Derivation of iPSCs

1. Derivation of iPSCs from fibroblasts

Each fibroblast line will be expanded to 5x10⁵ cells for electroporation with episomal reprogramming vectors (Okita, K., et al., A more efficient method to generate integration-free human iPS cells. Nat Methods, 2011. 8(5): p. 409-12). Electroporated cells will be serially diluted and grown 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.

Figure1: timeline for generating iPSCs from human fibroblasts

2. 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