukaryotic cell.
9. The method of claim 8 , wherein step (a) comprises contacting the cell with an amount of a retinoic acid effective to increase expression, activity, or both expression and activity of glycine-N-methyl transferase within the cell.
10. The method of claim 1 , wherein the SAM-to-SAH ratio is decreased to 0.1 or less.
11. The method of claim 1 , wherein the SAM-to-SAH ratio is decreased to 0.5 or less.
12. The method of claim 1 , wherein the SAM-to-SAH ratio is decreased to 1.00 or less.
13. An in vitro method for reprogramming comprising:
(a) specifically suppressing expression, activity, or expression and activity of DNA methyltransferases within a eukaryotic cell having a first phenotype; and simultaneously
(b) increasing expression, activity, or both expression and activity of glycine-N-methyl transferase within the eukaryotic cell;
(c) comparing the first phenotype to a phenotype of the cell obtained after steps (a) and (b); and
(d) selecting the cell from step (c), wherein differentiation potential has been restored to said cell.
14. The method of claim 13 , wherein step (a) comprises contacting the cell with a suppression-effective amount of a siRNA dimensioned and configured to suppress expression of DNA methyltransferases.
15. The method of claim 13 , wherein step (a) comprises contacting the cell with a suppression-effective amount of a siRNA dimensioned and configured to suppress or reduce 5-methylated cytosines in DNA in the cell by at least about 5%.
16. The method of claim 13 , wherein step (b) comprises contacting the cell with an amount of a retinoic acid effective to increase expression, activity, or both expression and activity of glycine-N-methyl transferase within the cell.
17. The method of claim 16 , wherein step (b) comprises contacting the cell with all-trans retinoic acid.
18. The method of claim 16 , wherein step (b) comprises contacting the cell with a compound that binds a retinoic acid receptor.
19. The method of claim 13 , further comprising, simultaneously with steps (a) and (b): specifically suppressing within the cell expression, activity, or expression and activity of 5,10-methylenetetrahydrofolate reductase.
20. The method of claim 13 comprising contacting the cell with a siRNA comprising the nucleic acid sequence of SEQ. ID. No. 14.