And 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 blood cell derived induced pluripotent stem cell (BC-iPSC) line,
wherein the BC-iPSC line is derived from non-expanded peripheral blood mononuclear cells (PBMCs) that are T-cells and comprises T-cell receptor gene rearrangement, and
wherein the BC-iPSC line does not show statistically significant differences between a first and a repeat G-band karyotype analysis, wherein the first or the repeat G-band karyotype analysis occurs during passages 10-23.
2 . The BC-iPSC line of claim 1 , wherein:
the BC-iPSC line has lower incidences of genomic aberrations compared to iPSC lines generated from fibroblast-derived iPSCs (Fib-iPSCs);
the BC-iPSC line exhibits less than 5% abnormal karyotype after 4-8 passages; and/or
the BC-iPSC line exhibits less than 2.8% abnormal karyotype after 4 passages.
3 . The BC-iPSC line of claim 1 , wherein the BC-iPSC line is produced by a reprogramming process comprising:
delivering a quantity of EBNA1 and reprogramming factors comprising Oct-4, Sox-2, Klf-4, l-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”) into isolated non-expanded PBMCs, wherein the reprogramming factors are encoded in one or more oriP/EBNA1 derived vectors; and
culturing the isolated non-expanded PBMCs in a reprogramming media for at least 4 days.
4 . The BC-iPSC line of claim 3 , wherein the one or more oriP/EBNA1 derived vectors comprise pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1.
5 . The BC-iPSC line of claim 3 , wherein the one or more oriP/EBNA1 derived vectors comprise:
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 l-Myc and Lin-28.
6 . The BC-iPSC line of claim 5 , wherein the quantity of EBNA1 is encoded by a fifth vector encoding EBNA1.
7 . A blood cell derived induced pluripotent stem cell (BC-iPSC) line,
wherein the BC-iPSC line is derived from non-expanded peripheral blood mononuclear cells (PBMCs) that are T-cells and comprises T-cell receptor gene rearrangement, and
wherein the BC-iPSC line exhibits less than 2.8% abnormal karyotype after 4 passages.
8 . The BC-iPSC line of claim 7 , wherein the BC-iPSC line exhibits less than 5% abnormal karyotype after 4-8 passages.
9 . The BC-iPSC line of claim 8 , wherein the BC-iPSC line does not show statistically significant differences between a first and a repeat G-band karyotype analysis, wherein the first or the repeat G-band karyotype analysis occurs during passages 10-23.