A non-redundant function of cyclin E1 in hematopoietic stem cells

Abstract

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.

Figure 1. Analysis of progenitors and primitive hematopoietic cells derived from cyclin E mice of mixed background. (A) LTC-IC assay performed with 250 000 BMCs isolated from 3 independent mice of the indicated genotypes. The graph reports the average number of colonies scored for the appropriate genotypes, ± stdv. (B) Representative pictures of the colonies obtained in an LTC-IC assay using BMCs derived from Ccne1^+/+ or Ccne1^−/− mice. Scale bar is 400 µm. (C) CAFC assay; 2 independent experiments are shown performed on Ccne1^+/+ or Ccne1^−/− derived bone marrow mononucleated cells (BMCs). Cobblestone areas were assessed after 5 wk of culture on stromal feeder layers. (D) CFU assay performed with 20 000 bone marrow mononucleated cells (BMCs) isolated from 3 independent mice of the indicated genotypes. Colonies were counted after 10 d of culture. The graph represents the average number of colonies scored. The error bar indicates the standard deviation (stdv). (E) FACS analysis of common lymphoid progenitors (CLP: Lin⁻, ILR7⁺, Kit^low, and Sca1^low) and common myeloid progenitors (CMP: Lin⁻, ILR7⁻, FcγRII/III^Low, Kit⁺, and Sca1⁻) in Ccne1 mice. Bar graphs are the average frequencies ± stdv.

Figure 2. FACS analysis of HSCs derived from Ccne1^−/− mice. (A) FACS analysis of bone marrow HSCs subpopulations belonging to the Lin⁻, Sca1⁺, Kit⁺ (LSK) population by using the Flk2 and CD34 surface markers. MP, myeloid progenitors (Lin⁻, Sca⁻ and Kit⁺); MPP, multipotential precursors (LSK, Flk2⁺, CD34⁺); ST, short-term HSCs (LSK, Flk2⁻, CD34⁺); LT, long-term HSCs (LSK, Flk2⁻, CD34⁻). (B) HSC analysis using the SLAM markers CD48 and CD150 to enrich in “dormant” HSCs (LSK, CD34⁻, CD48⁻ and CD150⁺). (C) Cell cycle analysis (Ki67/Hoechst) of the different HSC populations and progenitors. Five mice for each genotype were used in the experiments described in panels (A–C).

Figure 3. Cyclin E1 regulates HSCs following myeloablation. (A) Prospective identification of HSCs and progenitors in Ccne1 mice by FACS analysis of bone marrow mononucleated cells (BMCs) isolated from Ccne1 mice 24 h following 5-FU administration. On the left, FACS profiles of Lin⁻ cells stained with c-Kit and Sca1 are shown. The red box outlines the gating for the LSK cells used to define LT-HSC, ST-HSC and MPP (as described to in Fig. 2). Frequencies of the different LSK cells are represented in the bar graph on the right. The bar graph represents the average values relative to the different LSK population of cells; error bars are the standard deviation, n = 7. (B) Assessment of BMCs, at 1 d post-5-FU treatment (n = 7). Values are the average of the total count of cells isolated from 2 tibias and 2 femurs/mouse. (C) Cell cycle distribution by FACS analysis of the different HSC/progenitor populations of cells as shown in (A), stained for the proliferation marker Ki67 and the DNA dye Hoechst. (D) Analysis of quiescence (i.e., percentage of cells in G₀) based on the FACS profiles shown in (C). Bar graph is the average percentage of quiescent cells ± stdv, n = 7. (E) Analysis of the kinetics of accumulation of the LSK^low following a single injection of 5-FU. FACS profiles of Lin⁻ cells stained with c-Kit and Sca1 are shown on the left; the red box highlights the LSK^low gate used. Quantitation of LSK^low cells is shown on the right as average cells ± stdv, n = 3. (F) Assessment of BMCs 4 d post 5-FU treatment (n = 4). Values are the average of the total count of cells isolated from 2 tibias and 2 femurs/mouse. (G) A cohort of Ccne1^+/+ or Ccne1^−/− mice (n = 6) was injected with 5-FU (200 mg/Kg) at day 0. Peripheral white blood cells (WBCs) were counted at defined days; data were normalized to values measured before 5-FU treatment and plotted as average ± stdv. The count of white blood cells at day 14 is also reported in the inset. (H) Kaplan–Mayer survival curves of different cohorts of mice of the indicated genotypes subjected to 5-FU injections (150 mg/Kg) every 7 d, as indicated by the arrows. Ccne1^−/− = 8, Ccne1^+/+ = 5, and Ccne1^+/− = 4.

Figure 4. Cyclin E1 regulates quiescence of aged HSCs. (A) Prospective identification of HSCs and progenitors in cyclin E1 mice of C57/Bl6 background by FACS analysis. Bone marrow mononucleated cells (BMCs) were isolated from Ccne1^+/+ (n = 10) or Ccne1^−/− (n = 6) mice aged for 23 mo. Bar graphs report the average value, error bars are the standard deviation. HSCs, LSK (Lin⁻, Sca1⁺, Kit⁺); MPP, multipotential precursors, (LSK, Flk2⁺); LT/ST, short-term and long-term HSCs (LSK, Flk2⁻). (B) Cell cycle analysis of HSCs and myeloid progenitors (MP), cell cycle distribution was determined by FACS based on the Hoechst/Ki67 staining of the different cell populations. (C) FACS profiles of the analysis shown in (B)

Figure 5. Serial bone marrow transplants of Ccne1^+/+ or Ccne1^−/− BMCs (A) Ly5.2 BMCs from Ccne1^+/+ or Ccne1^−/− mice were transplanted in a 1:1 ratio with competitor cells derived from Ly5.1 C57/Bl6 mice into Ly5.2/Ly5.1 recipient mice. After long-term reconstitution (5 months), peripheral blood chimerism was assessed by FACS. BMCs derived from primary recipients were then transplanted into secondary recipients. The process was repeated until the 4th bone marrow transplant. Data were plotted as averages of the Ly5.2⁺ peripheral blood nucleated cells normalized to the total cells transplanted (Ly5.1⁺ + Ly5.2⁺ cells). Numbers within graph bars indicate the number of recipients used in each transplant. (B) GFP⁺ BMCs from Ccne1^+/+ or Ccne1^−/− mice were transplanted in a 1:1 ratio with competitor BMCs from C57/Bl6 mice into C57/129 F1 hybrids. The experiment was performed as in (A). After long-term reconstitution (5 mo) GFP⁺ cells were determined in peripheral blood by FACS analysis and reported as average percentage ± standard deviation. Numbers within graph bars indicate the number of recipients used in each transplant.