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. 2015 Nov 25:6:8928.
doi: 10.1038/ncomms9928.

STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling

Nadine Haetscher  1   2 , Yonatan Feuermann  1   3 , Susanne Wingert  1   2 , Maike Rehage  1   2 , Frederic B Thalheimer  1   2 , Christian Weiser  2 , Hanibal Bohnenberger  4 , Klaus Jung  5 , Timm Schroeder  6 , Hubert Serve  1   7   8 , Thomas Oellerich  1   7   8 , Lothar Hennighausen  3 , Michael A Rieger  1   2   7   8
Affiliations

STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling

Nadine Haetscher et al. Nat Commun. .

Abstract

Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b(-/-) mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.

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Figures

Figure 1
Figure 1. In vivo expansion of functional LT-HSCs in the absence of STAT5-regulated miR-193b.
(a) Differential miR expression in the presence and absence of STAT5 after stimulation with TPO. Expression fold change was calculated and normalized to the corresponding untreated (unstim.) cells. The data represent the mean expression of two independent experiments using LT-HSCs from 16 to 20 mice per group. (b) Percentage of LT-HSCs and MPPs in the LSK fraction of the BM from mice of the indicated age groups, N=4 miR-193b+/+ mice and N=6 miR-193b−/− mice at 2–3 months of age, N=6 mice/genotype at 6 months of age, N=8 miR-193b+/+ mice and N=4 miR-193b−/− mice at 12 months of age. Exemplary FACS plots display the CD150 and CD34 expression of LSK (Lineage− c-Kit+ Sca1+) BM cells from 12-month-old mice, representing LT-HSCs (CD150+ CD34lo) and MPPs (CD150− CD34+). (c) Experimental scheme of the primary and secondary transplantation of LT-HSCs from miR-193b−/−and miR-193b+/+mice into NSG mice. (d) Donor cell engraftment in the peripheral blood of primary recipients after competitive transplantation of LT-HSCs from 12-month-old miR-193b−/− and miR-193b+/+ mice was assessed via FACS. Mann–Whitney test. (e) FACS plots gated for donor LT-HSCs (left panel) and absolute numbers (right panel) after 16 weeks in both the femurs and tibiae of each primary recipient. Mann–Whitney test. (f) Donor cell engraftment in the peripheral blood of secondary recipients after transplanting 1 × 106 BM cells from primary transplanted mice. BM of one primary transplanted mouse was transplanted into two recipients. Mann–Whitney test. (g) FACS plots gated for donor LT-HSCs (left panel) and absolute numbers (right panel) after 24 weeks in both femurs and tibiae of each secondary recipient. Mann–Whitney test. (h,i) The distribution of donor stem and progenitor populations in primary (h) and secondary (i) recipient BM. Mann–Whitney test. The data are represented as the mean±s.d. *P<0.05; **P<0.01 and ***P<0.001.
Figure 2
Figure 2. An increased number of miR-193b-deficient LT-HSCs are in active cell cycle.
(a) Exemplary FACS plots and quantification of Ki67 and 7-AAD (DNA content) of LT-HSCs and MPPs to assess quiescent and cycling cells. (b) Time of the first division of LT-HSCs determined by video-microscopy-based cell tracking. The box plots represent the median with whiskers min to max. (c) A Kaplan–Meier survival curve of the mice injected with 5-fluorouracil (5-FU) at the indicated time points (arrows). N=7 miR-193b−/− mice, N=6 miR-193b+/+ mice. Gehan–Breslow–Wilcoxon test. All data are represented as the mean±s.d. *P<0.05; **P<0.01.
Figure 3
Figure 3. MiR-193b controls HSPC expansion by altering differentiation and not by influencing cell cycle or survival.
(a) Proliferation assay of sorted LT-HSCs in serum-free liquid culture (SCF, TPO, IL3, IL6 and EPO). N=3 independent experiments. (b,c) Lentiviral transduction of LT-HSCs with miR-193b or CTRL vectors and cell culture (SCF, TPO). N=3 independent experiments. (b) The percentage of transduced cells was determined via FACS. (c) Absolute number of cells after 9 days in culture. (d) Generation time and cell death determined using video-microscopy-based cell tracking. (e) Generation times in subsequent generations of LT-HSCs determined via single-cell tracking. (f) Cell death events assessed via single-cell tracking. (g) BM FACS analysis of BrdU incorporation in HSPC populations after 4 h of in vivo BrdU pulse. N=3 mice per genotype. (h) Experimental scheme for the in vitro differentiation assay. (i) Exemplary FACS plots of CD16/32/CD117 expression at day 5 of differentiation. (j) Cell distributions at days 5 and 7 determined according to their CD117 and CD16/32 expression. N=3 individual mice per genotype. All data are represented as the mean±s.d. *P<0.05; **P<0.01 and ***P<0.001.
Figure 4
Figure 4. The absence of miR-193b increases basal and cytokine-stimulated signalling in LT-HSCs.
(a) Functional annotation analysis using DAVID (KEGG pathways) of the upregulated and downregulated genes in sorted LSK cells derived from miR-193b−/− and miR-193b+/+ mice determined via RNA sequencing. Only clusters with a score >1 are displayed. (b) Representative histograms and quantification of phosphoflow cytometry analysing basal signalling pathways of HSPCs from miR-193b−/− and miR-193b+/+ mice. N=3 independent experiments. (c) Equal signalling intensities were observed in sorted LT-HSCs after 1 h of starvation (tonic signalling) as assessed by phosphoflow cytometry. N=4 independent experiments. (d) Representative histograms and quantification of phosphoflow cytometry of LT-HSCs stimulated with a myeloid cytokine cocktail for 20 min after starvation. The mean fluorescence intensity (MFI) was normalized to starved and unstimulated cells. N=4 independent experiments. (e) Quantification pSTAT5 and pAKT via phosphoflow cytometry and miR-193b expression via qPCR in LT-HSCs stimulated with a cytokine cocktail at various time points. The miR-193b expression was normalized to snoRNA202. N=3 mice per group (phosphoflow cytometry) and N=3 individual experiments (qPCR). All data are represented as the mean±s.d. *P<0.05; **P<0.01.
Figure 5
Figure 5. MiR-193b targets c-KIT expression and thereby modulates signalling in HSPCs.
(a) PamGene array volcano plots of tyrosine and serine/threonine kinases in BM cells derived from miR-193b−/− and miR-193b+/+ mice. Altered peptides and potential kinases (in brackets) are shown. N=4 independent experiments. (b) Target scan analysis of the 3′-untranslated region of murine c-Kit, including the location and conservation of the miR-193b-binding site. Sequence differences between the species are highlighted in red. (c) c-Kit mRNA expression upon miR-193b ectopic expression in LT-HSCs via qPCR. C-Kit mRNA expression was normalized to Gpdh mRNA. Transduction efficiencies were 94% and 90% for control and miR-193b, respectively. N=3 independent experiments. (d) FACS analysis of c-KIT surface expression upon miR-193b ectopic expression in LT-HSCs via FACS. A representative FACS plot and relative quantification of the c-KIT expression normalized to the CTRL (control) transduced cells are displayed. N=3 independent experiments. (e) Competitive transplantation of LT-HSCs that were lentivirally transduced with either miR-193b or CTRL 24 h prior transplantation. The transduction efficiency was 26% in both groups. Donor cell engraftment was measured in the peripheral blood after 4 weeks. N=5–6 mice per group. Mann–Whitney test. All data are represented as the mean±s.d. ***P<0.001.
Figure 6
Figure 6. Ectopic c-Kit expression can rescue miR-193b-mediated effects in HSPCs.
(a) Endogenous c-KIT protein expression in BM cells of miR-193b−/− and miR-193b+/+ mice measured via FACS. N=4 mice per group. (b) Rescue of HSPC survival by ectopic c-KIT expression. Re-expression of c-KIT lacking the 3′-untranslated region in miR-193b-transduced LT-HSCs via lentiviral co-infection and culture for several days. (c) Representative FACS plots and quantification of cultured cells at day 9. Ratio of double transduced cells (day 9/day 3) displayed as the mean±s.d. N=3 independent experiments. All data are represented as the mean±s.d. MFI, mean fluorescence intensity. *P<0.05; NS, not significant.
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