Efficient method for reprogramming blood to induced pluripotent stem cells

• US 10,221,395 B2
• Filed: 06/16/2016
• Issued: 03/05/2019
• Est. Priority Date: 06/16/2016
• Status: Active Grant

• Current Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Original Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Inventors: Sareen, Dhruv, Ornelas, Loren A., Svendsen, Clive
• Primary Examiner(s): Bertoglio, Valarie E
• Application Number:
  US15/184,241
• Publication Number:
  US 20170362574A1
• Time in Patent Office: 992 Days
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

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.

Claims

1. A method of generating blood cell derived induced pluripotent stem cells, comprising:

providing a quantity of peripheral blood cells;

fractioning the quantity of peripheral blood cells;

isolating a layer of peripheral blood mononuclear cells from the fractionated peripheral 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 peripheral blood mononuclear cells; and

culturing the peripheral blood mononuclear 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, SV4OLT 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 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 pCXWB-EBNA1.

4. The method of claim 1, comprising plating of the peripheral mononuclear 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 media suitable for culturing of embryonic stem cells (ESCs).

10. The method of claim 9, wherein the 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 peripheral mononuclear 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 peripheral mononuclear 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 peripheral 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 the peripheral blood cells are a sample drawn from a human subject.

19. The method of claim 3, wherein, the method comprises delivering 0.5 to 1.0 ug pCXLE-hOCT3/4-shp53, 0.5 to 1.0 ug pCXLE-hSK, 0.5 to 1.0 ug pCXLE-UL, 0.25 to 0.75 ug pCXWB-EBNA1 and 0.5 to 1.0 ug pEP4 E02S ET2K to 2 to 4×

10⁶ peripheral blood mononuclear cells.

20. The method of claim 3, wherein, the method comprises 0.83 ug pCXLE-hOCT3/4-shp53, 0.83 ug pCXLE-hSK, 0.83 ug pCXLE-UL, 0.5 ug pCXWB-EBNA1 and 0.83 ug pEP4 E02S ET2K and 3×

10⁶ peripheral blood mononuclear cells.

21. The method of claim 1, wherein the isolated peripheral blood cells comprise mononuclear cells substantially free of T-cells and B-cells.