Efficient method for reprogramming blood to induced pluripotent stem cells
Described herein are methods and compositions related to generation of induced pluripotent stem cells (iPSCs). Improved techniques for establishing highly efficient, reproducible reprogramming using non-integrating episomal plasmid vectors. Using the described reprogramming protocol, one is able to consistently reprogram non-T cells with close to 100% success from non-T cell or non-B cell sources. Further advantages include use of a defined reprogramming media E7 and using defined clinically compatible substrate recombinant human L-521. Generation of iPSCs from these blood cell sources allows for recapitulation of the entire genomic repertoire, preservation of genomic fidelity and enhanced genomic stability.
1. A method of generating blood cell derived induced pluripotent stem cells, comprising:
providing a quantity of blood cells;
delivering a quantity of EBNA1 and reprogramming factors comprising Oct-4, Sox-2, Klf-4, 1-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”) into the blood cells; and
culturing the blood cells in a reprogramming media for at least 4 days, wherein delivering the EBNA1 and reprogramming factors, and culturing in a reprogramming media generates blood cell derived induced pluripotent stem cells, wherein the reprogramming factors are encoded in four oriP/EBNA1 derived vectors comprising:
a first vector encoding Oct4, Sox2, SV40LT and Klf4,
a second vector encoding Oct4 and shRNA-p53,
a third vector encoding Sox2 and Klf4, and
a fourth vector encoding 1-Myc and Lin-28; and
wherein a fifth oriP/EBNA1 derived vector encodes EBNA1.
2. The method of claim 1 , wherein delivering a quantity of reprogramming factors comprises nucleofection.
3. The method of claim 1 , wherein the five oriP/EBNA1 derived vectors are pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCWB-EBNA1.
4. The method of claim 1 , comprising plating of the blood cells on a treated cell culture surface after delivering reprogramming factors into the blood cells, and culturing the blood cells in a reprogramming media on said treated cell culture surface.
5. The method of claim 4 , wherein the treated cell culture surface comprises plating of mouse embryonic feeders (MEFs).
6. The method of claim 4 , wherein the treated cell culture surface comprises an extracellular matrix protein.
7. The method of claim 6 , wherein the extracellular matrix protein comprises laminin.
8. The method of claim 7 , wherein laminin comprises L-521.
9. The method of claim 1 , wherein the reprogramming media comprises embryonic stem cell (ESC) media.
10. The method of claim 9 , wherein the ESC media comprises basic fibroblast growth factor (bFGF).
11. The method of claim 1 , wherein the reprogramming media comprises E7 media.
12. The method of claim 11 , wherein the reprogramming media comprises E7 media comprising L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF.
13. The method of claim 1 , wherein culturing the blood cells in a reprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days.
14. The method of claim 1 , wherein culturing the blood cells in a reprogramming media is for at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.
15. The method of claim 1 , wherein the blood cells are isolated from a subject possessing a disease mutation.
16. The method of claim 15 , wherein the disease mutation is associated with a neurodegenerative disease, disorder and/or condition.
17. The method of claim 15 , wherein the disease mutation is associated with an inflammatory bowel disease, disorder, and/or condition.
18. The method of claim 1 , wherein blood cells are non T-cell, non B-cell mononuclear cells.
19. The method of claim 1 , wherein the blood cells are a sample drawn from a human subject.
20. The method of claim 19 , wherein the sample is whole blood.
21. The method of claim 19 , wherein the sample is peripheral blood.
22. The method of claim 19 , wherein the sample is an isolated component of non T-cell, non B-cell mononuclear cells.
23. The method of claim 1 , wherein the blood cells are from a previously frozen sample thawed prior to providing a quantity of blood cells.
24. The method of claim 1 , wherein the blood cells are not expanded prior to delivering the quantity of EBNA1 and reprogramming factors.