Cell-intrinsic in vivo requirement for the E47-p21 pathway in long-term hematopoietic stem cells

Abstract

Major regulators of long-term hematopoietic stem cell (LT-HSC) self-renewal and proliferation have been identified, but knowledge of their in vivo interaction in a linear pathway is lacking. In this study, we show a direct genetic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC integrity in vivo under repopulation stress. Numerous studies have shown that E47 activates p21 transcription in hematopoietic subsets in vitro, and we now reveal the in vivo relevance of the E47-p21 pathway by reducing the gene dose of each factor individually (E47(het) or p21(het)) versus in tandem (E47(het)p21(het)). E47(het)p21(het) LT-HSCs and downstream short-term hematopoietic stem cells exhibit hyperproliferation and preferential susceptibility to mitotoxin compared to wild-type or single haploinsufficient controls. In serial adoptive transfers that rigorously challenge self-renewal, E47(het)p21(het) LT-HSCs dramatically and progressively decline, indicating the importance of cell-intrinsic E47-p21 in preserving LT-HSCs under stress. Transient numeric recovery of downstream short-term hematopoietic stem cells enabled the production of functionally competent myeloid but not lymphoid cells, as common lymphoid progenitors were decreased, and peripheral lymphocytes were virtually ablated. Thus, we demonstrate a developmental compartment-specific and lineage-specific requirement for the E47-p21 pathway in maintaining LT-HSCs, B cells, and T cells under hematopoietic repopulation stress in vivo.

Figure 1. Increased homeostatic proliferation in E47^hetp21^hetLT-HSCs in vivo

(A) BM from WT, E47^HET, p21^HET or E47^HETp21^HET mice was isolated and stained to resolve specific subsets of progenitor cells. Right panel, gating strategy used to identify LSKs (Lineage⁻c-kit⁺ Sca-1⁺), phenotypic LT-HSCs were identified as CD150⁺ flk2⁻LSK or CD150⁺ CD48⁻LSK, short-term HSCs (ST-HSC) as CD150⁻flk2⁻ LSK, total HSCs as flk2⁻ LSK and multipotent progenitors (MPP) as flk2⁺ LSK. Left panel, common lymphoid progenitors (CLP) were identified as Lin⁻ IL7R⁺ AA4.1⁺ Sca-1^low. (B) RNA extracted from sorted total LSKs was used to examine E47 and p21 transcript levels. Gene expression was normalized to β-actin and expression level of each gene is shown relative to WT LSK. Data are shown as mean ± SD of triplicates from two independent sorts. (C &D) WT, E47^het, p21^het or E47^hetp21^het mice were injected i.p. with 200 μL of 3 mg/mL BrdU twice a day for 2 days then sacrificed to examine proliferation status. (C) BM was either stained directly to identify total HSC (flk2⁻LSK) and MPP (flk2⁺ LSK); or was enriched for HSCs via depletion of Lineage⁺ cells followed by surface marker staining to identify LT-HSC (CD150⁺ flk2⁻ LSK) and ST-HSC (CD150⁻flk2⁻ LSK). Cells were then fixed and permeabilized followed by intracellular staining with anti-BrdU. Flow profiles shown are from one of 4–5 representative experiments. Grey histograms indicate PBS stained control. (D) Bar graph represents mean ± SD of data pooled from n=4–6 mice per genotype. (E) BM from WT, E47^het, p21^het or E47^hetp21^het mice was stained to enumerate LSK, total HSC, MPP or CLP numbers. Data represents 4–9 mice per genotype. *p<0.05

Figure 2. Response of E47^hetp21^het progenitors to two different hematopoietic stressors

(A) WT, E47^het, p21^het or E47^hetp21^het mice were injected i.p. with 150 mg/kg 5-FU and sacrificed after 16 hours. BM cells were stained to determine total HSC (flk2⁻LSK) or MPP (flk2⁺ LSK) numbers. Data is shown as mean ± SD of cell number relative to WT, n=4–8 mice per group. (B & C) Mice were treated with either PBS or 30 μg total LPS over 2 days and sacrificed 24 hours after last treatment. BM (B) or PB (C) cells were stained to identify mature (CD11b⁺ Gr-1^hi) or immature granulocytes (CD11b⁺ Gr-1^int). Data shown is representative of two independent experiments. *p<0.05

Figure 3. E47^hetp21^het LT-HSCs exhibit decreased self-renewal and persistence in vivo

(A) Serial transplantation was performed by transferring 2 × 10⁶ BM cells from CD45.2⁺ WT, E47^het, p21^het or E47^hetp21^het mice into sub-lethally irradiated CD45.1⁺ C57BL/6 recipient mice to examine LT-HSC self-renewal and persistence through three rounds of serial transfer. (B) Mice were sacrificed at sixteen weeks post-transplant and the number of donor-derived BM LT-HSCs (CD45.2⁺ CD150⁺ CD48⁻ LSK or CD45.2⁺ CD150⁺ flk2⁻ LSK) was enumerated in primary, secondary or tertiary recipients. Graph is shown as mean ± SD of data pooled from n= 4–8 recipient mice for each genotype. (C) Mice were sacrificed two weeks after primary transplantation and the frequency of CD45.2⁺ total HSCs was examined. Data is shown as mean ± SD from n= 2–3 recipient mice per group. *p<0.05

Figure 4. E47^hetp21^het LT-HSCs display hyperproliferation following transplantation stress

Serial transplantation was performed as described in Figure 3. Secondary recipient mice were transplanted with 2 × 10⁶ BM cells using primary recipient mice as donors. Sixteen weeks after transplantation, mice were sacrificed and BM cells stained to identify donor-derived total HSCs (CD45.2⁺ flk2⁻ LSK) and MPPs (CD45.2⁺ flk2⁺ LSK) followed by anti-BrdU staining to examine proliferation status (A) Flow cytometry profiles from one experiment representative of 3 experiments are shown. Shaded grey histograms indicate PBS stained control. (B) Bar graph represents mean ± SD of data from n=3–5 recipient mice per donor genotype. *p<0.05

Figure 5. E47^hetp21^het LT-HSCs display progressive in vivo decrease in lymphoid lineage reconstitution accompanied by normal myeloid lineage reconstitution

Serial transplantation was performed in Figure 3. Sixteen weeks after transplantation donor-derived cells were identified from spleen or peripheral blood (PB). (A) Left panel, gating strategy used to identify donor-derived B (CD45.2⁺ CD19⁺), T (CD45.2⁺ CD3⁺) and myeloid lineage (CD45.2⁺CD11b⁺) cells in spleen. Right panel, gating strategy used to identify donor-derived B (CD45.2⁺ CD19⁺), T (CD45.2⁺ CD3⁺) and myeloid lineage (CD45.2⁺ Gr-1⁺) cells in PB. Donor-derived reconstitution of lymphoid and myeloid lineage was examined in (B) spleen and (C) peripheral blood in BM recipients sixteen weeks after each round of transplantation. Graphs are shown as mean ± SD of data from n= 2–9 mice per recipient for each donor genotype. (D) Sixteen weeks after transplant, presence of donor-derived ST-HSC, MPP, LMPP and CLP in BM of secondary recipients was examined. Data are shown as mean ± SD from n= 5–9 mice per donor genotype. *p<0.05

Figure 6. Gene expression analysis in E47^hetp21^het HSCs

At day 14 after a single 5-FU i.v. injection, total HSCs (flk2⁻ LSK) were sorted from WT, E47^het, p21^het or E47^hetp21^het mice. RNA was extracted and cDNA was generated via RT-PCR and used to examine expression levels of cdk6, p18, and ikaros1 using quantitative real-time PCR (qPCR). Gene expression was normalized to β-actin. Data are shown as mean ± SD of triplicates from at least two independent sorts. *p<0.05