Derivation of iPSCs

Derivation of iPSCs from peripheral blood      

Erythroid progenitor cells will be isolated from 10ml fresh peripheral blood, and then expanded to 1x106 cells in 1-2 weeks. 3-5 x105 cells will be electroporated with episomal reprogramming vectors and grown in one well of 6-well plate for 5 weeks to generate iPSC colonies. At 3, 4 and 5 weeks, colonies will be picked and expanded to 5x105 cells and four liquid nitrogen stocks will be made.  Clones will be screened by PCR for expression of endogenous pluripotency genes (Oct3/4, Nanog) and the lack of expression of reprogramming factors. Embryoid body differentiation will be performed with each line and qPCR confirmation of all three germ layer markers will be performed.  At least 3 independent iPSC clones will be identified and frozen, or a second electroporation will be performed.  If 3 clones are not isolated, the charge will be prorated based on the number of iPSC clones generated.  The entire project should require 12-16 weeks.

Figure2: timeline for generating iPSCs from human peripheral blood