Fig. 1
Different effects of four MNC isolation methods on reprogramming of PB MNCs with episomal vectors. a Flow chart of optimized method for generation of integration-free iPSCs from human PB. Green ellipses highlight basic method. For figuring out best conditions, one factor was optimized while controlling others following indicated basic method. For each condition identified, at least three donors randomly selected and repeated three times per donor. b Number of living MNCs isolated from 1 ml of PB by four methods at day 0. c Number of living MNCs isolated from 1 ml of PB after 8 days in culture. d Number of TRA-1-60-positive colonies generated from 1 × 106 PB MNCs. e Number of TRA-1-60-positive colonies generated from 1 ml of PB by different isolating methods. PB MNCs cultured for 8 days before nucleofection. PB MNCs (1 × 106 cells) nucleofected and then seeded into each well. TRA-1-60 staining of iPSCs at 3 weeks after nucleofection of PB MNCs with episomal vectors. Data presented as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. OS, pEV SFFV-OCT4-E2A-SOX2; MK, pEV SFFV-MYC-E2A-KLF4; Shp53, pEV SFFV-Shp53;BCL-XL, pEV SFFV-BCL-XL; K, pEV SFFV- KLF4. HES hydroxyethyl starch, ECM erythroid culture medium, GCM granulocyte culture medium, G-CSF granulocyte-colony stimulating factor, SR1 StemRegenin1, iPS induced pluripotent stem, D day, PB peripheral blood