FIG. 1.

Determining the optimal concentration of CHIR99021 (CHIR) for HSC expansion. (A) c-Kit⁺ HSPCs were isolated from BM of WT mice and treated with increasing concentrations of CHIR for 3 h. β-catenin activity was examined by western blotting for non-p-β-catenin. (B, C) LSK-CD150⁺48⁻ HSCs were isolated from BM of WT mice and treated with indicated concentrations of CHIR for 3 h. β-catenin activity was assessed by FACS for intracellular non-p-β-catenin staining (B) and Axin 2, HoxB4 cyclin D1, and c-myc expression (C). (D–F) LSK-CD150⁺48⁻ HSCs were cultured in stem cell medium with indicated concentrations of CHIR. Two hundred HSCs were sorted for each group by FACS. Cells were cultured for 9 days with medium change every other day. The percentages and numbers of LSK HSPCs and LSK-CD150⁺48⁻ HSCs were examined by FACS (D); functional HSCs were evaluated by CHR assay (E) and serial transplantation (F). In (E, F), freshly isolated (uncultured) LSK-CD150⁺48⁻ HSCs were studied as controls. BM cells used for second transplantations in (F) were mixtures of BM cells collected from all nine recipient mice from the first transplantation in each group in (E). The percentage of CD45.1⁺ cells among mixed BM cells was 5.7% for the uncultured group, 19.2% for the 0 μM CHIR group, and 67.5% for the 0.5 μM CHIR group, respectively. Data in (A–D) are representative of three independent experiments. *P < 0.05; **P < 0.01. BM, bone marrow; CHR, competitive hematopoietic reconstitutive; HSC, hematopoietic stem cell; HSPCs, hematopoietic stem/progenitor cells; NS, not significant; WT, wild type. Color images are available online.