Histone H3K9 Methylation Features under Hypoxic Conditions after the HIF1A Knockdown in Mesenchymal Stromal Cells In Vitro.

The effects of HIF1A knockdown by RNA interference on the histone H3K9 methylation in human umbilical cord mesenchymal stromal cells in vitro under conditions of 24-h exposure to hypoxia (1% O2) were studied. Evaluation of transcriptional activity of genes involved in the regulation of H3K9 methylation (KDM3A, KDM4A, and EHMT2) and the cytofluorimetric analysis of the expression of the corresponding antigens and H3K9 methylation level demonstrated a pronounced stimulating effect of hypoxic exposure. Moreover, the expression of KDM4A and EHMT2 was regulated by HIF1A-mediated mechanism, unlike KDM3A; the level of the corresponding proteins depended on HIF1A. In addition, the HIF-1-dependent regulation of KDM3A, KDM4A, and EHMT2/G9a, and directly the H3K9 methylation level in mesenchymal stromal cells also took place under normoxia conditions.

Alteration of PBMC transcriptome profile after interaction with multipotent mesenchymal stromal cells under "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) have demonstrated a pronounced immunosuppressive activity, the manifestation of which depends on the microenvironmental factors, including O2 level. Here we examined the effects of MSCs on transcriptomic profile of allogeneic phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) after interaction at ambient (20%) or "physiological" hypoxia (5%) O2. As revealed with microarray analysis, PBMC transcriptome at 20% O2 was more affected, which was manifested as differential expression of more than 300 genes, whereas under 5% O2 220 genes were changed. Most of genes at 20% O2 were downregulated, while at hypoxia most of genes were upregulated. Altered gene patterns were only partly overlapped at different O2 levels. A set of altered genes at hypoxia only was of particular interest. According to Gene Ontology a part of above genes was responsible for adhesion, cell communication, and immune response. At both oxygen concentrations, MSCs demonstrated effective immunosuppression manifested as attenuation of T cell activation and proliferation as well as anti-inflammatory shift of cytokine profile. Thus, MSC-mediated immunosuppression is executed with greater efficacy at a "physiological" hypoxia, since the same result has been achieved through a change in the expression of a fewer genes in target PBMCs.

Oxygen Level Modifies the Expression of Genes Involved in the Epigenetic Regulation of Multipotent Stromal Cells In Vitro.

Changes in the transcriptional activity of genes involved in the epigenetic regulation of adipose tissue multipotent mesenchymal stromal cells were analyzed in vitro at different O2 levels. DNA microarray study showed that the most pronounced changes in gene expression, including genes responsible for the epigenetic regulation of mesenchymal stromal cells, occurred at 3% O2. A lower number of genes changed the expression at 1% O2, and a minimum response was observed at 5% O2 in comparison with standard culturing conditions (20% O2). The greatest number of differentially expressed genes were genes responsible for the regulation of histones; the genes encoding products that regulate chromatin, DNA, and RNA constituted a lower part. Thus, the degree of hypoxia can modify the response of multipotent mesenchymal stromal cells at the level of epigenetic regulators.

Osteogenic Commitment of MSC Is Enhanced after Interaction with Umbilical Cord Blood Mononuclear Cells In Vitro.

The effectiveness of stroma-dependent expansion of hematopoietic cells ex vivo may depend on the level of commitment of multipotent mesenchymal stromal cells (MSC). Markers of MSC osteodifferentiation and the level of soluble hematopoiesis regulators were determined during their interaction with umbilical cord blood mononuclears. After 72-h co-culturing, an increase in the expression of ALPL and alkaline phosphatase activity was revealed. In conditioned medium of co-cultures, the levels of osteopontin and osteoprotegerin were elevated and the levels of osteocalcin and sclerostin were reduced. Co-culturing of umbilical cord blood mononuclears with osteocommitted MSC was accompanied by more pronounced increase in the concentration of both positive (GM-CSF and G-CSF) and negative (IP-10, MIP-1α, and MCP-3) regulators of hematopoiesis. Thus, umbilical cord blood mononuclears induced the formation of early osteogenic progenitor phenotype in MSC ex vivo, providing the microenvironmental conditions necessary to support hematopoiesis. Preliminary osteocommitted MSC were more sensitive to the effect of umbilical cord blood mononuclears.

Сord blood hematopoietic stem cells ex vivo enhance the bipotential commitment of adipose mesenchymal stromal progenitors.

AIMS: Stroma-dependent ex vivo expansion of hematopoietic stem progenitor cells (HSPCs) is a valid approach for cell therapy needs. Our goal was to verify whether HSPCs can affect stromal cells to optimize their functions during ex vivo expansion. MAIN METHODS: HSPCs from cord blood (cb) were cocultured with growth-arrested adipose mesenchymal stromal cells (MSCs). Commitment-related transcriptional and secretory profiles as well as hematopoiesis-supportive activity of intact and osteo-induced MSCs were examined. KEY FINDINGS: During expansion, cbHSPCs affected the functional state of MSCs, contributing to the formation of early stromal progenitors with a bipotential osteo-adipogenic profile. This was evidenced by the upregulation of certain MSC genes of osteo- and adipodifferentiation (ALPL, RUNX2, BGLAP, CEBPA, ADIPOQ), as well as by elevated alkaline phosphatase activity and altered osteoprotein patterns. Joint paracrine profiles upon coculture were characterized by a balance of "positive" (GM-SCF) and "negative" (IP-10, MIP-1α, MCP-3) myeloid regulators, effectively supporting expansion of both committed and primitive cbHSPCs. Short-term (72 h) osteoinduction prior to coculture resulted in more pronounced shift of the bipotential transcriptomic and osteoprotein profiles. The increased proportions of late primitive CD133-/CD34+cbHSPCs and unipotent CFUs suggested that cbHSPCs after expansion on osteo-MSCs were more committed versus cbHSPCs from coculture with non-differentiated MSCs. SIGNIFICANCE: During ex vivo expansion, cbHSPCs can drive the bipotential osteo-adipogenic commitment of MSCs, providing a specific hematopoiesis-supportive milieu. Short-term preliminary osteo-induction enhanced the development of the bipotential profile, leading to more pronounced functional polarization of cbHSPCs, which may be of interest in an applied context.

Adipose-derived stromal cell immunosuppression of T cells is enhanced under "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) are characterized by immunomodulatory properties along with the high proliferative and paracrine activity, as well as multilineage potency. The effects of MSCs on the T cell adaptive immunity are of a special interest. Low O2 level (1-7 %) is known to be typical for the putative site of the MSC - T cell interactions. A comparative evaluation of the effects of adipose tissue derived MSC (ASCs) on the mitogen-stimulated T cells at the ambient (20 %) and tissue-related (5 %) O2 levels demonstrated reduced T cell activation by the HLA-DR expression, decreased pro-inflammatory and increased anti-inflammatory cytokine production in co-culture, inhibited T cell proliferation, with the effects increased at hypoxia. T cell interactions with ASCs resulted in the up-regulation of PDCD1, Foxp3, and TGFß1 known to play an important role in the immune response suppression, and in the down-regulation of genes involved in the inflammatory reaction (IL2, IFNG). These changes were significantly increased under hypoxia. At the same time, neither ASCs nor the reduced O2 level had negative effects on the viability of T cells.

Phenotype and Secretome of Monocyte-Derived Macrophages Interacting with Mesenchymal Stromal Cells under Conditions of Hypoxic Stress.

We studied the effect of short-term hypoxic stress on the phenotypic polarization of monocyte-derived macrophages and their secretory activity during interaction with mesenchymal stromal cells. In the presence of mesenchymal stromal cells, monocyte-derived macrophages exhibited the signs of M2 polarization, which was evidenced by increased expression of CD206 and CD163 markers, as well as increased transcription and translation of IL-6. Short-term hypoxic stress promoted a shift of macrophage phenotype from inflammatory M1 towards anti-inflammatory M2 in monoculture and co-culture with mesenchymal stromal cells. In addition to the immunoregulatory action, mesenchymal stromal cells demonstrated stromal activity and maintained high viability of monocyte-derived macrophages.

[PROFILE OF THE MARROW-DERIVED STROMAL PRECURSORS POPULATION IN C57BL/6N MICE FLOWN ON BIOSATELLITE BION-M1].

The CFU-F number, proliferative activity and spontaneous differentiation potential of stromal cells derived from the tibia marrow of C57BL/6N mice readapted to the 1-g gravity following a long-term flight on biosatellite Bion-M1 were evaluated. The CFU-F number, proliferative activity and spontaneous adipogenic and osteogenic differentiation of marrow-derived stromal cells from the space flown group were no different from the group of vivarium control. However, the proliferative activity and adhesion properties of the cells were down-regulated on day 7 of readaptation. These results suggest that space flight factors did not impact the stromal differon of the mouse marrow. The decline of stromal cells activity indicates the decompensation of their functions under 1g gravity.

Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.

[Human lymphocyte immunophenotype after interaction with mesenchymal stromal cells].

Human multipotent mesenchymalstromal cells were cocultured for 72h with allogeneic blood-borne mononuclear cells (MNCs) of different maturity (lymphocytes from adult peripheral blood and umbilical cord blood) and functional state (intact and PHA-activated human peripheral blood lymphocytes): After coculture with MMSCs the share ofB-cells among MNCs and cbMNCs decreased. The proportion ofT- and NK cells declined only among cbMNCs (p < 0.05). Only T-NK subpopulation reduced among MNC and cbMNC T-cells. The drop of CD8+ cells was detected in coculture of MMSCs and PHA-MNCs. Activated HLA-DR+ cells declined, CD25+ cells increased compared to monoculture of PHA-MNCs. MMSCs supported MNC, PHA-MNC and cbMNC viability. These results are important for understanding the physiology of the allogeneic cell-to-cell interaction in connection of potential clinical application of allogeneic cell products of blood-borne and stroinal origin.