Inhibition of TBRI restores self-renewal and clonogenic capacities of MM HSPCs. (A) BM HSPCs of 6 MM patients were preincubated for 1 hour with a vehicle, SD-208 (0.5μM) or TGFβ in combination with SD-208 and grown in the presence of SCF, TPO, and FLT-3L. On days 0, 2, 3, 6, and 7, cell aliquots were taken and viable cell concentrations were determined and compared with BM HSPCs of healthy donors (P < .05). To determine the frequency of long-term culture-initiating cells 6 × 10³ HSPCs from the BM of 5 MM patients were preincubated with vehicle or SD-208 and plated (22 replicates per concentration: 180, 60, 20, 7.5 cells/well) onto irradiated confluent AFT024 feeder layers subcultured in 96-well plates and maintained in liquid medium. After 5 weeks, the liquid medium was replaced by methylcellulose medium and secondary CFC were scored after 2 weeks. The absolute frequency of LTC-ICs was calculated using Lda STAT software. (B) LTC-IC frequency of HSPCs of healthy donors (□), vehicle-treated HSPCs of MM patients (■), and SD-208–treated HSPCs of MM patients () is shown. Purified HSPCs from the BM of 6 MM patients were seeded into semisolid ready-to-use methylcellulose growth medium at a concentration of 5 × 10² cells/mL after preincubation with vehicle or SD-208. (C) Colony numbers were counted after 2 weeks under an inverted light microscope. Proportions of colonies (BFU-E/CFU-E, CFU-G/CFU-M/CFU-GM, CFU-GEMM) derived from HSPCs of healthy donors (□), vehicle-treated HSPCs of MM patients (■), and SD-208–treated HSPCs () are shown. Cell-cycle analyses of HSPCs obtained from the BM of 4 MM patients were performed after 7 days in liquid medium supplemented with SCF, TPO, and FLT-3L and treatment with either vehicle or SD-208 using the PE BrdU Flow Kit and flow cytometric analysis. (D) The bar chart displays the mean percentages of HSPCs of healthy donors (□), vehicle-treated HSPCs of MM patients (■), and SD-208–treated HSPCs of MM patients () in G₀/1 phase and S phase. Error bars represent SEM. The Student t test or one-way ANOVA was used to detect statistically significant differences. *P < .05; **P < .01.