Induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells: Engineering innovations, translational hurdles and clinical prospects in immune therapy

Figure 4 Manufacturing workflow of induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells. The figure outlines the manufacturing workflow of induced pluripotent stem cell-derived chimeric antigen receptor natural killer (iPSC-CAR-NK) cells. Somatic cells, including adult human dermal fibroblasts and peripheral blood mononuclear cells, are first isolated and reprogrammed into iPSCs using synthetic mRNA or Sendai viral vectors. Candidate iPSCs are then cultured and expanded under current Good Manufacturing Practice-compliant, feeder-free, chemically defined culture conditions, followed by characterization and quality-control testing, including pluripotency validation, karyotype and genomic-stability analysis, sterility testing, mycoplasma testing, and viral detection. NK cell differentiation is initiated by CHIR99021 and bone morphogenetic protein 4 or by co-culture with OP9 stromal cells to induce cluster of differentiation 34-positive (CD34+) hematopoietic progenitor cells. These cells are further differentiated into NK precursor cells under stimulation with interleukin-3 (IL-3), IL-7, IL-15, and stem cell factor, followed by IL-15-mediated activation and expansion into mature NK cells with high expression of CD56, CD16, and natural killer group 2 member D. CAR introduction is performed using third-generation lentiviral vectors, with centrifugation-assisted infection to enhance transduction efficiency, or by non-viral electroporation. Finally, CAR-positive NK-cell populations are sorted and enriched by flow cytometry using fluorochrome-conjugated anti-CAR antibody staining combined with CD56 and CD16 staining. iPSCs: Induced pluripotent stem cells; NK: Natural killer; CAR: Chimeric antigen receptor; miRNA: MicroRNA; CD: Cluster of differentiation; IL: Interleukin; SCF: Stem cell factor; NKG2D: Natural killer group 2 member D.