Candidate compounds promote MK maturation and subsequent PLP production in vitro and in vivo. (A) Enlargement of imMKCL surface area upon exposure to candidate compounds. imMKCLs were incubated with SR1 (0.75 μM), TCS 359 (3.3 μM), Wnt-C59 (1.1 μM), or vehicle. (B) Representative electronic micrographs of imMKCLs showing extensive DMS development in the presence of the candidate compounds. imMKCLs were incubated for 6 days with each candidate compound or vehicle. Scale bars, 2μm. (C) PAC-1 binding to human donor platelets and imMKCL-derived PLPs was quantified in the absence or presence of 100 μM ADP and 40 μM TRAP-6 using flow cytometry. The change in mean fluorescence intensity (ΔMFI) was calculated as agonist (+) − agonist (−). (D) Candidate compounds promote PLP production from CB MNC-derived MKs. MNCs were isolated from human CB and differentiated into MK lineage. The CB MNC–derived MKs were incubated for 7 days with each candidate compound or vehicle. PLP yield per initial CD41a⁺ MK in the presence of vehicle (DMSO alone) was assigned a value of 1.0. (E) Candidate compounds promote PLP production from MKs differentiated directly from iPSCs (direct iPSC-derived MKs). iPSCs (TkDN 3-4 clone) were first differentiated into hematopoietic progenitor cells in the presence of feeder cells, followed by MK lineage differentiation without intervention. Direct iPSC-derived MKs were incubated for 10 days with each candidate compound or vehicle. PLP yield per initial cell in the presence of vehicle (DMSO alone) was assigned a value of 1.0. (F) Schema of mice experiments to evaluate the in vivo thrombopoietic effect of the candidate compounds. Platelets were depleted by the intravenous (i.v.) administration of anti-GPIbα (200 ng/g body weight) to male C57BL/6JRj mice. The mice were then divided into 4 groups and administered 100 μg/kg CH-223191, Wnt-C59, or TCS 359, or PBS (intraperitoneally [i.p.]) on the days indicated. Blood samples were collected before and after anti-GPIbα injection. (G) Promotion of in vivo platelet recovery in an anti-GPIbα–induced thrombocytopenic mouse model by the candidate compounds. Data are expressed as the mean ± S.D. from 3 to 5 independent experiments. *P < .05, **P < .01, ***P < .001 vs vehicle or PBS, 2-way ANOVA, followed by multiple comparisons (G) or 1-way analysis (C-E). N.S., not significant.