Supplementary MaterialsS1 Fig: Strategy #1 to reprogram V9V2 T cells into iPSCs. and the producing iPSC colony. (d) A result summary of iPSC generation using reprogramming strategy #2.(TIF) pone.0216815.s002.tif (7.3M) GUID:?43CD6C34-ED46-4D4B-8385-899EB3129E41 S3 Fig: Recognition of T-iPSC lines using TCRG gene clonality assay. To identify iPSC lines derived from T cells, genomic DNA was extracted and PCR was carried out using the expert mixes provided in the TCRG gene clonality assay kit. The yellow arrows show positive amplified products.(TIF) pone.0216815.s003.tif (2.9M) GUID:?B179790F-D87F-4A0B-B91C-8253E1ED1D7A S4 Fig: Verification of EPI-001 T-iPSC origin. To confirm the origin of T-iPSC collection, genomic DNA EPI-001 was extracted as template. PCR was carried out using primers specific for rearranged TCRG (a) and TCRD (b). The sequences of amplicons were compared with the ones in Gene database at NCBI.(TIF) pone.0216815.s004.tif (3.0M) GUID:?CFECDA83-76F3-4FAA-84E4-C907823FD0A0 S5 Fig: Characterization of T-iPSCs. (a) A high resolution image of a T-iPSC collection, GDTA/NF-iPSC#1. (b) Manifestation EPI-001 of pluripotent markers OCT4, SOX2 and NANOG in GDTA/NF-iPSC#1 as analyzed by RT-PCR. Fibroblast-like cells (FLCs) derived from iPSC lines, GDTA/NF-iPSC#1 and PBC-iPSC#9, using a previously reported protocol (and genes and TCR manifestation are the hallmarks of T cells[22]. While it is definitely demanding to accurately recapitulate the process of somatic recombination of and genes and genes and that such T cell-derived iPSCs can be re-differentiated into T cells, that may re-express the same antigen-specific TCR[23, 24]. Using this strategy, many antigen-specific T cells can be generated from an iPSC collection. But EPI-001 the feasibility of using such strategy to generate T cells from GDF6 T cell-derived iPSCs ( T-iPSCs) remains unexplored. Furthermore, to express multiple NKRs in T cells, genetic engineering could be a possible approach. However, limited genetic payload and limited size and number of changes that can be safely made in the genome of an immune cell remain the practical constraints to utilize such an approach for delivering and integrating multiple genes[2]. We hypothesized that EPI-001 genetic modification might be unneeded if we were able to induce the manifestation of NKRs in the process of differentiating T-iPSCs into mimetic T cells. Therefore, in view of the above-mentioned options, we designed a simple two-step strategy to generate functionally enhanced mimetic T cells from iPSCs (Fig 1): In step 1 1, V9V2 T cells are reprogrammed to generate T-iPSCs; in step 2 2, T-iPSCs are differentiated into NKR-expressing mimetic V9V2 T cells using an NK cell-promoting protocol. Here, we shown that this two-step strategy is definitely feasible. The T-iPSC-derived mimetic V9V2 T cells are endowed with an array of NKRs and are potent to target a broad range of cancers. Open in a separate windowpane Fig 1 A schematic of a two-step strategy to derive mimetic T cells endowed with NKRs from iPSCs.In step 1 1, V9V2 T cells are reprogrammed to generate T cell-derived iPSCs ( T-iPSCs) carrying the rearranged and genes; in step 2 2, T-iPSCs are differentiated to V9V2 T cells that communicate NKRs using an NK cell-promoting differentiation protocol. Reprogramming V9V2 T cells into T-iPSCs We tested three reprogramming strategies to generate iPSCs from V9V2 T cells (S1 Fig, S2 Fig, Fig 2 and S1 Table). To activate and increase V9V2 T cells for iPSC generation, we cultured peripheral blood mononuclear cells (PBMCs) from a healthy donor using zoledronic acid (Zol) and interleukin-2 (IL-2). Total cell number improved and cell clumps appeared in the PBMC cultures over time (S1B Fig and Fig 2B), which show the expanding of V9V2 T cells. More than 60% of one-week cultured cells were T.