The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging.
Disclosures
No relevant conflicts of interest to declare.
Hypoxia mediates low cell-cycle activity and increases the proportion of long-term–reconstituting hematopoietic stem cells during in vitro culture
