Short-Term Interaction with Endothelial Cells Enhances Angiogenic Activity of Growth-Arrested Mesenchymal Stromal Cells In Vitro and In Ovo.

We compared angiogenic effects of conditioned medium from mesenchymal stromal cell (MSC) monoculture and co-culture of MSC with endothelial cells (EC). Conditioned medium from 24-h EC-MSC co-cultures significantly stimulated the proliferation and migration of EC in monoculture and growth of the vascular network of the chorioallantoic membrane of the quail embryo in ovo in comparison with the conditioned medium from MSC monoculture. Conditioned medium from the co-culture contained increased levels of angiogenic factors (FGF-2, MCP-1, PDGF-AB/BB, IL-6, IL-8, etc.), which could explain the revealed effects. We hypothesized that a similar mechanism of EC-mediated enhancement of functional activity of MSC could be involved in reparative angiogenesis in the target tissues in vivo.

Functional Activity of Non-Proliferating Mesenchymal Stromal Cells Cultured at Different Densities.

We analyzed the state of intracellular compartments and production of cytokines in MSC depending on the culture density. MSC were growth-arrested with mitomycin C and seeded at a density of 300-7000 cell/cm2. MSC in low-density cultures had 2-fold higher levels of transmembrane mitochondrial potential (MitoTracker Red) and endogenous ROS (CMH2DCFDA), lysosomal compartments were less acidified (LysoTracker Green DND26), the production of immunoregulatory and angiogenic mediators VEGF, IL-6, IL-8, MCP-1, TGF-ß was more intensive. It was assumed that culture density can be an effective tool for phenotypic polarization of MSC providing directional changes in their properties.

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.

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.

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.

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.

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.

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.

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.

[IMPACT OF ACTIVE OXYGEN FORMS INDUCTION ON EXPRESSION OF MOLECULAR ADHESION AND PARACRINE ACTIVITY OF MESENCHIMAL STROMAL CELLS].

Impact of low doses of active oxygen forms (AOFs) on the paracrine activity of mesenchimal stromal cells (MSCs) was studied. Photodynamic treatment (PDT) was shown to be a method for controlled generation of intracellular AOFs. Active oxygen forms generated at a dose of 0.25J/cm² do not impact significantly the MSCs mitochondrial activity or viability and can be recognized as regulatory. This was the first discovery that low-intensity PDT modulates substantially the MSCs paracrine activity which was manifested by an increased secretion of proinflammatory cytokines (IL-6, IL-8), vascular endothelium growth factor (VEGF), and suppressed secretion of the transforming growth factor (TGFß). While expression of intercellular adhesion molecule ICAM-1 increases and of Thy-1 antigen (a common MSCs marker) decreases, no changes occur to expression of chemokine receptor CXCR4 or other adhesion molecules (H-CAM, MCAM and VCAM-1). Our data make it clear that low-dose PDT is the most important regulator of the MSCs function. Key words: active oxygen forms, mesenchimal stromal cells, paracrine activity, photodynamic exposure.

[EFFECT OF STROMAL CELLS AND OXYGEN CONCENTRATION ON THE MAINTENANCE OF CORD BLOOD HEMATOPOIETIC PRECURSORS].

The paper analyses the morphological and functional features of cord blood cells (CBCs) in the co-culture with multipotent mesenchymal stromal cells (MMSCs) from human adipose tissue under tissue-related oxygen. We have established that MMSCs effectively maintained viability of CBCs at different oxygen concentrations (1, 5 and 20%). According to the data obtained, the oxygen concentration affected the number of colony-forming units (CFUs) formed by CBCs in selective medium. In particular, not co-cultured CBCs the 1 and 5% O2 than at 20% O2. After co-culture with MMSCs, the CFUs numbers were similar at 1 and 20% O2 and increased by 30 at 5% O2. Tissue related O2 concentrations had an impact on the proportion of lineage-resctricted CFCs among CBCs: the number of more committed progenitors--CFU-G and CFU-M, increased, and multi and bipotent--CFU-GEMM and CFU-GM, decreased at low oxygen concentrations. This effect was more pronounced after co-culture compared to that of initial CBCs. Thus, the presence of stromal cells and tissue-related oxygen jointly and severally influenced CBCs in vitro.

WNT-associated gene expression in human mesenchymal stromal cells under hypoxic stress.

The effect of short-term stress on the expression of 84 Wnt-related genes in mesenchymal stromal cells (MSCs) was analyzed. In MSCs permanently expanded at 5% O2, Dkk1, RHOA, CSNK1A1, CSNK2A1, GSK3ß, and PORCN genes were most highly expressed. FZD1, FZD3, SFRP1, and SFRP4 genes were up-regulated after short-term hypoxic stress (0.1% O2), whereas the expression of LEF-1 and RUVBL1 genes was significantly inhibited (down-regulated). The data suggest that Wnt signal transduction was suppressed under hypoxic stress, whereas the noncanonical Wnt signaling prevailed under the "physiological hypoxia" (5% O2).

[Hypoxic stress as a trigger of multipotent mesenchymal stromal cell activation].

Multipotent mesenchymal stromal (stem) cells (MSCs) are a heterogeneous cell population of different commitment and is actively involved in the physiological and regenerative tissue remodeling. MSC mobilization from local tissue depots and their activation in the tissue damage foci are the key issues in the study of mechanisms of MSC features. Short-term (up to 72 h) hypoxic stress that is considered as a constitutive feature of the damage foci, may contribute to the activation of MSC potential. This review is analyzed the data on the impact of short hypoxic stress ex vivo on the viability, functional activity of MSCs and possible molecular mechanisms of these effects.

[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.

[EFFECTS OF HYPOXIA AND GROWTH FACTORS ON THE ANGIOGENIC ACTIVITY OF MULTIPOTENT MESENCHYMAL STROMAL CELLS].

The effects of fetal calf serum (FCS) growth factor concentration and cell growth phase on production of angiogenic mediators by mesenchymal stromal cells (MSCs) at different O2 levels (20 and 5%) was studied. For this purpose vascular endothelial growth factor (VEGF-A) production was measured in MSC-conditioned medium (CM); besides, branching vessels as well as vessel end points (ramification) in the chorioallantoic membrane of Japanese quail eggs (Coturnix coturnix japonica) were counted following MSC-CM application. During the standard cultivation (20% O2; 10% FCS) the total number of vessels was 1.6 times higher comparing with hypoxic condition (5% O2; 10% FCS) due to increase in ramification, the number of branching vessels did not change. Maximal (double) increase in the total vessel number was observed when CM from MSCs after hypoxia plus serum deprivation was added. VEGF-A synthesis linearly increased with FCS concentration both at 20% and 5% O2. In all cases VEGF-A level was higher at hypoxia. No direct correlation between the VEGF-A concentration and total number of vessels was noted indicating that hypoxia possibly stimulates synthesis of additional angiogenic factors to enhance vascular growth despite the drastic serum deprivation. At 20% oxygen, exponentially growing MSCs showed the highest angiogenic activity and the ramification increased in 1.6 times. Depending on O2, MSCs produced angiogenic factors required at different stages of vascularization. Specifically, mediators of ramification were accumulated in the standard conditions (20% O2) and factors stimulating growth of branching vessels--in hypoxia.

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs. METHODS: Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide. RESULTS: We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs. CONCLUSIONS: Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state. GENERAL SIGNIFICANCE: These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.

[Modification of silicon nanoparticles with silver or gold attenuates its biocompatibility in vitro].

The effects of crystalline silicon nanoparticles covered with gold or silver on the viability and state of organelles of cultured human peripheral blood mononuclear cells have been investigated. Exposure to nanosized Si, Si/Ag, Si/Au provoked an increase in the leved of reactive oxygen species in the cells, but did not cause significant reduction in cell viability. Si/Au nanoparticles reduced activity of lysosomes and mitochondria, Si/Ag--only mitochondria. We have concluded that the surface modification by gold or silver may reduce the biocom-atibility of the crystalline silicon nanoparticles.

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.

[Bone marrow mononuclear cells from murine tibia after the space flight on biosatellite "Bion-M1"].

Cellularity, viability and immunophenotype of mononuclear cells derived from the tibial marrow of C57bL/6 mice were measured after the 30-day "Bion-M1" space flight and subsequent 7-day recovery. Cell number in the flight group was significantly less than in the group of vivarium control. There was no difference in the parameter between the flight and control groups after the recovery. Viability of mononuclear cells was more than 95% in all examined groups. Flow cytometric analysis failed to show differences in bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1); however, the flight animals had more large-sized CD45+ mononuclears than the control groups of mice. These results indicate that spaceflight factors did not have significant damaging effects on the number or immunophenotype of murine bone marrow mononuclears. These observations are consistent with the previously made assumption of a moderate and reversible stress reaction of mammals to space flight.