MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output

Abstract

The production of blood cells depends on a rare hematopoietic stem-cell (HSC) population, but the molecular mechanisms underlying HSC biology remain incompletely understood. Here, we identify a subset of microRNAs (miRNAs) that is enriched in HSCs compared with other bone-marrow cells. An in vivo gain-of-function screen found that three of these miRNAs conferred a competitive advantage to engrafting hematopoietic cells, whereas other HSC miRNAs attenuated production of blood cells. Overexpression of the most advantageous miRNA, miR-125b, caused a dose-dependent myeloproliferative disorder that progressed to a lethal myeloid leukemia in mice and also enhanced hematopoietic engraftment in human immune system mice. Our study identifies an evolutionarily conserved subset of miRNAs that is expressed in HSCs and functions to modulate hematopoietic output.

Fig. 1.

Identification of miRNAs enriched in HSCs. (A) Microarray experiment comparing miRNA expression in the LKS compartment (FACS plot in Upper and subset boxed in red) with total bone marrow from C57BL6 mice. Number of miRNAs significantly enriched in total BM vs. the LKS compartment, unchanged between the two groups, or significantly enriched in LKS vs. total BM cells are shown (Lower). BM cells were pooled from 10 mice and sorted, and RNA was extracted for the microarray. (B) qPCR was used to determine the fold enrichment of the 11 miRNAs preferentially expressed in LKS cells vs. total BM. Data represent two independent experiments. (C) Expression levels of the 11 HSPC miRNAs were measured in hematopoietic cells from the different bone-marrow compartments shown in Fig. S1 and represent different stages of blood-cell development. (D) LKS-gated cells were further sorted by FACS into three groups according to their expression of the signaling lymphocytic activation molecule (SLAM) markers CD150 and CD48. Total RNA was collected from each group, and the relative expression levels of miRNAs from each LKS subpopulation were assayed by qPCR. Data represent two independent experiments, each starting with pooled BM from 20 mice. All data were normalized to sno202 and represent the mean + SEM.

Fig. 2.

Gain-of-function approach to assess the impact of HSC miRNAs on long-term hematopoietic reconstitution. (A) HSC miRNAs were cloned into an MSCV-based retroviral vector (MG) using an miR-155 arms and loop format. (B) Competitive BM reconstitutions were performed using C57BL6 mice to assess the engraftment potential of BM expressing each of the 11 HSC miRNAs (CD45.1 cells) compared with control BM (CD45.2 cells). GFP was used to identify cells containing a vector. A 4-mo time course showing the percentage of CD45.1+GFP+ cells in the peripheral blood of each group of mice is shown. The experiment was broken into two batches (Upper and Lower), each with its own negative control. (C) Lineage analysis of the CD45.1+GFP+ peripheral blood cells at the 4-mo time point was assessed by determining the percentages of B220+ (B cell), CD3+ (T cell), and CD11b+ (myeloid cell) in each group. Data represent the mean + SEM. Experiments had four mice per group.

Fig. 3.

miR-125b causes a dose-dependent MPD. (A) The three constructs used to enforce expression of miR-125b are shown. K562 cells were transduced with each retrovector, and miR-125b levels were assayed by qPCR. (B) Blood concentrations of the indicated cell types are shown for mice expressing MG, MG-125b1, MG-125b2, and in a separate experiment (separated by a dashed line), MG and MG-125b(f) 2-mo postreconstitution. (C) FACS plot showing a representative FACS plot of Gr1+CD11b+ cells in the peripheral blood of mice overexpressing miR-125b2, miR-125b1, or control vector 2 mo postreconstitution. Asterisk denotes a P value < 0.05.

Fig. 4.

MPDs caused by miR-125b progress to myeloid leukemia in a dose-dependent manner. (A) Survival of mice expressing different levels of miR-125b. (B) Liver and spleen from miR-125b2–expressing mice with leukemia 4.5 mo after reconstitution. Representative H&E-stained tissue sections of each respective organ taken from miR-125b2–expressing mice are shown on Right. (C) Percentage of leukemic blasts in the peripheral blood of control or miR-125b2–expressing mice 3.5 and 4.5 mo postreconstitution, and representative Wright-stained blood smears from miR-125b2 mice at each time point. Examples of blasts are indicated by red arrows. (D) Survival of mice after i.v. transfer of malignant MG-125b1 (four different donors) or MG control bone marrow to Rag2^−/−γc^−/− recipients (n = 3–6 mice per group). Asterisk denotes a P value < 0.05.

Fig. 5.

Evolutionarily conserved miRNA expression and function in human CD34+ HSPCs. (A) Human CB was sorted into CD34+ (HSPC+) and CD34− (HSPC−) fractions. Expression of the miRNAs enriched in mouse HSCs was measured by qPCR. Data are normalized to RNU48 and presented as mean + SEM (n = 3 different donors). (B) Human CB CD34+ cells were transduced with control or miR-125b1–expressing lentiviral vectors at an MOI of ~4 or 40, and miR-125b levels were assayed by qPCR. (C) Representative FACS plots showing an increased ratio of human to mouse CD45+ WBCs in the peripheral blood of miR-125b–expressing HIS mice 10 wk after CD34+ cell injection. (D) A gray dot represents each mouse. (E) Human cells in the peripheral blood of high-dose (MOI 40) HIS mice were analyzed by FACS for the expression of different lineage markers including CD19 (B cells) and CD33 (myeloid). Data are represented as the mean ± SEM. (F) A representative FACS plot of hCD45+ and hCD34+ cells in the BM of miR-125b–expressing and control vector HIS mice 12 wk post-CD34+ injection. (G) A gray dot represents each mouse. Asterisk denotes a P value < 0.05.