Methods of reprogramming cells to a less differentiated state
The present invention relates in part to methods for producing tissue-specific cells from patient samples, and to tissue-specific cells produced using these methods. Methods for reprogramming cells using RNA are disclosed. Therapeutics comprising cells produced using these methods are also disclosed.
1. A method for reprogramming a cell to a less differentiated state, comprising:
(a) culturing a differentiated cell with a reprogramming medium;
(b) transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and
(c) repeating step (b) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state,
wherein steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium.
2. The method of claim 1 , wherein the reprogramming medium is substantially free of immunosuppressants.
3. The method of claim 1 , wherein the cell is a fibroblast.
4. The method of claim 3 , wherein the cell is derived from a biopsy.
5. The method of claim 4 , wherein the biopsy is a full-thickness dermal punch biopsy.
6. The method of claim 1 , wherein the cell culture has a starting cell density of less than and 20,000 cells/cm 2 .
7. The method of claim 1 , wherein the cell culture has a starting cell density of between 2,000 cells/cm 2 and 20,000 cells/cm 2 .
8. The method of claim 1 , wherein the method further comprises a step of rinsing the cell after transfection.
9. The method of claim 1 , wherein the reprogramming medium comprises a cell-adhesion molecule.
10. The method of claim 9 , wherein the cell-adhesion molecule is selected from one or more fibronectin and vitronectin.
11. The method of claim 10 , wherein the cell-adhesion molecule is recombinant.
12. The method of claim 1 , wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding at least one member of the group: Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein.
13. The method of claim 1 , wherein the one or more synthetic RNA molecules contains at least one member of the group: a pseudouridine residue and a 5-methylcytidine residue.
14. The method of claim 1 , wherein the amount of one or more synthetic RNA molecules transfected in earlier transfections is between about 20 ng/cm 2 and 250 ng/cm 2 .
15. The method of claim 1 , wherein the amount of one or more synthetic RNA molecules transfected in later transfections is between about 100 ng/cm 2 and 600 ng/cm 2 .
16. The method of claim 1 , wherein the method comprises at least 5 transfections and the amount of one or more synthetic RNA molecules transfected is about 25 ng/cm 2 for the first transfection, about 50 ng/cm 2 for the second transfection, about 100 ng/cm 2 for the third transfection, about 200 ng/cm 2 for the fourth transfection, and about 400 ng/cm 2 for the fifth transfection.