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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
First Claim
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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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