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Characterization of Progenitors with the Potential to Differentiate into Mesenchymal and Hematopoietic Lineages

Within the bone marrow microenvironment mesenchymal and hematopoietic cells are anatomically and functionally related. In our previous study we hypothesize that cells expressing smooth muscle a-actin promoter (aSMA) directed transgene represent mesenchymal progenitors of adult bone tissue and confirmed that aSMA+ cells have the potential for terminal differentiation into mature osteoblast lineage cells in vitro and in vivo. We now proposed that aSMA+ cells also comprise hematopoietic progenitors that may differentiate toward hematopoietic lineages. This is in line with recent studies that identified progenitor cells expressing both mesenchymal and hematopoietic markers, challenging the long-existing postulation of early separation between those two lineages. In order to trace phenotypic and functional characteristics of aSMA+ cells, we generated aSMACreERT2 transgenic mice and we characterized its expression by crossing it with the Ai9 reporter transgenic line to generate aSMACreERT2/Ai9 (SMA9) mice. In a time-course of in vivo tamoxifen induction we were able to identify aSMA+ cells within the bone marrow compartment, comprising less than 0.1% of bone marrow cells 4 days after the first injection and reaching approximately 0.3% among bone marrow cells 65 days after the first tamoxifen injection. Flow-cytometric analysis confirmed that majority of cells express CD45 together with other hematopietic lineage markers, such as CD11b, Gr-1, Ter119, B220 or CD3. However, around 10% of cells were CD45+ but negative for all other lineage markers. Three weeks (19 days) after the first tamoxifen injection those cells expressed immature markers Sca-1 and CD34, but were negative for endothelial markers CD106 and CD31. Supposedly, those cells generate mature hematopoietic cells that are released into circulation, since we found around 0.15% aSMA+ cells in the peripheral blood, all expressing hematopoitic markers. Moreover, sorted aSMA+ cells gave rise to myeloid/monocyte colonies in the methylcellulose cultures. In the parabiotic pairs between the wild-type and transgenic SMA9 mouse, we could detect aSMA+ cells in circulation as well as rare aSMA+ cells within the spleen of the wild-type counterpart. On the other hand, CD45- bone marrow population expressed mesenchymal markers such as PDGFb and leptin receptor and increased in the percentage with the time after tamoxifen induction. Interestingly, certain percentage of CD45low cells also expressed mesenchymal markers together with Sca-1. Our findings provide evidence of the potency of aSMA+ population to give rise to mature mesenchymal as well as hematopietic cells. In further experiments we aimed to identify intracellular signaling molecules that control the bifurcation between progenitor commitments to those lineages.

progenitor cells; hematopietic lineages; mesenchymal lineages

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