Regulation of hematopoietic stem cells by their mature progeny

Abstract

Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb(Plt4/Plt4) mice show altered expression of TPO-responsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.

Fig. 1.

Deficiencies in the TPO/Mpl pathway in Myb^Plt4/Plt4 mice. (A) Platelet counts and (B) circulating serum TPO levels in 8- to 12-wk-old mice of each genotype; TP indicates data from mice 16 wk after bone marrow transplant with bone-marrow donors as indicated. n = 6–21 mice per genotype. (C) Mpl expression measured by quantitative real time RT-PCR in cell populations purified by flow cytometry. Data shown are expression of Mpl relative to Hprt expression, measured by the ΔΔCt method. n = 5–8 mice per genotype. (D) Thymidine incorporation in lineage-negative (Lin⁻) cells from mice of the indicated genotypes after 4 d of culture in TPO. n = 5 for Mpl^+/+, n = 2 for Mpl^−/− mice. Data are shown as means ± SDs. ***P < 0.001; **P < 0.01; *P < 0.05; and ns, not significant, for comparisons between Mpl^+/+ Myb^+/+ and Mpl^+/+ Myb^Plt4/Plt4 data.

Fig. 2.

Myb^Plt4/Plt4 mice have more prospective ST-HSCs and MPPs. (A) Representative flow cytometric analyses of viable Lin⁻ cells (Upper) and LSK cells (Lower) from bone marrow of mice of the indicated genotypes. The gates used to distinguish prospective LT-HSCs (CD34⁻ Flt3⁻ LSK), ST-HSCs (CD34⁺ Flt3⁻ LSK), and MPPs (CD34⁺ Flt3⁺ LSK) are shown. Percentages of gated cell populations within the parent populations are indicated. (B) Graphs show mean ± SD of the number per femur of total LSKs and LSK subsets. n = 5–6 mice per genotype. ***P < 0.001; ns, non significant.

Fig. 3.

Common gene-expression signature in Myb/p300 deficient and Mpl^−/− LSK cells. (A) Heat map visualization of genes that are differentially expressed in Myb^Plt4/Plt4, p300^Plt6/Plt6, and Mpl^−/− LSK cells in the same direction (either up or down) compared with wild type (P < 0.05). Genes are distributed horizontally and grouped vertically according to genotype. The scale represents log₂-fold change differences for each mutant compared with wild type. (B) Quantitative real-time RT-PCR of gene expression in MPPs (black bars), ST-HSCs (dark gray bars), and LT-HSCs (light gray bars) purified by flow cytometry from Myb^+/+ mice (^+/+) and Myb^Plt4/Plt4 mice (Plt4/Plt4). Expression is presented as relative to that in Myb^+/+ ST-HCS, set as 1. Data, expressed as mean and SD, represent one experiment with two to five independent cDNA samples. CD34 and Flt3 were used to define the populations, but not shown to be differentially expressed on the microarrays. All other genes were identified as down-regulated by microarray analysis. Genes that were unable to be detected as expressed in a particular subset are marked with #. ***P < 0.001; *P < 0.05; ns, not significant in comparison with Myb^+/+ to Myb^Plt4/Plt4. (C) Hematopoietic expression pattern of genes selected as down-regulated in Myb^Plt4/Plt4, p300^Plt6/Plt6 and Mpl^−/− LSK cells. The scale represents normalized expression across the cell types for each gene. Cell types are murine, as defined in Chambers et al. (33), and sourced from http://franklin.imgen.bcm.tmc.edu/loligag/, where SP HSC is side population LSK, N Ery, nucleated erythrocyte; Gran, granulocytes; Mono, monocytes; T CD8 A and T CD4 A, activated T cell; T CD8 N and T CD4 N, naive T cell; NK Cell, natural killer cell.

Fig. 4.

Cycling is enhanced in Myb^Plt4/Plt4 cells. (A) Representative flow cytometric analyses of bone marrow from wild-type, Mpl^−/−, Myb^Plt4/Plt4, and TPO^Tg mice showing LSK and Lin-S-K⁺ gates (Upper) and K_i-67/DAPI staining profiles for LSK cells (Lower). (B) Mean percentages of LSK cells that are in G0, G1, or S-M phase of the cell cycle. n = 4–8 mice of each genotype. (C) Relative expression of Mpl in hematopoietic progenitor populations as determined by qRT-PCR. Expression is relative to Hprt expression as determined by the ΔΔCt method. n = 3 independent samples per genotype. (D) Mean percentages of Lin⁻ Sca-1⁻ c-Kit⁺ cells that are in G0, G1, or S-M phase. n = 6–10 mice of each genotype. Data shown are mean ± SD. ***P < 0.001; ns, not significant.

Fig. 5.

Age-related deficits in Myb^Plt4/Plt4 bone marrow. Counts of (A) LSK cells per femur and (B) LT-HSCs per femur from Myb^Plt4/Plt4 and Myb^+/+ mice at 7 wk, 9 mo, and 13 mo of age. (C) Ratio of the number of bone marrow-derived colony-forming cells in Myb^Plt4/Plt4 and Myb^+/+ mice at 13 mo vs. 7 to 10 wk of age. Bone-marrow cells were cultured in SCF/IL3/EPO. Meg, megakaryocyte; G/M/Eo, Granulocyte/Macrophage/Eosinophil. Data shown are mean ± SD. n = 3–9 mice per genotype and age. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.