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.

Changes Induced by Inflammatory-Activated Immune Cell Microenvironment in the Paracrine Profile of MSC.

We studied the influence of activated innate and adaptive immune cells on the production of growth factors by human adipose tissue multipotent mesenchymal stromal cells (MSC). MSC showed immunosuppressive properties in vitro: decreased activation and proliferation of stimulated immune cells. T-cell interaction with MSC resulted with increased secretion of EGF, PDGF-AB/BB, FGF-2, and VEGF growth factors. Co-culturing with natural killer cells also stimulated TGFα production. The intensity of the effect varied depending on the type of immune cells. Natural killer caused a more significant increase in PDGF-AB/BB and FGF-2 secretion, while VEGF secretion increased stronger after co-culturing with T cells. The obtained data indicate the possibility of increasing reparative potential of MSC under the influence of inflammatory microenvironment.

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.

Simulation of Microgravity and Coculturing with Hematopoietic Cells Oppositely Modulate Wnt Signaling in Mesenchymal Stromal Cells.

The osteogenic potential of mesenchymal stromal cells (MSCs) can determine bone homeostasis and the physical characteristics of bones. Microgravity reduces the ability of these cells to differentiate in osteogenic direction. It has been shown that the addition of hematopoietic stem and progenitor cells (HSPCs) to MSC culture in vitro can have the opposite effect. The aim of this study was to identify transcriptional changes in 84 genes associated with Wnt signaling in MSCs during microgravity simulation and interaction with HSPCs. The results indicate an increase in the non-canonical Wnt signaling activity during coculturing of MSCs and HSPCs, while simulated microgravity enhances the canonical component of this signaling pathway. These changes may underlie the modulation of osteogenic potential of MSCs in hematopoietic niche under microgravity.

Bone loss recovery in mice following microgravity with concurrent bone-compartment-specific osteocyte characteristics.

Space missions provide the opportunity to investigate the influence of gravity on the dynamic remodelling processes in bone. Mice were examined following space flight and subsequent recovery to determine the effects on bone compartment-specific microstructure and composition. The resulting bone loss following microgravity recovered only in trabecular bone, while in cortical bone the tissue mineral density was restored after only one week on Earth. Detection of TRAP-positive bone surface cells in the trabecular compartment indicated increased resorption following space flight. In cortical bone, a persistent reduced viability of osteocytes suggested an impaired sensitivity to mechanical stresses. A compartment-dependent structural recovery from microgravity-induced bone loss was shown, with a direct osteocytic contribution to persistent low bone volume in the cortical region even after a recovery period. Trabecular recovery was not accompanied by changes in osteocyte characteristics. These post-space-flight findings will contribute to the understanding of compositional changes that compromise bone quality caused by unloading, immobilisation, or disuse.

Secretory Activity of Mesenchymal Stromal Cells with Different Degree of Commitment under Conditions of Simulated Microgravity.

Microgravity negatively affects the bone tissue which manifested in a decrease in mineral density of the bones during long-term space flights. Impairments of bone homeostasis are determined among other things by changes in secretory activity of heterogeneous populations of low-committed precursors, such as mesenchymal stromal cells (MSC) and osteoblasts. We studied the effect of microgravity modeling during 10 days on paracrine activity of osteogenically committed and intact MSC. Cell response to simulated microgravity depended on the degree of commitment. The response of osteogenically committed MSC was less pronounced and manifested in increased production of sclerostin. In intact MSC, an increase in IL-8 and VEGF secretion and a decrease in osteoprotegerin level were detected. These changes can underlie the shift of bone homeostasis towards bone resorption.

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.

Replicative Senescence and Expression of Autophagy Genes in Mesenchymal Stromal Cells.

Cell senescence leads to a number of changes in the properties of mesenchymal stromal cells (MSCs). In particular, the number of damaged structures is increased producing negative effect on intracellular processes. Elimination of the damaged molecules and organelles occurs via autophagy that can be important in the context of aging. Cultivation under low oxygen level can be used as an approach for enhancement of MSC therapeutic properties and "slowing down" cell senescence. The goal of this work was to study some morphological and functional characteristics and expression of autophagy-associated genes during replicative senescence of MSCs under different oxygen concentration. The study revealed changes in the regulation of autophagy at the transcriptional level. Upregulation of the expression of autophagosome membrane growth genes ATG9A and ULK1, of the autophagosome maturation genes CTSD, CLN3, GAA, and GABARAPL1, of the autophagy regulation genes TP53, TGFB1, BCL2L1, FADD, and HTT was shown. These changes were accompanied by downregulation of IGF1 and TGM2 expression. Increase of the lysosomal compartment volume was observed in the senescent MSCs that also indicated increase of their degradation activity. The number of lysosomes was decreased following prolonged cultivation under low oxygen concentration (5%). The replicative senescence of MSCs under conditions of different oxygen levels led to the similar modifications in the expression of the autophagy-associated genes.

Combined Effects of Irradiation and Hindlimb Suspension on Erythroid Lineage Precursors from Rat Bone Marrow.

Erythroid precursors from the femoral bone marrow of Wistar rats were characterized after 30-day hindlimb suspension, fractionated γ-radiation, and their combination. After hindlimb suspension, the total content of myeloid CFU decreased; activity of erythroid differon also considerably suppressed, which manifested in a decrease in the number of erythroid burst-forming units and area of colonies formed by erythrocyte precursors. After irradiation and combined exposure to these two factors, no significant differences from the control were revealed; optical density of formed colonies slightly increased in all experimental groups. Thus, suppression of the erythroid lineage was most pronounced during hindlimb unloading. The combined effect of radiation and hindlimb suspension produced no appreciable negative effect on erythropoiesis in rat bone marrow.

Extracellular Matrix Proteins and Transcription of Matrix-Associated Genes in Mesenchymal Stromal Cells during Modeling of the Effects of Microgravity.

We studied the effects of simulated microgravity (10 days) on the production of extracellular matrix proteins and expression of extracellular matrix-associated genes in human mesenchymal stem cells. A decrease in collagen production, reduced expression of TIMP-1, TIMP-3, and MMP-11 genes, and enhanced expression of tenascin and laminin subunit were revealed. The results attest to activation of proteolytic processes in the matrix of mesenchymal stromal cells and weakening of cell adhesion to extracellular matrix under conditions of simulated microgravity.

Differential Expression of Bipotent Commitment-Related Genes in Multipotent Mesenchymal Stromal Cells at Different O2 Levels.

The transcriptomic profile associated with osteo- and adipogenic differentiation in growth-arrested multipotent mesenchymal stromal cells (MSCs) from human adipose tissue was analyzed in vitro at 20% (standard laboratory) and 5% (tissue-related) O2 levels. Compared with day 7, at 5% O2 on day 14 spontaneous upregulation of osteo- (RUNX2, SP7, BGLAP, and SPP1) and adipogenic differentiation (CEBPA, PPARG, and ADIPOQ) genes in MSCs was observed (p < 0.05). Thus, upon expansion under tissue-related O2, MSCs demonstrated a bipotent transcriptomic profile, which may contribute to the improvement of their hematopoiesis-supportive function.

Alteration of Hypoxia-Associated Gene Expression in Replicatively Senescent Mesenchymal Stromal Cells under Physiological Oxygen Level.

Mesenchymal stromal cells (MSCs) are a population of adult stem cells that modulate functional state of neighboring tissues. During cell aging, the biological activity of MSC changes, which may affect tissue homeostasis. It is known that reducing the oxygen level in vitro to physiological values typical to a particular cell niche leads to attenuation of some morphological and functional changes associated with aging. This work aimed to study gene expression in MSCs involved in response to physiological hypoxia using a replicative aging model under physiological (5%) and atmospheric (20%) oxygen in cultures. Our results show that significant reduction of proliferative activity of MSCs is observed after 20 passages (~50 cell generations). Regardless of the oxygen, in senescent cells PKM2, SERPINE1, and VEGFA were upregulated while ANKRD37, DDIT4, HIF1A, and TXNIP were downregulated. Also, ADORA2B, BNIPL, CCNG2, EGLN1, MAP3K1, MXI1, and P4HA1 were downregulated under hypoxia. The effect of oxygen was more pronounced at earlier passages both on the cellular and transcription levels. Irrespective of the passage, genes ANGPTL4, GYS1, PKM2, SERPINE1, and TP53 were downregulated under hypoxia. Also, decreased expression was observed for ADM, F10, HMOX1, P4HB, PFKL, SLC16A3 in earlier passages, and for HK2 - in later passages. Upregulation was only observed for ANKRD37, both at early and late cultures.

Secretome of Cultured Human Endothelial Cells in Simulated Microgravity.

The secretome of human umbilical vein endothelial cells (HUVEC) cultured under static conditions and in modeled microgravity for 24 h was studied by chromatography-mass spectrometry. In the secretome of cells exposed to microgravity, we identified a group of microtubule proteins including many structural elements of microtubules and regulatory proteins interacting with Rho-GTPases. Hence, reorganization of actin cytoskeleton and microtubules induced by microgravity is under complex regulation mediated by Rho proteins.

Evaluation of committed and primitive cord blood progenitors after expansion on adipose stromal cells.

Umbilical cord blood mononuclear fraction is a valuable source of hematopoietic stem and progenitor cells (CB HSPCs). The rarity of this population is a serious limitation of its application in cell therapy. Ex vivo expansion enables to significantly amplify the number of hematopoietic precursors of different commitment. Here, we expand CB MNCs in co-culture with human adipose tissue-derived stromal cells (ASCs) to enrich HSPCs and describe phenotypic features of newly formed hematopoietic populations. The CD34+-HSPCs demonstrated 6-fold enrichment with 9000 CFUs per 50 × 103 HSPCs on average. A part of the floating HSPCs were bearing lineage markers, while others were primitive precursors (CD133-/CD34+). Among ASC-associated HSPCs, two subsets of cord blood-borne cells were revealed: СD90+/СD45- and СD90+/СD45+. The proportion of CD3+/CD8+ and NK-T as well as CD25+ and HLA-DR+ Т cells among СD90+/СD45- cells was significantly higher compared to MNCs and floating HSPCs. More than 80% of CD45+/СD90+ HSPCs were identified as late primitive precursors (CD133-/CD34+). Thus, CB MNC expansion in the presence of ASCs provides the generation of both lineage committed lymphoid progenitors and CD34+/CD133- primitive HSPCs. Substantially enriched with primitive precursors, ASC-associated HSPCs could be considered as a perspective tool for a long-term restoration of hematopoiesis in various hematologic disorders.

Expression of Adhesion Molecules in Activated Endothelium after Interaction with Mesenchymal Stromal Cells.

The studied the expression of intercellular adhesion molecules and transcription of the corresponding genes in intact and activated endothelial cells both in monoculture and during interaction with mesenchymal stromal cells. It was found that the levels of integrin-α1 and VE-cadherin mRNA increased during co-culturing. TNFα-induced activation of endothelial cells enhanced expression of integrin-α1 both at the mRNA and protein synthesis stages and had no effect on the level of VE-cadherin. Direct contact with mesenchymal stromal cells did not eliminate the effect of endothelial cell activation, but expression of integrin-α1 and VE-cadherin in activated endothelial cells tended to decrease.

Endothelial Cells Modulate Differentiation Potential and Mobility of Mesenchymal Stromal Cells.

We studied the effect of endothelial cells on in vitro migration and differentiation potential of multipotent mesenchymal stromal cells. Down-regulation of stemness genes OCT4, SOX2, and chondrogenic differentiation regulator SOX9 gene and upregulation of osteogenesis master-gene RUNX2 in mesenchymal stromal cells were observed in the presence of intact and TNFα-activated endothelial cells, which indicated an increase in commitment of mesenchymal stromal cells.The medium conditioned by endothelial cells stimulated migration activity of mesenchymal stromal cells; migration rate increased significantly in conditioned medium from activated cells in comparison with medium from non-activated cells. It was concluded that the interaction with endothelial cells modulated functional activity of mesenchymal stromal cells; moreover, activated endothelial cells produced more pronounced effects on differentiation potential and migration activity of mesenchymal stromal cells both in direct contact and through paracrine regulation.

Effect of 30-Day Hindlimb Unloading and Hypergravity on Bone Marrow Stromal Progenitors in C57Bl/6N Mice.

We studied the effect of 30-day hindlimb unloading and subsequent simulated hypergravity on the cellularity and proliferative, clonogenic, and differentiation potential of bone marrow stromal progenitors in mice. Clonogenic and differentiation activity of stromal cells decreased after unloading; proliferative and differentiation activity of bone marrow stromal progenitors increased after hypergravity simulation. Our findings demonstrated negative effect of unloading on functional activity of mouse bone marrow stromal progenitors. Short-term hypergravity after unloading produced a stimulating effect on the bone marrow stromal progenitors.

The Differential Expression of Adhesion Molecule and Extracellular Matrix Genes in Mesenchymal Stromal Cells after Interaction with Cord Blood Hematopoietic Progenitors.

The dynamics of the expression of genes encoding adhesion molecules, molecules of the connective tissue matrix, and its remodeling enzymes was studied in multipotent mesenchymal stromal cells (MSCs) from human adipose tissue after interaction with cord blood hematopoietic progenitors (HSPCs). An upregulation of ICAM1 and VCAM1, directly proportional to the coculture time (24-72 h), was found. After 72 h of culturing, a downregulation of the genes encoding the majority of matrix molecules (SPP1; COL6A2,7A1; MMP1,3; TIMP1,3; and HAS1) and cell-matrix adhesion molecules (ITGs) was revealed. The detected changes may ensure the realization of the stromal MSC function due to improvement of adhesion and transmigration of HSPCs into the subcellular space.

Myeloid Precursors in the Bone Marrow of Mice after a 30-Day Space Mission on a Bion-M1 Biosatellite.

The content of myeloid stem CFU in bone marrow karyocytes from the tibial bone of C57Bl/6 mice was evaluated after a 30-day Bion-M1 pace flight/ground control experiment and subsequent 7-day recovery period. After the space flight, we observed a significant decrease in the number of erythroid progenitors in the bone marrow, including common myeloid precursor - granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte CFU. After 7-day readaptation, CFU level in flight animals did not recover completely. In the ground control, the count of erythroid burst-forming units was higher than in vivarium animals. Comparison of the changes observed in fight and ground experiments demonstrated effects associated space flight factors and manifesting in suppression of the bone marrow erythropoiesis.

Expression of focal adhesion genes in mesenchymal stem cells under simulated microgravity.

The expression of 84 focal adhesion genes of multipotent mesenchymal stromal cells (MMSCs) after 96-h microgravity simulation at 3D clinorotation was studied. The upregulation of ITGA6, ITGA7, BCAR1, GRB2, CAV1, and DIAPH1 and the downregulation of ITGA11, ITGAV, ITGB1, PTEN, PTK2 (FAK), ARHGAP5, DOCK1, ROCK2, and AKT3 was found. These changes at the transcriptional level may be a cause of the reduction of the osteogenic potential of MMSCs and their ability to migration and adhesion in microgravity.