MiR-199b-5p Differentially Modulates Hematopoietic Stem Cell Self Renewal in Steady State and Stress Hematopoiesis

The emergence of microRNAs (miRNAs) is providing novel insights into the molecular control networks underlying hematopoiesis and diseases that stem from aberrations in hematopoiesis. We, amongst others, have shown that miR-199b is significantly downregulated in acute myeloid leukemia (AML) and is a promising prognostic factor for FAB-M5 subtype. Herein, to determine miR-199b's role in steady state and stress hematopoiesis, we utilized our recently generated miR-199b knockout (KO) mice (CRISPR approach) to assess frequencies of hematopoietic stem progenitor cells (HSPCs) in WT and miR-199b KO mice through flow cytometry. Under steady state conditions, deletion of miR-199b resulted in significant reduction of Lin^- Sca-1⁺ c-Kit⁺ (LSK), long-term hematopoietic stem cells (LT-HSCs) (Lin⁻ CD117⁺ Sca1⁺ CD150⁺ CD34^low/− CD48⁻) and increases in multipotent progenitors (MPPs) (Lin⁻ CD117⁺ Sca1⁺ CD150⁻ CD34⁺). However, 5FU induced stress hematopoiesis resulted in significant increases in frequencies of LSK cells, LT-HSCs, and Lin^- Sca-1^- c-Kit⁺ (KL) cells compared to wild-type (WT) litter mates. Furthermore, silencing of miR-199b resulted in a significant increase of granulocyte monocyte progenitors (GMP), which was reversed during stress hematopoiesis. The highest expression levels of miR-199b were restricted to the LT-HSC fraction. Mir-199b levels remained moderately high in MPPs and LSK cells. Primarily within the LSK population, miR-199b expression was most pronounced in LT-HSCs and to a lesser extent, MPPs, indicating that miR-199b may play a functional role in LT-HSC activity. Gene profiling studies in HSPCs identified Integrin alpha 8 (Itga8) as a conserved target of miR-199b. We then examined Itga8 transcript levels in HSCs from miR-199b knockout (-/-) mice and found significant elevation of Itga8 transcripts compared to WT mice. One possible mechanism for the differential expansion of miR-199b-/- HSPCs under steady state and stress is an increased or decreased rate of cell cycling. To test this, we examined cell proliferation using Ki67 and Hoechst 33342 markers. MiR-199b-/- LT-HSC at steady state showed significantly decreased proliferation whereas miR-199b-/- LT-HSCs under 5FU induced stress showed significant expansion when compared to wild-type. Next, we tested the adhesion abilities of LT-HSCs from miR-199b KO mice under both steady state and stress hematopoiesis. At steady state, miR-199b-/- LT-HSCs significantly elevated adhesion to vitronectin and fibronectin. Interestingly, like HSPC frequencies, stress hematopoietic-derived miR-199b-/- LT-HSCs showed significantly decreased adhesion to both vitronectin and fibronectin. These findings imply that miR-199b intrinsically regulates HSC behavior, including cell adhesion. To elucidate whether miR-199b's effects on LT-HSCs are mediated by Itga8, we performed an Itga8 knockdown in WT and miR-199b-/- LT-HSCs. Cell proliferation assessed by Ki67 positive cells revealed that downregulation of Itga8 in miR-199b KO LT-HSCs reverts to a miR-199b-induced decrease of LT-HSC proliferation. Intriguingly, we observed that silencing of Itga8 reversed the miR-199b-induced increase of cell adhesion. To corroborate that miR-199b-/- LT-HSCs are fully functional, we performed a competitive repopulation assay. BM derived lineage negative hematopoietic progenitor cells were further purified via immunomagnetic selection for SLAM marker purified (CD150+, CD244+ and CD48-) to enrich the LT-HSC fraction. MiR-199b-/- or miR-199b+/+ LT-HSCs (n=100 LT-HSCs) were transplanted intravenously together with 2.5 X 10⁵ competitor recipient cells. Overall donor BM cellularity was markedly increased in LT-HSCs derived from 5FU treated miR-199 KO mice and overall donor BM cellularity was markedly decreased in LT-HSCs derived from steady state miR-199 KO mice. This suggested that under stress, miR-199b-/- LT-HSCs self-renew extensively after transplantation to repopulate the recipient. The analyses of HSPCs in BM of primary recipients revealed a significant increase in donor LT-HSCs in recipients that were transplanted with LT-HSCs derived from the 5FU induced stress model. Intriguingly, this didn't translate into subsequent increases in MPP, GMP or MEPs, suggesting that miR-199b may govern the self renewal of LT-HSCs and also have progenitor cell specific roles as evident from its expression during early hematopoiesis.

Figure

View largeDownload slide

Figure

View largeDownload slide

Close modal