Optimizing culture conditions to improve MK maturity and platelet production. (A-B) Bar graphs comparing previously published culture medium (CM) and bespoke medium (AMK) showing the number of viable MK per starting hiPSCs at day 20 posttransduction (A) and purity (percentage of CD41⁺CD42⁺ cells) (B). Mean ± 1× SEM; n = 3. Results are shown for a control hiPSC line (gray/lavender bars) and 2 clinical-grade hiPSC lines (blue bars). (C) Cytospins of day 20 MKs from the control QOLG and clinical-grade DELTA-3 lines cultured in either CM or AMK stained using Rapid Romanowsky. Scale bars, 50 μm. Black arrows indicate polynucleated MKs. Examples of cytospins performed for each experiment. (D) DMS assessment. Left: fluorescent immunohistochemistry images of DELTA-3 FoP MKs cultured in AMK or CM as indicated and stained for GPIbβ (CD42C, green) and nuclei stained using 4′,6-diamidino-2-phenylindole (DAPI; blue), classified according to their DMS development as per Aguilar et al²⁴  (supplemental Methods). Magnification ×60. Left bar graph: the percentage of MKs at each level of maturity for MKs grown in CM or AMK. There is an increase in both class III and IV MKs in AMK and a significant increase in the average mean pixel intensity (right graph) for GPIbβ. **P ≤ .005. Analysis of variance (ANOVA) combining data from all classes. Data from DELTA-3; n = 3, 125 images. (E) Transmission electron microscopy images of mature DELTA-3 MKs cultured in either CM or AMK. MKs cultured in CM (left) have a heterogeneous granule population including α granules (arrowheads) but lack a DMS. MKs grown in AMK (right) show both granules and the beginning of a DMS network (black arrow). Scale bars, 500 nm. Twelve images were examined for CM and 9 for AMK. (F) Proplatelet formation was assessed in MKs derived from both LIPSC-GR1.1 and DELTA-3 and cultured in CM or AMK. Representative immunohistochemistry images of LIPSC-GR1.1 MKs showing α tubulin (green), F-actin (red), and DAPI 1 μg/mL (blue). Magnification ×60. The percentage of MKs showing at least 1 proplatelet extension (left graph) and the percentage of multibranched proplatelet forming MKs (right graph) was increased in AMK. Ten images were examined per sample from n = 2 biological replicates. Error bars indicate the range in values. Mean ± 1× SD. (G) Platelet production by mature MKs transferred to high-glucose cytokine-free medium (RPMI 1640) for 72 hours was assessed by flow cytometry. The percentage of viable (by Calcein-AM) platelet-size CD41⁺CD42⁺ particles produced by QOLG, LIPSC-GR1.1, and DELTA-3 increased compared with leaving the MKs in cytokine-free AMK when mature. An increase in platelets produced per MK ranging from 2.84-fold to 7.3-fold was seen for iLIPSC-GR1.1.2 and iDELTA-3.7, respectively. Error bars indicate the range in values. Mean ± 1× SD; n = 2. (H) Viability by Calcein-AM of the platelet-size CD41⁺CD42⁺ particles also significantly increased for QOLG and iLIPSC-GR1.1.2. Error bars indicate the range in values. Mean ± 1× SD; n = 2.