Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24Bst/+ mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24Bst/+ cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.
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S.J.M. is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, and the director of the Hamon Laboratory for Stem Cells and Cancer. This work was supported by the Cancer Prevention and Research Institute of Texas and the National Institute on Aging (R37 AG024945). R.A.J.S. was supported by fellowships from the Leukemia & Lymphoma Society (5541-11) and the Canadian Institutes of Health Research (MFE-106993). J.A.M. was supported by the UT Southwestern K12 Pediatrics Training Grant (K12-HD068369). We thank A. Pineda, K. Cowan, E. Daniel, M. Acar, H. Oguro, J. Peyer, K. Rajagopalan, M. Agathocleous and E. Piskounova for technical support and advice; N. Loof and the Moody Foundation Flow Cytometry Facility, L. Hynan and J. Reisch for advice regarding statistics; J. Shelton for histology; and R. Coolon, S. Manning, M. Gross and K. Correll for mouse colony management.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Isolation of haematopoietic progenitor cell populations by flow cytometry and histograms showing protein synthesis in vivo relative to HSCs from the same mice.
a–e, One hour after OP-Puro administration to mice we observed no effect on bone marrow cellularity (one femur and one tibia (a); n = 7 PBS treated and n = 9 OP-Puro treated mice) or the frequencies of CD150+CD48-LSK HSCs (b; n = 4 PBS treated, n = 6 OP-Puro treated mice), annexin V+ bone marrow cells (c; n = 4 PBS treated mice, n = 6 OP-Puro treated mice), annexin V+ HSCs (d; n = 4 PBS treated mice, n = 6 OP-Puro treated mice), or HSCs in S/G2/M phase of the cell cycle (e; n = 3 mice per treatment; a–e each reflect two or three independent experiments). f–i, Representative flow-cytometry plots showing the markers and gating strategies used to isolate CMPs46, GMPs46, and MEPs46 (f), pro-B47, pre-B47 and IgM+ B cells (g), Gr-1+ myeloid cells (h), CD3+ T cells (h) and CD71+Ter119+ erythroid progenitors (i). Each panel also shows OP-Puro incorporation histograms for each cell population relative to HSCs after 1 h of OP-Puro incorporation in vivo. The level of background fluorescence from PBS treated controls is overlaid in black. j, Data from Fig. 1h showing protein synthesis in various haematopoietic-cell populations relative to unfractionated bone marrow cells on a log2 scale (n = 15 mice from 9 independent experiments). All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance in a–e. The statistical significance of differences relative to HSCs in j was assessed using a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons. Asterisks indicate statistical comparison to HSCs (*P < 0.05, **P < 0.01, ***P < 0.001).
Extended Data Figure 2 OP-Puro-containing polypeptides are not degraded within 30 min, the degradation that occurs over 24 h is blocked by bortezomib, and OP-Puro administration does not induce cell death.
a, OP-Puro fluorescence in haematopoietic cells after 1 h of OP-Puro administration in vivo followed by a 30-min ex vivo incubation on ice or at 37 °C (n = 11 mice from 4 independent experiments). b, OP-Puro fluorescence in haematopoietic cells 24 h after OP-Puro administration in vivo. Treatment with bortezomib 1 h before OP-Puro administration increased OP-Puro fluorescence in every cell population 24 h later (n = 3 independent experiments; total number of mice per treatment are shown in the panel). c, Frequency of annexin V+ cells in each cell population 1 h after OP-Puro administration in vivo relative to the same cells from untreated mice (n = 7 mice per treatment from 2 independent experiments). All data represent mean ± s.d. To assess the statistical significance of treatment effects within the same cell population we performed two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). To assess the statistical significance of differences between HSCs and other cell populations in a, we performed a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (†P < 0.05, ††P < 0.01, †††P < 0.001).
Extended Data Figure 3 Cyclophosphamide and GCSF treatment drives certain cells into cycle and increases protein synthesis.
a, Frequency of dividing cells in S/G2/M phases of the cell cycle (>2N (>diploid) DNA content; n = 5 mice from 3 independent experiments). b, Total protein isolated from 50,000 unfractionated bone marrow cells or Gr-1+ cells in G0/G1 or S/G2/M measured by BCA assay (n = 3). c, Total RNA content in 15,000 cells from each stem- or progenitor-cell population (n = 3 mice). d, The frequency of cycling (KI-67+) HSCs increased dramatically after treatment with cyclophosphamide (Cy) and GCSF (n = 5 untreated mice and n = 6 mice treated with cyclophosphamide and GCSF, from 2 independent experiments, P < 0.001). e, Frequency of KI-67+ cells in haematopoietic-cell populations before and after treatment with cyclophosphamide and GCSF (n = 5 untreated mice and n = 6 mice treated with cyclophosphamide and GCSF for BM, HSC and MPP, n = 3 mice per treatment for other cell populations). f, g, Protein synthesis in G0/G1 and S/G2/M cells from untreated mice or mice treated with cyclophosphamide followed by two days of GCSF (n = 10 mice per treatment from 6 independent experiments). These data are the same as shown in Fig. 3b, d, e, shown together in this panel for comparison. The data are plotted on a linear scale in f and on a log2 scale in g. All data represent mean ± s.d. To assess the statistical significance of treatment effects within the same cell population (b, e–g) we performed two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). To assess the statistical significance of differences between HSCs and each other cell population (a, c, f, g) we performed a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (†P < 0.05, ††P < 0.01, †††P < 0.001).
Extended Data Figure 4 Differences in protein synthesis among haematopoietic stem and progenitor cells are not fully explained by differences in cell division, cell diameter, pS6 levels, rRNA or total RNA content.
a–f, Scatter plots show the relative rates of protein synthesis (per hour) in each cell population (from Fig. 1h) plotted against the frequency of dividing cells (a; from Extended Data Fig. 3a), cell diameter (b; from Fig. 3f), 18S rRNA content (c; from Fig. 3g), 28S rRNA content (d; from Fig. 3g), total RNA content (e; from Extended Data Fig. 3c) and pS6 levels (f; from Fig. 5a normalized to β-actin). For each parameter, regressions were performed using all populations excluding HSCs and 95% confidence intervals were determined. R2 values are shown in each plot. Rates of protein synthesis are plotted on a linear scale (left panels) and on a log2 scale (right panels). Note that HSCs were outliers with respect to each regression. CD150+CD48−LSK cells were used to determine HSC rates of protein synthesis, cell diameter and percentage S/G2/M, and CD48−LSK cells (HSCs and MPPs) were used to determine 18S, 28S, total RNA and pS6 levels (as these measurements required more cells). All data represent mean ± s.d.
Extended Data Figure 5 Rpl24Bst/+ mice have normal frequencies of lymphoid and myeloid lineage progenitors and do not express increased p53 or p21Cip1 in adult haematopoietic cells.
a, Bone marrow (2 femurs and 2 tibias; n = 5 wild-type and n = 6 Rpl24Bst/+ mice from 4 experiments), spleen (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments), and thymus cellularity (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). b, White blood cell, red blood cell and platelet counts (n = 5 wild-type and n = 6 Rpl24Bst/+ mice from 4 experiments). c–f, The frequencies of B (c), myeloid (d) and T (e) lineage cells in the bone marrow and spleen (f) of Rpl24Bst/+ and control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). g, h, The frequencies of T-lineage progenitors in the thymus of Rpl24Bst/+ and control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). Double negative (DN)1 and early T-lineage progenitor (ETP) cells were CD4−CD8−CD44+CD25−; DN2 cells were CD4−CD8−CD44+CD25+; DN3 cells were CD4−CD8−CD44−CD25+; and DN4 cells were CD4−CD8−CD44-CD25−. i, The frequencies of annexin V+ HSCs and MPPs in Rpl24Bst/+ versus littermate control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 3 experiments). j, Western blot analysis for Rpl24 and β-actin using 30,000 cells from each haematopoietic-cell population. Differences in β-actin between lanes represent differences in β-actin content per cell (one representative blot from two independent experiments). k, Donor- B-cell, T-cell and myeloid-cell engraftment when 5 × 105 donor bone marrow cells were transplanted along with 5 × 105 recipient bone marrow cells into irradiated recipient mice (n = 4 independent experiments with a total of 17 recipients for wild-type cells and 20 for Rpl24Bst/+ cells). These transplant recipients are the same as those shown in Fig. 4e. l, The frequency of donor cells in the bone marrow 20 h after transplanting 1 × 105 donor LSK cells from Rpl24Bst/+ or wild-type control mice into irradiated recipient mice (n = 3 recipients per donor). The horizontal line represents the level of background detected in an untransplanted control. m, Western blot analysis for p53 using 5 × 105 Lineage− bone marrow cells from wild-type or Rpl24Bst/+ mice, or 5 × 105 bone marrow cells from a p53−/− mouse (one representative blot from two experiments). n, Western blot analysis for p21Cip1 using 285,000 LSK cells from the bone marrow of adult wild-type or Rpl24Bst/+ mice, or 142,500 LSK cells from a wild-type mouse that received 540 rad of total body irradiation 3 to 4 h before being euthanized (one representative blot from three independent experiments). All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).
Extended Data Figure 6 Rpl24Bst/+ and Pten-deficient progenitors form colonies with normal cellularity but Rpl24Bst/+ impairs the development of haematopoietic neoplasms after Pten deletion.
a, b, The percentage of HSCs (a) or bone marrow cells (b) that formed colonies in methylcellulose within 14 days of culture (n = 3 mice per genotype in 3 independent experiments with 16 HSCs or 3,200 bone marrow cells tested per mouse per experiment). c, d, The average number of cells per granulocyte-monocyte (GM) or granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colony derived from single HSCs (c) or bone marrow cells plated at clonal density (d) (n = 4 independent experiments). e, The average number of cells per GM or GEMM colony derived from individual HSCs of the indicated genotypes 15 days after plating (n = 2 independent experiments). f, Frequency of annexin V+ CD4+CD8+ thymocytes (n = 5 independent experiments). g, Representative histograms of OP-Puro fluorescence in HSCs of the indicated genotypes. h, Mass of spleens and thymuses 2 weeks after pIpC administration (n = 7 independent experiments). i, Representative photographs of thymuses 2 weeks after pIpC administration to wild-type, Mx1-Cre; Ptenfl/fl, Rpl24Bst/+ and Mx1-Cre; Ptenfl/fl; Rpl24Bst/+ mice. j, HSCs in the spleen 2 weeks after pIpC administration (n = 7 independent experiments). k, Haematoxylin and eosin stained spleen and thymus sections from mice 2 weeks after pIpC administration or when they were killed owing to illness. All data represent mean ± s.d. In a–d and f, two-tailed Student’s t-tests were used to assess statistical significance relative to wild-type; *P < 0.05, **P < 0.01. To assess statistical significance in e we performed a one-way ANOVA followed by Tukey’s t-tests for multiple comparisons (relative to wild-type, *P < 0.05; ***P < 0.001; and relative to p53+/− , †P < 0.05, ††P < 0.01). To compare the statistical significance of differences among genotypes in h and j we performed a one-way ANOVA followed by Dunnett’s test for multiple comparisons relative to Pten-deficient (**P < 0.01, ***P < 0.001).
Extended Data Figure 7 Rpl24Bst/+ and Pten-deficient MPPs have relatively normal reconstituting activity.
a, b, 100 donor CD150−CD48−LSK MPPs from Mx1-Cre; Ptenfl/fl versus control mice (a; n = 3 independent experiments with a total of 13 recipients per genotype) or Rpl24Bst/+ versus control mice (b; n = 3 independent experiments with a total of 14 recipients of wild-type cells and 13 recipients of Rpl24Bst/+ cells) were transplanted along with 3 × 105 recipient-type bone marrow cells into irradiated recipient mice. Donor-cell engraftment levels in the peripheral blood were assessed at 3, 5 and 7 weeks after transplantation. All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance relative to wild-type; *P < 0.05.
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Signer, R., Magee, J., Salic, A. et al. Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014). https://doi.org/10.1038/nature13035
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