Method for rejuvenating cells
The invention relates to a method for reprogramming cells from aged donors or senescent cells to pluripotent cells that have lost marks of senescence. In particular, the invention relates to an ex vivo method for preparing induced pluripotent stem cells (iPSCs) from a target cell population comprising cells from aged donors or senescent cells, said method comprising the steps of culturing said target cell population under appropriate conditions for reprogramming said cells into iPSCs, wherein said appropriate conditions comprises increasing expression in said target cells, of at least the following reprogramming factors: Oct4, Klf4, Sox2, c-Myc, Lin28 and, optionally Nanog.
1. An ex vivo method for preparing rejuvenated induced pluripotent stem cells (iPSCs) from senescent cells, said method comprising the steps of:
a) providing said senescent cells and,
b) culturing said target cell population under appropriate conditions for reprogramming said target cell population into iPSCs, wherein said appropriate conditions comprise increasing expression in said target cell population, of at least the following combination of reprogramming factors:
i. a reprogramming factor encoded by one gene of the Oct gene family,
ii. a reprogramming factor encoded by one gene of the Klf gene family,
iii. a reprogramming factor encoded by one gene of the Sox gene family, iv. a reprogramming factor encoded by one gene of the Myc gene family,
v. Lin28,
vi. and Nanog,
wherein the method results in preparation of rejuvenated iPSC cells in which the expression of p21 CIPI and p16 IN1K4A is downregulated to a level found in human embryonic stem cells (hESC).
2. The method of claim 1 , wherein said combination of reprogramming factors comprises Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog.
3. The method of claim 1 , wherein said senescent cells are:
a. human senescent cells;
b. human senescent cells obtained from an adult subject that is at least 50 years old; or
c. human senescent cells obtained from a subject suffering from age-related disorders leading to a high proportion of senescent cells.
4. The method of claim 1 , wherein said method does not further comprise a step of silencing senescence effectors in said senescent cells.
5. The method of claim 4 , wherein said senescence effectors include p21 CIP and/or p16 INK4a and/or p53.
6. The method according to claim 1 , wherein said conditions for increasing expression of the reprogramming factors comprise
a) introducing one or more expression vectors comprising coding sequences of said combination of reprogramming factors into said senescent cells; or,
b) directly delivering an effective amount of each precursor RNA of the reprogramming factors into said senescent cells.
7. The method of claim 6 , wherein said step of introducing is performed by transfecting said senescent cells with said one or more expression vectors.
8. An in vitro method for rejuvenating senescent cells, said method comprising the step of
reprogramming said cells to induced pluripotent stem cells iPSCs, by increasing expression in said cells of at least a combination of the following reprogramming factors:
i. a reprogramming factor encoded by one gene of the Oct gene family,
ii. a reprogramming factor encoded by one gene of the Klf gene family,
iii. a reprogramming factor encoded by one gene of the Sox gene family,
iv. a reprogramming factor encoded by one gene of the Myc gene family, Lin28,
v. and Nanog.
9. The method of claim 8 , wherein said step of reprogramming includes a step of culturing said cells under appropriate conditions for increasing expression of the following reprogramming factors: Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog.
10. The method of claim 3 , wherein said human senescent cells are human fibroblast senescent cells.
11. The method according to claim 1 , wherein said senescent cells provided at step (a) are comprised in a cell composition obtained from an adult subject that is at least 50 years old.
12. The method of claim 8 , wherein said senescent cells are comprised in a cell composition obtained from an adult subject that is at least 50 years old.