Meis1 preserves hematopoietic stem cells in mice by limiting oxidative stress

Abstract

The transcription factor Meis1 is expressed preferentially in hematopoietic stem cells (HSCs) and overexpressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. In the present study, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over the long term, whereas steady-state hematopoiesis was sustained in the absence of Meis1 in inducible knock-out mice. BM cells of Meis1-deficient mice showed reduced colony formation and contained significantly fewer numbers of long-term HSCs, which exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species in HSCs and decreased expression of genes implicated in hypoxia response. Finally, reactive oxygen species scavenging by N-acetyl cysteine or stabilization of hypoxia signaling by knockdown of the von-Hippel-Lindau (VHL) protein led to reversal of the effects of Meis1 deletion. The results of the present study demonstrate that Meis1 protects and preserves HSCs by restricting oxidative metabolism.

Figure 1

Meis1 deletion leads to loss of LT-HSCs. Meis1-flox/CreER or control mice were treated with tamoxifen as described in the text. (A) Representative flow cytometry plots of CD48 and CD150 staining (gated on LSK fraction) showing significantly fewer LT-HSCs in the BMs of Meis1-deleted mice compared with controls. Bar graph depicts mean ± SEM of the percentage of LT-HSCs in lineage-depleted BM of control and Meis1-deleted mice (n = 7). (B) Bar graph representing colony numbers in methylcellulose medium. Data are shown as means ± SEM of total colony numbers/10 000 cells (n = 4). (C) Line graphs of blood counts (vertical axis) over time (horizontal axis) since tamoxifen treatment. Identities of the various groups are indicated in the legend on the right, with 5 mice in each group. Also shown is a representative gel image of PCR on tail blood 12 weeks after vehicle or tamoxifen treatment. (D) Transplantation experiment demonstrating that the loss of LT-HSCs with Meis1 deletion was cell autonomous. Flow plots are as in panel A, but depict donor LT-HSCs (recipients were lethally irradiated). The bar graph shows peripheral blood chimerism in recipients.

Figure 2

Meis1 deletion leads to loss of HSC function. (A) Meis1-flox/CreER or control mice were treated with tamoxifen, as described in the text, and transplanted into lethally irradiated CD45.1 mice. (B) Representative flow cytometry plots on the left depict peripheral blood chimerism at 4 weeks after transplantation. Representative flow cytometry plots on the right depict the percentages of LSKs and LT-HSCs in the BM of transplantation recipients at 4 months. Shown are donor cells. (C) Recipient mice were killed at 4 months and whole BM was transplanted into lethally irradiated secondary recipient mice. Shown are representative flow cytometry plots depicting chimerism in the BM of secondary recipients. The recipients of Meis1-deleted BM died of hematopoietic failure.

Figure 3

Meis1-deleted HSCs are deficient in maintenance of hematopoiesis. (A) Competitive repopulation assay was performed as described in the text. (B) Summary of peripheral blood CD45.2 chimerism (vertical axis) in recipient mice over time (horizontal axis) since transplantation. The groups are labeled to the right of the respective curve. Data are shown as means ± SEM (n = 5-8 per group). Also shown on the lower right is a representative PCR result on peripheral blood of recipient mice treated with vehicle (V) or tamoxifen (T). Bands labeled to the right represent, respectively, in numerical order floxed, wild-type, and deleted Meis1 alleles, showing that we achieved complete deletion of Meis1 by tamoxifen treatment. (C) Chimerism in the various lineages at 4 months. (D) Flow cytometry plots showing chimerism in the LT-HSC fraction of the BM of recipient mice at 4 months.

Figure 4

Meis1 deletion leads to loss of quiescence in LT-HSCs. (A) Meis1-flox/CreER and control mice were treated with tamoxifen and, 1 week later, BM was analyzed by flow cytometry for cell-cycle analysis. Flow cytometry plots depict results of pyronin Y (vertical axis, RNA stain) and Hoechst (horizontal axis, DNA stain) staining gated on the LSK and LT-HSC fractions. Left lower quadrant represents cells in G₀, left upper quadrant G₁, and upper right S phase. Data are summarized in the bar graph to the right (n = 2 control and 3 Meis1 deleted). (B) Mice were treated as in panel A and then injected with BrdU before being killed. Shown are representative histograms of anti-BrdU staining gated on the LT-HSCs, as defined previously. (C) Flow cytometry plots of annexin V and 7-amino-actinomycin D gated on the LSK fraction of control and Meis1-deleted mice. (D) Meis1-deleted and control mice were treated with weekly doses of 5-FU. Graph shows the percentage survival (vertical axis) over time (horizontal axis) with the horizontal-axis labels representing the dates of 5-FU injection.

Figure 5

Meis1 regulates oxidative stress. (A) Representative flow cytometry plot showing results of dichlorofluorescein diacetate staining (a measurement of ROS levels) gated on LT-HSCs. (B) Bar graph depicting results of colony-formation assay performed with Meis1-flox/CreER lineage-depleted cells that were treated in vitro with vehicle (ethanol) or 4-OHT in the presence of the ROS scavenger N-acetyl cysteine (NAC), 1 of 2 shRNA constructs directed against VHL (shVHL1 and shVHL2), scrambled shRNA, or cobalt chloride (treatment conditions labeled on the bottom of the graph). Colony numbers (vertical axis) were normalized to those formed by vehicle-treated cells in each experiment. Data represent means ± SEM of triplicate colonies from 1 representative experiment. (C) Lineage-negative BM cells of Meis1-flox/CreER and control mice were treated with 4-OHT for 48 hours and then analyzed by whole genome microarrays. Depicted are gene-set enrichment analysis plots of the analysis showing enrichment in the control cells compared with Meis1-deleted of gene sets associated with leukemias that express high levels of Meis1 (top panel) and those associated with hypoxia-response (bottom panel). The gene sets are labeled on the top of each plot. (D) Western blot (top panel) showing levels of Hif-1α and actin (loading control) in whole BM of control (first 2 lanes) and Meis1-deleted mice. Bottom panel depicts Western blot results of Hif-1α and actin in vehicle- or 4-OHT–treated cells transduced with lentivirus expressing shRNA against VHL or a nontargeting construct.

Figure 6

Negative feedback of Pbx1 by Meis1. (A) Western blot showing Meis1, Pbx1, and actin (loading control) levels in control and Meis1-deleted lineage-negative BM. (B) Bar graph depicting Meis1 and Pbx1 mRNA levels in control and Meis1-deleted, lineage-negative BM. mRNA levels were measured by real-time quantitative RT-PCR using TaqMan primer/probes and normalized to β-actin (n = 2). (C) Lineage-negative BM cells were harvested from tamoxifen-treated Meis1-flox/CreER (Meis1-deleted) and Rosa26CreER (control) mice. Cells were transduced with lentivirus expressing 1 of 2 Pbx1-shRNAs or a control construct, and transduced cells were selected by culture in puromycin for 72 hours. Colony assay was then performed as described in the text. Bar graph depicts means ± SEM of triplicate colony counts from one representative experiment.