We elaborated a method of preparing cytogenetic preparations of cultured multipotent mesenchymal stromal cells from the adipose tissue. It was found that karyotypic changes (monosomy, translocations) appear in some samples during culturing. Clones with changed karyotype were detected in 11-14-passage cultures from 2 of 7 individuals. The percent of aberrant cells in cultures from different individuals varied from 1.5 to 5.95 per 100 cells, which attested to karyotype instability. These data substantiate the need for cytogenetic control of cells before their transplantation into donor organism and further investigation of chromosome variability in stem cells.

The use of synthetic osteoplastic materials not always provides the required amount of the bone tissue. Transplantation of tissue-engineering constructs containing osteogenic precursor cells can be an alternative high-technology implantation method. Here we present the results of a pilot clinical study demonstrating safety of this method, accelerated healing of the operation wound, formation of young bone tissue after transplantation, and the possibility of mounting implants after 3 months in case of sufficient amount of the bone for primary fixation.

BackgroundMesenchymal stromal/stem cells derived from human umbilical cord (UC-MSCs) uniquely combine properties of embryonic and postnatal MSCs and may be the most acceptable, safe, and effective source for allogeneic cell therapy e.g. for therapeutic angiogenesis. In this report we describe pro-angiogenic properties of UC-MSCs as manifested in vitro.MethodsUC-MSCs were isolated from human Wharton’s jelly by enzymatic digestion. Presence of soluble forms of VEGF-A in UC-MSC-conditioned media was measured by ELISA. Effects of the conditioned media on human umbilical vein-derived endothelial EA.hy926 cells proliferation were measured by MTT-assay; changes in cell motility and directed migration were assessed by scratch wound healing and transwell chamber migration assays. Angiogenesis was modeled in vitro as tube formation on basement membrane matrix. Progressive differentiation of MSCs to endothelioid progeny was assessed by CD31 immunostaining.ResultsAlthough no detectable quantities of soluble VEGF-A were produced by UC-MSCs, the culture medium, conditioned by the UC-MSCs, effectively stimulated proliferation, motility, and directed migration of EA.hy926 cells. In 2D culture, UC-MSCs were able to acquire CD31+ endothelial cell-like phenotype when stimulated by EA.hy926-conditioned media supplemented with VEGF-A165. UC-MSCs were capable of forming unstable 2D tubular networks either by themselves or in combinations with EA.hy926 cells. Active spontaneous sprouting from cell clusters, resulting from disassembling of such networks, was observed only in the mixed cultures, not in pure UC-MSC cultures. In 3D mode of sprouting experimentation, structural support of newly formed capillary-like structures was provided by UC-MSCs that acquired the CD31+ phenotype in the absence of exogenous VEGF-A.ConclusionThese data suggest that a VEGF-A-independent paracrine mechanism and at least partially VEGF-A-independent differentiation mechanism are involved in the pro-angiogenic activity of UC-MSCs.

Stem cells from human fetal thymus ectomesenchyma capable of forming colonies during in vitro culturing were isolated and characterized. Selection of culturing conditions showed that the growth and phenotypical characteristics of these cultures depended on seeding density and presence of basic fibroblast growth factor in the medium. After nonspecific inhibition of proliferation clonogenic cultures of thymic mesenchymal stem cells differentiated into myoblasts, formed characteristic myotubes, and expressed specific myogenesis markers. Colonies of thymic mesenchymal stem cells differentiated into chondrogenic, osteogenic, and adipogenic lines under conditions described for bone marrow mesenchymal stem cells. Cytofluorometric analysis of surface epitopes of thymic mesenchymal stem cells showed that the majority of cells expressed mesenchymal markers Thy-1, CD44, and CD105. Testing for CD34, CD38, CD45, and HLA-DR were negative in all cases. The main cell population (70-95%) did not express MHCl antigens during long-term culturing.

In the central nervous system and in the liver, the macrophage populations are represented exclusively by descendants of the hematopoietic progenitor cells of the yolk sac. The reasons for such differential distribution of macrophages are not fully understood. We found that, as can be judged by corresponding changes in the expression of CD86 and CD163 markers, the transient macrophages of monocytic lineage are more sensitive to activating stimuli. The two macrophage populations have distinct patterns of gene expression, which is particularly noticeable for M1- and M2-associated genes. For instance, Kupffer cells more readily develop and longer maintain the elevated expression levels of Il4, Il10, and Il13 upon the activation; by contrast, the macrophages of monocytic lineage express Il1b, Il12a, and Tnfα upon the activation. The obtained results allow us to conclude that the in vitro activated Kupffer cells of the liver are committed to M2 phenotype, whereas the in vitro activated monocyte-derived macrophages show a typical M1 behavior. These observations are likely to reflect the situation in the in vivo microenvironments.

The system of NMDA glutamate receptors in human adipose tissue multipotent stromal cells and SH-SY5Y human neuroblastoma cells was used as a model for studies of NMDA receptor expression during neurodifferntiation. Glutamate NMDA receptors were detected in multipotent stromal cells of human adipose tissue. The expression of NRI subunits of NMDA receptors increased significantly after 6-day incubation of multipotent stromal cells of human adipose tissue with 10 microM retinoic acid. Only NR1 subunits of NMDA receptors were expressed in SH-SY5Y neuroblastoma cells. Incubation with retinoic acid did not promote the appearance of mRNA of other subunits (NR2A-D, NR3). The results indicate that expression of NMDA receptors can serve as an indicator of neuronal differentiation of cells and as a marker of the efficiency of neuronal differentiation protocol.

BackgroundIn many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the ‘small for size’ liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats.ResultsThe data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages – the resident Kupffer cells with CD68+CX1CR3−CD11b− phenotype.ConclusionsThe delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the ‘proregeneratory’ M2 macrophage-specific marker CD206 in the course of regeneration.Electronic supplementary materialThe online version of this article (10.1186/s12865-018-0260-1) contains supplementary material, which is available to authorized users.

Proliferation of hepatocytes is known to be the main process in the hepatectomy-induced liver regrowth; however, in cases of extensive loss it may be insufficient for complete recovery unless supported by some additional sources e.g. mobilization of undifferentiated progenitors. The study was conducted on rat model of 80% subtotal hepatectomy; the objective was to evaluate contributions of hepatocytes and resident progenitor cells to the hepatic tissue recovery via monitoring specific mRNA and/or protein expression levels for a panel of genes implicated in growth, cell differentiation, angiogenesis, and inflammation. Some of the genes showed distinctive temporal expression patterns, which were loosely associated with two waves of hepatocyte proliferation observed at 2 and 7 days after the surgery. Focusing on genes implicated in regulation of the progenitor cell activity, we came across slight increases in expression levels for Sox9 and two genes encoding tumor necrosis factor-like cytokine TWEAK (Tnfsf12) and its receptor Fn14 (Tnfrsf12a). At the same time, no increase in numbers of cytokeratin 19-positive (CK19+) cells was observed in periportal areas, and no CK19+ cells were found in hepatic plates. Since CK19 is thought to be a specific marker of both cholangiocytes and the hepatic progenitor cells, the data indicate a lack of activation of the resident progenitor cells during recovery of hepatic tissue after 80% subtotal hepatectomy. Thus, proliferation of hepatocytes invariably makes the major contribution to the hepatic tissue recovery, although in the cases of subtotal loss this contribution is distinctively modulated. In particular, induction of Sox9 and TWEAK/Fn14 regulatory pathways, conventionally attributed to progenitor cell activation, may incidentally stimulate mitotic activity of hepatocytes.