Fig 1.
Cell-autonomous regulation of HSC function by GSK-3 and mTORC1.
(A) HSCs were cultured for 3 d, and cell number relative to day 0 was determined visually. (B) Viability of HSCs cultured in control medium or CR after 7 d culture, determined by Trypan Blue exclusion. (C, D) HSCs were cultured for 7 d and then transplanted with competitor cells into lethally irradiated hosts. Peripheral blood was collected at 24 weeks post-transplant, and multilineage potential of donor-derived (CD45.1+) cells was determined by flow cytometry for lineage-specific markers as indicated (C). Flow cytometry data shown are from one recipient representative of CR-cultured HSCs. Long-term engraftment of freshly isolated HSCs or of HSCs cultured in vehicle or CR was determined by flow cytometry for donor-derived (CD45.1+) cells following competitive transplant (D). Chimerism data shown were collected 24 weeks after transplant. Each symbol represents the results from an individual mouse. Red dotted line indicates 5% threshold defining recipients as positive for donor-derived engraftment. Error bars represent S.E.M.
Fig 2.
mTORC1 activity and global translation during HSC maintenance.
(A-D) Representative flow cytometry histograms of pS6 (A) and p4E-BP1 (C) from 1.5 h time point, and their median fluorescence intensities relative to DMSO (B, D), measured by phospho-flow cytometry in the LSK-CD48- fraction of lineage-depleted bone marrow cells at the indicated times after cells were isolated and placed into culture medium. Asterisks indicate statistical significance for comparison of treatment to DMSO within the same time point. (E, F), c-Kit+ cells were cultured for 24 h followed by RNA isolation for RT-PCR analysis. RT-PCR for ribosomal protein (E) and ribosomal RNA (F) gene expression relative to DMSO. (G) Lineage-depleted bone marrow cells were cultured for 24 h, and then rate of protein synthesis relative to DMSO was measured by incorporation of OP-Puro followed by flow cytometric analysis of the LSK-CD48- fraction. Error bars represent S.E.M. Statistical significance for gene expression analyses was assessed using a one-way ANOVA followed by Dunnett’s test for multiple comparisons. Statistical significance for pS6 and p4E-BP1 median fluorescence intensities was assessed using a one-way ANOVA followed by Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.005.
Fig 3.
Targeting S6K and eIF4E downstream of mTORC1 in HSC maintenance.
(A) Strategy to test inhibition of S6K or eIF4E in HSC culture: If the primary effect of rapamycin in HSC maintenance is to inhibit S6K and/or eIF4E, then direct inhibitors of these downstream effectors should be able to replace rapamycin and suppress lineage commitment. (B) Flow cytometric analysis of pS6 in the LSK-CD48- fraction of lineage-depleted bone marrow cells treated with PF-4708671. (C) Rate of translation relative to vehicle in LSK-CD48- cells treated with 4EGI-1, measured by OP-Puro incorporation. (D,E) LSK cells were cultured for 7 d and then transplanted with competitor cells into lethally irradiated hosts. Donor-derived engraftment was determined by flow cytometry for CD45.1+ cells in peripheral blood from recipients of cultured HSPCs following competitive transplant. Chimerism data shown were collected 8 weeks after transplant. Flow cytometry data shown are from one recipient representative of each transplant group (D). Each symbol represents the result from an individual mouse, and red dotted line indicates 5% threshold defining recipients as positive for donor-derived engraftment (E). CP, CHIR99021 + PF-4708671; C4, CHIR99021 + 4EGI-1; CP4, CHIR99021 + PF-4708671 + 4EGI-1. Error bars represent S.E.M. Statistical significance was assessed using a one-way ANOVA followed by Dunnett’s test for multiple comparisons. *** p < 0.0005.
Fig 4.
Mitochondrial mass and activity in maintained HSCs.
(A, B) Flow cytometric analysis of mitochondrial mass (median fluorescence intensity of Mitotracker Green) relative to DMSO (A) and mitochondrial membrane potential (JC-1) (B) in the LSK-CD48- fraction of lineage-depleted bone marrow cells following 16 h culture. Flow cytometric data shown are representative results of 3 experiments. Error bars represent S.E.M. Vm, mitochondrial membrane potential. Statistical significance was assessed using a one-way ANOVA followed by Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.005.
Fig 5.
Autophagy as a molecular mark of HSC maintenance.
(A) RT-PCR for a pro-autophagic gene signature relative to DMSO in c-Kit+ cells following 24 h culture. Data shown are average of 4 experiments. (B-D) HSCs were cultured for 24 h and then fixed for immunofluorescence analysis of LC3 staining. Sample image of LC3-punctum-positive cell at 400× magnification (B). White arrowhead indicates LC3 punctum. Scale bar, 10 μm. Quantification of percentage of LC3-puncta-positive cells (C) and of average number of LC3 puncta per cell (D). For immunofluorescence analyses, n = 85–150 cells counted per treatment group per experimental replicate. Data shown are average of 3 experiments, where each symbol represents the average from one replicate. Error bars represent S.E.M. Statistical significance was assessed using a one-way ANOVA followed by Dunnett’s test for multiple comparisons. ** p < 0.005, *** p < 0.0005.