Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways

Abstract

Hematopoietic stem cell (HSC) self renewal and lineage commitment depend on complex interactions with the microenvironment. The ability to maintain or expand HSCs for clinical applications or basic research has been substantially limited because these interactions are not well defined. Recent evidence suggests that HSCs reside in a low-perfusion, reduced-nutrient niche and that nutrient-sensing pathways contribute to HSC homeostasis. Here we report that suppression of the mTOR pathway, an established nutrient sensor, combined with activation of canonical Wnt-β-catenin signaling, allows for the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that the combination of two clinically approved medications that together activate Wnt-β-catenin and inhibit mTOR signaling increases the number (but not the proportion) of long-term HSCs in vivo.

Conflict of interest statement

The authors have no conflicts of interest.

Figures

Figure 1. Inhibition of GSK-3 and mTOR preserves HSPCs

(a) Experiment design: Mouse c-Kit+ or LSK cells were cultured in cytokine-free medium with inhibitors (CR) or vehicle for 7 d, then either plated on OP9 stromal cells or transplanted into lethally irradiated mice. FCM was performed after 3 weeks (OP9) or 4 months (transplantation, Figure 2). (b) Sorted LSK cells were cultured for 7 d, then plated on OP9 cells. Markers for myeloid (OP9), T and B lineages (OP9-DL1) (left) were measured at 3 weeks by FCM. Representative FCM data on left, quantification of myeloid, T and B lineages on right.

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).

Figure 3. Human long-term HSCs maintained in cytokine-free culture

(a) Human umbilical cord blood (UCB) CD34+ cells were cultured in cytokine-free medium with or without Chiron+rapamycin (CR) for 7 d, then competitive serial transplants were performed by transplanting all the cells into sub-lethally irradiated NSG recipients. Engraftment was assessed as percent of human CD45+ cells in BM after 4 months. (b) Representative FCM data (CD45+) are shown for BM from primary recipients of human CD34+ cells that were directly transplanted (uncultured) or cultured in vehicle (DMSO), CR, or conventional cytokines. (c) Progenitors, lymphoid (T-cell and B-cell), and myeloid lineages derived from human CD34+ cells that were directly transplanted (uncultured) or cultured in CR for 7 d before transplant. Similar numbers of each lineage were detected by FCM. (d) Four months after primary transplantation, BM was transplanted into 2° NOD-SCID recipients and chimerism was assessed after 4 months as in A.

Figure 4. Maintenance of long-term HSCs in culture requires β-catenin

(a) BM was harvested from mice with a conditional deletion of β-catenin (using cre-ERT2) or cre- controls, and LSK cells were cultured in cytokine-free medium with or without inhibitors (CR) for 7 d. Cultured cells were then plated on OP9 cells and assessed by FCM after 3 weeks as in Fig. 1. (b) C-Kit+ cells were harvested from mice with a conditional deletion of β-catenin (using mx-cre) or cre- controls and cultured in cytokine-free medium with or without inhibitors (CR) for 7 d, then competitive transplants were performed by transplanting all the cells in each group mixed with 3X105 fresh BM cells into lethally irradiated recipients. Chimerism in BM was measured by FCM for CD45.1+LSK,CD150+CD48−Flk2− cells after 4 months. For panels (a) and (b), depletion of β-catenin was confirmed by western blot (Supplementary Fig. 5c,d). (c) To assess cell cycle status, c-Kit+ cells were cultured for 4 d and analyzed by FCM for LSK markers, DAPI staining, and Ki67. Representative FACS data are shown for control (DMSO) vs. CR cultured cells. (d) To assess apoptosis, c-Kit+ cells were cultured as in A and analyzed by FCM for LSK, 7AAD, and Annexin V.

Figure 5. Lithium and rapamycin increase the number of long-term HSCs in vivo

(a) Mouse c-Kit+ cells were cultured for 7 d in cytokine-free medium with or without lithium and rapamycin (LR) and transplanted into lethally irradiated recipients. Survival of recipients receiving cells cultured with vehicle (blue diamond), lithium (red square), rapamycin (purple circle), or LR (green triangle) is shown as a Kaplan-Meier plot. (b) Representative FCM of donor-derived phenotypic HSCs in BM (upper panels) and myeloid and lymphoid lineages in peripheral blood (lower panels) for recipients of LR cultured cells. (c) Mice received lithium and rapamycin for 3 weeks. Cellularity of BM in vehicle control, lithium, rapamycin, and LR treated mice was measured as the number of nucleated cells/mouse recovered from one femur and tibia. (d) Absolute number of phenotypic HSCs (LSK,CD34−Flk2−CD150+CD48−) in BM in control, lithium, rapamycin, and LR treated mice. (e) Mice were treated with lithium and rapamycin for 3 weeks. BM was harvested, limiting dilution assay was performed, and competitive rescue units for control and LR are shown. (f) Western blot analysis for β-catenin, phosphorylated-S6, S6, phosphorylated-4EBP in BM cells harvested from control, lithium, rapamycin, and LR treated mice.