*3.1. Cells Isolated From the Bone Marrow of Mice Display Features of Mesenchymal Stem Cells*

MSCs isolated from the bone marrow of mice were characterized by adhesiveness and morphology. Light microscopy showed that MSCs were attached to the surface of culture flasks and were polymorphic cells with a fibroblast-like morphology. Cells actively proliferated and formed a monolayer (Figure 1a).

Phenotyping of MSCs of two to three passages by flow cytometry showed that most of them (83–96%) expressed CD73, CD90.1, and CD105 receptors that are markers of MSCs (Figure 1b, upper panel). At the same time, no expression was detected for hematopoietic cell markers CD45 and CD34 (Figure 1b, lower panel).

Analysis of the adipogenic potency of the obtained MSCs revealed that, at the end of 20 days of cultivation, single adipocytes were present in the control culture (Figure 1c, upper left panel); adipocytes located either individually or in small, usually loose groups of 5–10 cells were detected in the induced culture (Figure 1c, middle and upper right panel). Adipocytes differed in morphology and were at different stages of fat droplet accumulation: both mature oval or round adipocytes containing

many large lipid vacuoles were found, as well as fibroblast-like cells, in which fat inclusions were smaller and occupied only part of the cytoplasm.

**Figure 1.** Characterization of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. (**a**) The morphology of MSC isolated from the red bone marrow of mice is polymorphic cells that exhibit a fibroblast-like shape. The cells were seeded on culture flasks, and, after staining the nuclei with hematoxylin, they were visualized by light microscopy, scale bar, 100 μM (left), and 25 μM (right). (**b**) These are typical results of MSCs receptor analysis by flow cytometry. For immunophenotyping of MSCs, adhesive cells of the second and third passages were stained with phycoerythrin (PE)-labeled antibodies against CD73, CD90.1, CD105, or PE-labeled antibodies of the corresponding isotype controls, and unlabeled rat antibodies against CD45 or CD34 followed by rabbit antirat fluoresceine isothiocyanate (FITC) conjugate. The isolated MSCs expressed CD105, CD73, and CD90 (upper panel), but did not express CD45 and CD34 (lower panel). (**c**) The adipogenic (upper panel) and osteogenic (lower panel) differentiation of MSC isolated from the bone marrow of mice. MSCs were grown into an adipogenic or osteogenic medium; the medium was changed twice a week for 20 days (adipogenesis) or 21 days (osteogenesis). Then, MSCs were stained to detect neutral fat inclusions or alkaline phosphatase activity, respectively. Single adipocytes (upper left panel) and osteocytes (lower left panel) were present in the control cultures; arrows indicate spontaneous differentiation, scale bar, 100 μM; induced cultures MSC, arrows indicate fat cells (middle and upper right panel) and osteocytes (middle and lower right panel), (scale bar, 25 μM, and 10 μM, respectively).

A study of the osteogenic differentiation of MSCs showed that, after 21 days of cultivation, the cells in the control culture formed a confluent monolayer of uneven density with cells of fibroblast-like morphology. The cytochemical reaction to alkaline phosphatase, a marker of osteogenic cells, identified just a few cells containing this enzyme in the control culture (Figure 1c, lower left panel). In contrast, in the induced culture, most cells that contained alkaline phosphatase significantly exceeded those in the control culture (85±13 vs. 9±8, respectively, p<0.001). Cells with the positive reaction had a fibroblast-like shape and formed clusters of various size and density, in many cases closely contacting each other. The reaction intensity varied in different cells and often was very high (Figure 1c, middle and lower right panel), thus showing that the cells underwent osteogenic differentiation. Therefore, MSCs isolated from mouse bone marrow exhibited moderate capability to differentiate into mature multilocular adipocytes. They also could pass initial stages of osteogenic differentiation. It can be concluded that the isolated and multiplied cells corresponded to the generally accepted minimal characteristics of MSCs [45].
