Efficient method for reprogramming blood to induced pluripotent stem cells
First Claim
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, anda fourth vector encoding 1-Myc and Lin-28; and
wherein a fifth oriP/EBNA1 vector encodes EBNA1.
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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.
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Citations
21 Claims
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1. A method of generating blood cell derived induced pluripotent stem cells, comprising:
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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; andculturing 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification