Isolation and differentiation of iPSC-HPCs into BCMA iPSC–T cells with a mature CD8αβ⁺ cell phenotype. (A, top) Representative gating strategy for isolation of HPCs (CD34⁺ CD43⁺/CD14⁻ CD235a⁻) from BCMA-specific EBs sorted using FACS. (A, bottom) Summary of 3 independent analyses (N = 3; mean ± SD). (B) Summary of the increase in total cell numbers during differentiation of BCMA-iPSC HPCs (N = 3) or EBV-specific iPSC HPCs from the initial number of cells (5 × 10³; day 0) (∗P < .05). (C) Representative flow cytometric analyses demonstrating a gradual differentiation of BCMA iPSC-HPCs (day 14 > 10 > 7 > 3) into CD3⁺ and CD8⁺ T lymphocytes using retronectin-coated culture plates in the presence of Fc-DLL4. (D) Representative flow cytometric analysis comparing phenotype of primary T cells (CD3⁺ CD45⁺ TCR αβ⁺ / CD4⁺ CD8α⁺ CD8β⁺) in healthy donor’s peripheral blood mononuclear cells (top) and BCMA iPSC–T cells (CD3⁺ CD45⁺ TCR αβ⁺/CD4⁻ CD8α⁺ CD8β⁺) on day 21 of T-cell differentiation (bottom). (E) Summary of overall BCMA iPSC–T-cell (N = 3; mean ± SD) phenotype on day 21 of the T-cell differentiation process. (F) Representative flow cytometric analyses of BCMA iPSC–T cells on day 21 of differentiation showing T-cell activation (CD38 and CD69) and costimulatory molecule (CD28 and 41BB) expression without immune checkpoint (CTLA-4, PD1, LAG3, and TIM3) expression. (G) Summary analyses of T-cell activation, costimulatory molecule, and immune checkpoint expression on BCMA iPSC–T cells (N = 3; mean ± SD) on day 21 of differentiation.