E.A. Markina - Junior Research Scientist, FSC RF - IMBP RAS, Moscow E-mail: goncharova-tim@list.ru E.R. Andreeva - Ph.D. (Biol.), Leading Research Scientist, FSC RF - IMBP RAS (Moscow) E-mail: andreeva_er@mail.ru

Fibroblast-like cells in rodent bone marrow (later called multipotent mesenchymal stromal cells (MMSCs)) having been dis-covered by A.Ya. Friedenstein and his coworkers and at now days become an attractive object for investigation and are often used for studying the adult stromal cell biology. MMSC quantity in murine bone marrow amounts to 0,001 - 0,0001% of the total number of mononuclear cells. A characteristic feature of this population is that quote difficult to get homogeneous culture of MMSCs due to the admixture of hematopoietic cells which form tight contacts with stromal elements. MMSCs from murine tibia compact bone are very similar by their characteristics to bone marrow MMSCs (Lin-CD45-, CD31-, Sca-I+). Several papers have shown, that cultivation of cells, including MMSCs from different tissues, at low oxygen conditions (5%) improves parameters of cultured cells such as proliferative activity, metabolism, vitality compared with cells in normal conditions (20% О2). The goal of this research was to compare morphofunctional characteristics of MMSCs from murine tibia compact bone and bone marrow after expansion in selective medium MesenCult, which ensures preferential growth of stromal precursors in standard (20%) and hypoxic (5% O2) conditions. Mononuclear cells for MMSC analysis were isolated from bone marrow and compact bone. Cells were cultivated in selective medium MesenCult, which ensures preferential growth of stromal precursors in standard (20%) and hypoxic (5% O2) conditions. Over cultivated MMSCs were observed using phase-contrast microscopy. The functional state of MMSCs was characterized by increase in growth and differentiation capacities. The osteodifferentiation was confirmed by histochemical detection of alkaline phosphatase (ALP) activity (marker of early osteocommitment), adipodifferentiation was detected after staining of intracellular lipid droplets with Oil Red O. The number of bone marrow mononuclears isolated from 2 femur and 2 tibia bones was 186*106-189,6*106. The number of compact bone mononuclears, isolated from 2 femur and 2 tibia bones, was 1,56*106-1,66*106. While cultivating bone marrow MMSCs, the confluence was estimated in high density zones instead of whole dish because of uneven distribution of cells. 80% of confluence was reached after 16 days at standard conditions (20% О2), and after 11 days at hypoxia (5% О2). In case of compact bone MMSCs, 80% of confluence was reached after 14 days under standard conditions (20% О2), and after 10 days at hypoxic one (5% О2). Osteocommitted ALP-positive MMSCs were detected both in uninduced control and after osteoinduction at both О2 concentrations. In control group the number of stained cells was similar either under 20% or 5% O2. After osteoinduction the number of ALP-positive cells was more at hypoxic conditions. Compact bone MMSC response to osteogenic stimulus was more clearly defined than that of bone marrow MMSC. Lipid conclusions were found in MMSC both in control, and after adipoindaction regardless of О2 concentration. After adipoinduction the number of cells with Oil Red O-positive lipid droplets as well as droplets itself increased. For bone marrow MMSCs the effect was more clearly defined at 20% О2. Compact bone MMSCs responded on adipogenic stimulus similarly at both 20% and 5% О2. Thus, cultivation in MesenCult selective medium at low tension of oxygen provides faster growth of stromal population from bone marrow and compact bone compared with MMSCs cultured at 20% О2. In addition, more murine bone marrow and compact bone MMSCs osteodifferentiate after expansion in MesenCult medium at hypoxic conditions. Compact bone adipodifferentiation doesn-t depend on oxygen concentration. Adipodifferentiation of bone marrow MMSCs is better at 20% О2. That allows us to consider compact bone MMSCs as a full-value alternative to murine bone marrow MMSCs in labora-tory practice. This study was supported by the Program of Basic Research of IBMP RAS.

 

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