Figure 2. Maintenance of long-term HSCs ex vivo by inhibition of GSK-3 and mTOR.
(a) Mouse c-Kit+ cells were cultured in cytokine-free medium for 7 d with the inhibitors as indicated, then competitive serial transplants were performed. Chimerism in bone marrow (BM) was measured by FCM after 4 months. The data are from 3 independent experiments. (b–d) LSK cells were cultured in cytokine-free medium for 7 d, then competitive serial transplants were performed in primary (b), secondary (c), and tertiary recipients (d). Bone marrow was harvested after 4 months for analysis of chimerism and for transplant to subsequent recipients. (e) Limiting dilution experiments were performed with three doses of cultured c-Kit+ test marrow (CD45.1+) from control or CR treated cells combined with 2×105 competing cells (CD45.2+) transplanted into groups of at least nine recipients per dose. Chimerism at 4 months for each dose is represented as the percentage of mice negative for engraftment in BM for vehicle control (green triangle), CR treated cells (red square), and uncultured cells (blue diamond).