| Popis: |
TCR stimulation is necessary for T cells to be reprogrammed to T-iPSCs. A, Schematic representation of PBMC derived T-cell reprogramming into iPSC. Whole PB T cells or the sorted populations were stimulated by αCD3/28 beads, and transduced with Sendai virus containing four Yamanaka factors and SV40 large T antigen. Approximately after 20–25 days’ culture in iPSC culture condition, the number of established iPSC colonies were counted. B, A bar graph indicates the numbers of AP-positive ES cell-like iPSC colonies derived from 1E+5 T cells with or without TCR stimulation. N = 3 for each donor. SeV containing four Yamanaka factors (OSKM) or OSKM and SV40 were used. C, Gating strategy to sort four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA). PBMC were stained by antibodies against CD3, CD8, CD62L, CD45RO, CCR7, and CD45RA. CD8+ T cells were first gated on the basis of CD45RO and CD62L expression, followed by the second gate based on CD45RA and CCR7 expression. Four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA) were sorted according to the gates indicated in the flow cytometry panels. D, A summary table showing which subset (Naïve, CM, EM, and EMRA) were reprogrammed to iPSCs by different stimulation methods. Sorted CD8+ T-cell subsets (Naïve, CM, EM, and EMRA) were stimulated by different methods (OKT-3 or αCD3/28 beads) before SeV infection. E, Representative AP, phase contrast, and bright field images were shown to describe the methods of iPSC colonies enumeration from different subsets (Naïve, CM, EM, and EMRA) of CD8 T cells reprogrammed into iPSC in each experiments following transduction of Sendai virus containing four Yamanaka factors and SV40 large T antigen. Each well was seeded with 1E+5 T cells. F, A bar graph indicates the numbers of AP-positive ES cell-like colonies derived from 1E+5 sorted CD8+ naïve/CM/EM/EMRA T cells activated by αCD3/28 beads. N = 3 for each donor. |
