MicroRNA miR-125a controls hematopoietic stem cell number - PubMed (original) (raw)

. 2010 Aug 10;107(32):14229-34.

doi: 10.1073/pnas.0913574107. Epub 2010 Jul 8.

Jun Lu, Rita Schlanger, Hao Zhang, Judy Y Wang, Michelle C Fox, Louise E Purton, Heather H Fleming, Bradley Cobb, Matthias Merkenschlager, Todd R Golub, David T Scadden

Affiliations

MicroRNA miR-125a controls hematopoietic stem cell number

Shangqin Guo et al. Proc Natl Acad Sci U S A. 2010.

Abstract

MicroRNAs influence hematopoietic differentiation, but little is known about their effects on the stem cell state. Here, we report that the microRNA processing enzyme Dicer is essential for stem cell persistence in vivo and a specific microRNA, miR-125a, controls the size of the stem cell population by regulating hematopoietic stem/progenitor cell (HSPC) apoptosis. Conditional deletion of Dicer revealed an absolute dependence for the multipotent HSPC population in a cell-autonomous manner, with increased HSPC apoptosis in mutant animals. An evolutionarily conserved microRNA cluster containing miR-99b, let-7e, and miR-125a was preferentially expressed in long-term hematopoietic stem cells. MicroRNA miR-125a alone was capable of increasing the number of hematopoietic stem cells in vivo by more than 8-fold. This result was accomplished through a differentiation stage-specific reduction of apoptosis in immature hematopoietic progenitors, possibly through targeting multiple proapoptotic genes. Bak1 was directly down-regulated by miR-125a and expression of a 3'UTR-less Bak1 blocked miR-125a-induced hematopoietic expansion in vivo. These data demonstrate cell-state-specific regulation by microRNA and identify a unique microRNA functioning to regulate the stem cell pool size.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: A patent has been filed on data presented in this article. D.T.S. is a founder, consultant, and stockholder in a company focused on stem cell research, Fate Therapeutics.

Figures

Fig. 1.

Fig. 1.

Dicer deletion abolishes functional and immuno-phenotypic HSPCs. (A) Peripheral blood chimerism by control and mutant BM in a 1:1 competitive transplantation assay. The seven arrows indicate pIpC injections. Each dot on the line indicates the average donor-type cell percentage (%CD45.2+) at the indicated time points (d, days; w, weeks after pIpC injection). n = 15. (B) Lineage contribution by donor-type cells 20 wk after pIpC injections. Lineages analyzed include myeloid (Mac-1+), B (B220+), and T cells (CD3+). Error bars indicate SD. *P < 0.05. (C) Representative FACS plot showing donor-type LKS cells in recipient BM 6 mo post-pIpC injections. (D) Intracellular flow cytometry for activated caspase-3 in three BM populations including the Lin-c−Kit+Sca+ (LKS), Lin-c−Kit+Sca− (L-K+S-), and Lin-c−Kit−Sca+ (L-K-S+) cells.

Fig. 2.

Fig. 2.

MiR-99b-let-7e-miR-125a cluster expression enhances long-term multilineage reconstitution. (A) Heatmap of microRNA expression profiles of hematopoietic cells. Population designation: Sca+, Lin-c−Kit−Sca+; BM, whole BM; K, Lin-c−Kit+Sca-; LKS, Lin-c−Kit+Sca+; LT, Lin-c−Kit+Sca+CD34-Flk2-; MPP, Lin-c−Kit+Sca+CD34+Flk2+; and ST, Lin-c−Kit+Sca+CD34−Flk2+. Each column represents an independent sample. Populations were sorted from pooled BM cells from multiple animals on multiple days. Red color indicates higher expression; blue for lower. (B) Bar graph of data in A for miR-125a, miR-99b, and let-7e. (C) Primed wild-type donor marrow (5FU) was transduced with retrovirus expressing either control vector or miR-99b-let-7e-miR-125a (cluster) in addition to GFP. Contribution to peripheral blood by control or cluster-transduced BM cells (GFP+) at indicated time is shown. The first time-point indicates GFP+% in the culture 2 d posttransduction. wks: weeks; mths: months. *P < 0.05. (D) Multilineage differentiation into myeloid (Mac-1+), B (B220+), and T lineages (CD3+) among GFP+ cells 5 mo posttransplantation. C and D: n = 4–7 each. Error bars indicate SD.

Fig. 3.

Fig. 3.

miR-125a enhances HSC function. The miR-99b-let-7e-miR-125a cluster or individual miRNA was transduced and transplanted as in Fig 2C. Peripheral blood contribution by transduced BM cells was analyzed 4 mo posttransplantation. (A) Representative FACS plots. (B) Quantification of data shown in A. (C) Comparison of contribution to blood formation by control or miR-125a alone (125a). The first time point (3 d) indicates GFP+% in the culture posttransduction. (D) Multilineage differentiation by transduced BM cells. For B to D, n = 1–5 per group per time point shown. (E) Contribution to blood formation by transduced BM cells that were cultured ex vivo for 18 d. n = 4 for control (Ctrl) and n = 5 for miR-125a (125a). *P < 0.05. (F) Serial methylcellulose colony formation in the presence of a control or miR-125a-specific antagomir. Representative fields from primary (10) and secondary (20) cultures. (G and H) Purified HSPCs were transduced with control or miR-125a and transplanted. Peripheral blood contribution in recipients was quantified. See

SI Materials and Methods

for population definition. Each animal received either (H) 100 SLAM, (G) 1,000 LKS, or 10,000 progenitors together with 2.5 × 105 supporting BM cells. n = 5 except for miR-125a–transduced CMP (n = 4). Error bars reflect SD.

Fig. 4.

Fig. 4.

MiR-125a inhibits immature hematopoietic cell apoptosis and targets Bak1. (A) Selective protection against apoptosis by miR-125a in lineage-negative cells. BM cells were analyzed for lineage markers (Lin), AnnexinV, and 7-AAD with flow cytometry. AnnexinV histograms show Lin− and Lin+ populations after gating for 7-AAD− cells. Lin− population is defined as the lowest 3% cells expressing lineage markers. n = 5. (B) Quantification of data in A. Percentage of AnnexinV+ (AV+) cells present in 7AAD−Lin−GFP+ cells are shown. n = 5. *P < 0.05. (C) HL-60 cells were transduced with miR-125a or a control vector (Ctrl). Western blot was probed for Bak1 and β-actin. (D) Luciferase reporters of WT (wild-type) or Mut (mutant for miR-125a site) Bak1 3′UTR was analyzed in the presence of miR-125a or a control vector in 293T cells. Normalized luciferase activities are shown. Error bars represent SD. (E and F) Primed wild-type donor marrow (5FU) was cotransduced with miR-125a (DsRedExpress+) and a virus for either control or Bak1 (GFP+). Cells were (E) cultured in vitro for 6 d or (F) transplanted (n = 4) and analyzed 4 wk afterward. The percentage of cotransduced cells (GFP+DsRedExpress+) in culture or in peripheral blood is shown. *P < 0.05.

References

    1. Cozzio A, et al. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors. Genes Dev. 2003;17:3029–3035. - PMC - PubMed
    1. Krivtsov AV, et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006;442:818–822. - PubMed
    1. Park IK, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003;423:302–305. - PubMed
    1. Yilmaz OH, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006;441:475–482. - PubMed
    1. Zhang J, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature. 2006;441:518–522. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources