Figure 3. Optimization of the PBMNC reprogramming protocol.

(A) Depletion of CD3⁺ and 19⁺ lymphoid cells increases reprogramming efficiency. Whole PBMNCs or CD3⁻/CD19⁻ cells (T/B cell-depleted PBMNCs) were cultured for 4 days before nucleofection with episomal vectors expressing OS (OCT4 and SOX2), MK (MYC and KLF4) and B (BCL-XL). 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection and numbers of iPSC colonies per 1 ml of PB were calculated by normalization to the amount of starting peripheral blood. Data shown are presented as mean ± SEM (n = 4). * indicates P<0.05. (B) Culturing PB CD3/19⁻ cells for 4 days allows maximum reprogramming. CD3/19⁻ cells (T/B cells-depleted) PBMNCs were cultured for 0 to 8 days before nucleofection with episomal vectors expressing OS, MK and B. 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection. Graphed data are presented as mean ± SEM (n = 6). * indicates P<0.05.