N. N. Butorina

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.

Clonal growth and differentiation of mesenchymal stromal cells from rat liver at different stages of embryogenesis

Fetal liver, during its hematopoietic activity, contains mesenchymal stromal cells (MSCs) generating its hematopoietic microenvironment. These cells are clonogenic and capable of multilineage differentiation; however, little is known about how their properties alter during embryogenesis. We compared the cloning efficiency of MSCs from rat fetal liver at 14, 16, and 20 days of development, as well as their capacity for osteo- and adipogenesis in vitro and chondrogenesis in vivo by ectopic transplantation of intact liver. The relative content of clonogenic MSCs in liver cell suspension was highest in 16-day fetuses and lowest in 20-day fetuses. Cells from 14-day fetuses exhibited high osteogenic and less apparent adipogenic and chondrogenic potential; cells from 20-day fetuses displayed weak adipogenic capacity and no osteo- or chondrogenic ability. These results show the correlation of MSC content and the cell differentiation potential with hematopoietic dynamics in developing rat liver. It may be thought that the changes we observed are related to the loss of hematopoietic activity and liver getting of definitive functions.

Spontaneous myogenesis in the primary culture of fetal rat liver

187 Fetal liver stroma is known as a source of mesenchymal stromal cells (MSCs), which exhibit osteogenic, adipogenic, and chondrogenic potencies [1]. It was reported that stromal cells of this organ can undergo myogenic differentiation in an induction medium [2], when cultured under specific conditions [3] and upon fusion with skeletal muscle cells [4]. We discovered that myosymplasts may spontaneously form in the primary culture of fetal rat liver.

Mesenchymal Stromal Cells of Rat Spleen during Pre-and Postnatal Ontogeny: Comparative Analysis of Clonal Growth, Phenotype and Differentiation Potencies

Comparison of mesenchymal stromal cells of embryonic and adult rat spleen showed that splenic cells from 20-day rat fetuses exhibit the capacity for clonal growth, express surface antigens CD73, CD90, and CD106, and have weak osteogenic and adipogenic potencies, while splenic cells from adult animals are characterized by lower cloning efficiency, rapid decrease of proliferative activity during passaging, the absence of CD73 and CD90 expression, and are incapable of osteogenesis The observed changes are probably related to extinction of myelopoiesis in the spleen during the postnatal ontogeny.

Cell Composition of the Primary Culture of Fetal Liver

Fetal liver is known as a source of multipotent mesenchymal stromal cells. These cells are routinely isolated by adhesion to plastic, but thus prepared culture is contaminated by other cells. For instance, primary cell culture of from rat fetal liver, apart from fibroblasts with phenotypic characteristics of mesenchymal stromal cells, contained skeletal muscle precursors, myofibroblasts, and epitheliocytes expressing cytokeratin-19 (the latter was also detected in some fibroblast-like cells probably undergoing epithelio-mesenchymal transition). During passaging, fibroblasts become practically the only cell type in the culture.

Spontaneous and induced myogenesis in cell cultures from rat fetal liver

Fetal liver stroma consists of different cell populations. We found that the liver of 17- and 20-day rat fetuses contained skeletal muscle precursors that expressed MyoD. In primary cultures of liver cells from 15-, 17- and 20-day fetuses, spontaneous myotube formation was observed. The antigenic profile of these myogenic elements assayed by immunocytochemistry and PCR unambiguously indicated their skeletal muscle nature. Examination of major myogenic gene expression demonstrated that myogenic potencies cells from liver depended on the stage of fetal development cell cultivation. It was shown that fetal liver MSCs were capable of myotube formation in induction medium with 5-azacytidine. The results of our study show that 15- to 20-day prenatal rat liver contains mainly preexisting skeletal muscle precursors expressing MyoD and, probably, inducible muscle precursors.

Influence of primary adhesive interactions with fibronectin on clonal growth and osteogenic potential of rat mesenchymal stromal cells

Interactions with the extracellular matrix play an important role in the regulation of cell growth and differentiation. Fibronectin (Fn) is a major component of the extracellular matrix. The impact of Fn and its domains on the adhesion and osteogenic potency of rat mesenchymal stromal cells (MSC) have been evaluated. The examination of bone marrow and fetal liver MSC adhesion dynamics showed that after 7 days of cultivation on Fn the number of adhered clonogenic cells derived from both sources was comparable with their number on plastic but their content in suspension usually was decreased. The population of fetal liver MSC differed from bone marrow-derived MSC in the larger number of cells that adhered for the first 7 days. We found that the primary adhesion of MSC to Fn affected their differentiation potency. Bone marrow MSC cultured on Fn had reduced activity of alkaline phosphatase compared with cells grown on plastic. They deposited a significantly less amount of calcium salts in osteogenic medium. The cultivation of MSC on Fn fragments demonstrated that the crucial role in the inhibition of osteogenesis is played by the Fn cell-binding domain.

Embryonic Sources of Definitive Hemopoietic Stem Cells

A review of one of the key problems of experimental hematology: the origin of hemopoietic stem cells in the development of vertebrates (amphibians, birds, and mammals). The appearance and functioning of two independent sources of hemopoietic stem cells (extra- and intraembryonic) were considered in amphibians, birds, and mammals. The contribution of each source to the formation of definitive hemopoietic tissue was analyzed. It was shown for amphibians and birds that intraembryonic organs such as the dorsolateral plate and the mesenchyme of dorsal aorta are involved in the formation of adult hemopoietic tissue, while the extraembryonic organs such as ventral islets and the yolk sac are devoid of true stem cells and provide only for the primary, transient hemopoiesis. New data have been considered concerning the previously unknown intraembryonic hemopoietic organ in mammals, a region of aorta–gonad–mesonephros arising in embryogenesis simultaneously with the yolk sac. Two extreme views on the involvement of stem cells of all these organs in the formation of definitive hemopoiesis have been considered. The data are provided on the interaction of the embryonic hemopoietic stem cells and the hemopoietic microenvironment of adult recipients.

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.