E. I. Domaratskaya

Mesenchymal stem cells: Sources, phenotype, and differentiation potential

Mesenchymal stem cells present in the bone marrow and some other organs are primitive pluripotent precursors of osseous, cartilaginous, adipose, and other mesenchymal tissues. The recently revealed capacity of these cells for differentiation into nonmesenchymal derivatives is of considerable theoretical and practical interest. However, many aspects of the biology of these cells remain obscure despite active research. This review considers possible sources and methods for the isolation of mesenchymal stem cells, their potential for proliferation and differentiation in different directions, and outlooks of their therapeutic application. A model of parent-progeny relationships of stromal cells is proposed, and the problems of regulation of proliferation and differentiation of mesenchymal precursors as well as their role in the maintenance of regeneration and tissue functioning are discussed.

Blood and clonogenic hemopoietic cells of newts after the space flight

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight.

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.

Analysis of sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to 5-fluorouracil

The sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to the cytotoxic effect of 5-fluorouracil (5-FU) was compared in vivo and in vitro. Cells from both tissues demonstrated a similar resistance to 5-FU in vitro; however, stromal stem cells from fetal liver proved notably more sensitive to 5-FU compared to marrow CFU-f in vivo. Cells forming colonies of different size were identified in stem cell populations from both tissues. Cells giving rise to small colonies had a higher resistance to 5-FU both in vivo and in vitro.

Stem cells as bone marrow residents

In addition to being a source of hematopoietic and mesenchymal stromal cells, bone marrow is known to be the source of stem cell populations, which express pluripotent markers and are capable of differentiating into cells of three germinative layers (ectoderm, mesoderm, and endoderm). Recently, a new population of so-called “very small embryonic like cells” (VSELs) has been identified in the bone marrow in addition to well-known pluripotential cells, such as MIAMI, MSC, and MAPS. The presence of stem cells with a wide spectrum of differentiation capacities in the bone marrow allows an alternative interpretation of the phenomenon of plasticity and the possibility of their switch from a canonical to a nontrivial path of differentiation. The phenotypic features of VSELs, their differentiation capacities, ontogenetic origin, and relationships with other types of stem cells are studied. The role of bone marrow stem cells and induced pluripotential stem cells in regeneration processes and their possible therapeutic application are discussed.

Alkylating damage by dipin of hematopoietic and stromal cells of the bone marrow

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBA × C57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably decreased and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).

Effect of Mesenchymal Stromal Cells and Conditioned Media on Healing of Skin Wound

We studied the effect of mesenchymal stromal cells and conditioned media on healing of full-thickness skin wound in rats. Cell transplantation to the wound bed did not accelerate wound closing and had no effect on the severity of inflammation, but increased vasculariza - tion of the granulation tissue in 14 days after injury. After injection of conditioned medium to the wound, less pronounced inflammation or enhanced epithelialization was observed. The angiogenic effect was observed only after repeated administration of conditioned medium and was associated with slower regeneration, probably due to skin traumatization by repeated injections. At the same time, fetal skin fibroblasts stimulated angiogenesis only after transplantation in high doses and the medium conditioned by these fibroblasts had no effect on wound healing.

Comparative study of bone marrow mesenchymal stromal cells at different stages of ontogeny

The aim of this study was to analyze the changes that occur in the population of bone marrow mesenchymal stromal cells (MSCs) during the individual development of an organism. For this purpose, the basic characteristics of MSCs (the content of clonogenic cells, immunophenotype, and potencies to differentiate in vitro and in vivo) in the prenatal, early postnatal, and late postnatal ontogeny of the rat were compared. It is shown that the cloning efficiency of bone marrow MSCs in 10-day-old and adult rats is comparable and hundreds of times smaller than that of bone cells of 20-day-old fetuses with a bone marrow rudiment. The activity of alkaline phosphatase, a marker of osteogenic cells, was found in the majority of colonies formed by MSCs of postnatal bone marrow but not by the fetal bone. By the CD90 expression and potencies for in vitro adipogenesis, the stromal cells from the fetal bone and bone marrow of 9- to 10-day-old rats were comparable with those of the mature bone marrow MSCs but differed from them by the small number of CD73-bearing cells and a weaker ability to osteogenesis in an inductive environment. The analysis of the fate of MSCs from the studied sources after their transplantation to adult rats showed that their ectopic transplantation as part of tissue fragments into the kidney results in the formation of bone tissues and hematopoietic stroma. In diffusion chambers with MSCs that were precultured in vitro, transplantation into the peritoneal cavity led to osteogenesis and chondrogenesis. However, no significant differences in the potencies of bone marrow MSCs for differentiation in vivo depending on the developmental stage have been found. Thus, during ontogeny, bone marrow MSCs enhance the expression of CD73 and the ability to osteogenesis in vitro, whereas the expression of CD90 and the potencies for adipogenesis in induction medium and differentiation in different directions in vivo do not change significantly.

Comparative analysis of subpopulations of mesenchymal stromal cells of the bone marrow and fetal liver differing in sensitivity to 5-fluorouracil

It is shown that the effect of 5-fluorouracil (5-FU) on mesenchymal stromal cells (MSCs) of the bone marrow and fetal liver of the rat enriches the population with cells with a reduced proliferative potential. It is shown that, in the course of passaging, the 5-FU-resistant MSCs of the bone marrow and fetal liver lose their ability to differentiate in the osteogenic direction more and less rapidly, respectively, than the 5-FU-sensitive MSCs.

Clonal growth, phenotype, and differentiation potential of mesenchymal stromal cells derived from the rat fetal bone

394 Studies on mesenchymal stromal cells (MSCs) are a focus of modern biology. Initially, MSCs were isoo lated from the bone marrow, spleen, and thymus in the form of colonyyforming units of fibroblasts (CFUUF) [1]; later, MSCs were found in all organs of transient and definitive hematopoiesis. Correlation between the contents of CFUUF and hematopoietic stem cells in these organs at various stages of ontogeny may show indirectly consecutive change of location of MSCs, which prepare a niche for hematopoietic cells [2]. It is possible that, during ontogeny, MSCs are modified to some extent. Specifically, in the prenatal period, stroo mal cells of the human bone marrow have a higher proliferative activity and differentiation potential compared to those of adult donors [3, 4]; however, they have a lower capability for maintaining hematoo poiesis [5]. The problem of changes in MSC properties in ontogeny has been poorly studied. Studies on the features of these cells in hematopoietic organs of developing or mature individuals will shed light on maturation of the hematopoietic system. It is of spee cific interest to describe MSCs, located in the fetal bone, prior to the start of active hematopoiesis and to compare them with stromal cells of the mature bone marrow. This was the purpose of our study. Histological study was performed in sections of the femoral bone of 200day fetuses of Wistar rats. We did not observe clear bone marrow cavity in the femoral bone because the epiphyses consisted of cartilage tiss sue, and the diaphyses were filled with nets of bone trabecules, between which, fragments of disintegrated cartilage, multiple mesenchymal cells, and a few hematopoietic cells were located. Cells separated from the bone after its treatment with 0.1% collagenase and seeded into the αMEM medium containing 10% fetal calf serum at a density of (1–2) × 10 5 cells/mL, formed a confluent fibroblast monolayer after 33day growing. Discrete colonies formed after seeding at a density of (1–4) × 10 3 cells/mL and 6 to 12day grow ing. The efficiency of CFUUF cloning was more than two orders of value higher compared to cells from the bone marrow of adult rats. Moreover, the activity of alkaline phosphatase, which is specific for osteogenic cells and present in most colonies formed by cells of mature bone marrow, was only rarely observed in the culture of fetal bone CFUUF (table). The differences in cloning efficiency of stromal cells obtained …