Fig. 2
Optimized culture conditions for generation of integration-free iPSCs. a ECM can improve reprogramming in both normoxic and hypoxic conditions. b Adding StemRegenin1 (SR1) or granulocyte-colony stimulating factor (G-CSF) to ECM does not affect reprogramming. PB MNCs cultured for 8 days before nucleofection with episomal vectors expressing OS, MK. PB MNCs (1 × 106 cells) nucleofected and then seeded into each well. Numbers of TRA-1-60-positive iPSC colonies counted 3 weeks after nucleofection. c Reprogramming efficiency of PB MNCs from four healthy volunteers and two polycythemia patients. d Hypoxia (3%) increases reprogramming efficiency under both ECM and GCM conditions. e Reprogramming efficiency of all healthy volunteers at different ages. PB MNCs cultured for 8 days before nucleofection with episomal vectors expressing OS, MK. PB MNCs (1 × 106 cells) nucleofected and then seeded into each well. Numbers of TRA-1-60-positive iPSC colonies counted 3 weeks after nucleofection. f Number of living MNCs decreased after 10 days of culture with ECM. g Ratio of living MNCs changed after 10 days of culture with ECM. h Culturing PB MNCs for different numbers of days affected reprogramming efficiency. PB MNCs cultured for 4–10 days before nucleofection with episomal vectors expressing OS, MK. PB MNCs (1 × 106 cells) nucleofected and then seeded into each well. Numbers of TRA-1-60-positive iPSC colonies counted 3 weeks after nucleofection. i AP staining photographs after different days of culture with SFFV promoter episomal vectors. Data representative of three experiments (mean ± SEM). *P < 0.05; ***P < 0.001. ECM erythroid culture medium, GCM granulocyte culture medium, PRV polycythemia vera, D day