FIG. 6.

TGFBI reduces clonogenic capacity of HSPCs. Clonogenic assays with CB-derived HSPCs with increased TGFBI (A, C) or decreased TGFBI expression. (B, D) CAFC assays (A, B) show frequency of CAFC in MSC/HSPC co-culture, relative to input cells. LTC-CFC assays (C, D) show colony-forming capacity of the MSC/HSC co-culture progeny. At indicated time points (week 2, 4, or 6), part of the NA HSPCs or SA HSPCs from MSC/HSPC co-cultures, was plated in methylcellulose medium. LTC-CFC were analyzed after 12–14 days. (A) HSPCs overexpressing TGFBI in CAFC assays show decreased CAFC frequencies at week 6 of MSC/HSPC co-culture. Shown frequencies are normalized to control conditions at week 2 (n = 3). (B) HSPCs with TGFBI knockdown in CAFC assay show decreased CAFC frequencies at week 4 and 6 of MSC/HSPC co-cultures. Shown frequencies are normalized to control conditions for each week (n = 3). (C) HSPCs overexpressing TGFBI in LTC-CFC assays show reduced colony outgrowth. Shown are mean ± SEM (n = 3). (D) HSPCs with TGFBI knockdown in LTC-CFC show increased colony outgrowth from the stromal adherent HSPC fraction after 6 weeks of MSC/HSPC co-culture (W6SA). Shown are mean ± SEM (n = 3). *P < 0.05, sh4/sh5 indicates that sh4 or sh5 was used in independent experiments. CAFC, cobblestone-area-forming cell; LTC-CFC, long-term culture-colony forming cell; HSC, hematopoietic stem cell; NA, nonadherent; SA, stroma adherent.