Figure 6.

Basic media switch enables significant cell expansion upon NK-cell differentiation. (A) Screening of indicated basic media to support NK-cell differentiation and proliferation from iPSC-HPCs. All media were supplemented with SCF, FLT3L, IL-7, and IL-15. Cells remained in the original differentiation wells throughout (media switch on day 14). (B) Defined APEL2 medium with factors does not support longer-term cell expansion after transfer of HPCs to independent culture wells, regardless of cell density (n = 3 per data point). (C) Media switch at transition point between precursors and NK cells enables cell expansion upon differentiation (n = 6 similar conditions). Note the logarithmic scale. Right panel: Flow cytometric analysis at end of time course. (D) Resulting protocol with indicated media changes and added signaling molecules. S = SCF, F = FLT3L. Refer to Figure 5A for treatments over the first 7 days. (E) RNA-seq analysis highlighting NK-cell-specific cluster with selected marker genes and enrichment terms (also see Supplementary Table S2). (F) Killing assays using NK cells derived from 3 iPSC lines. (G) Illustration linking differentiation paradigms of the present study to context-dependent strategies for upscaling. Ball sizes are to reflect cell numbers.