Regulatory Effects of Senescent Mesenchymal Stem Cells: Endotheliocyte Reaction.

Currently, there is a growing focus on aging and age-related diseases. The processes of aging are based on cell senescence, which results in changes in intercellular communications and pathological alterations in tissues. In the present study, we investigate the influence of senescent mesenchymal stem cells (MSCs) on endothelial cells (ECs). In order to induce senescence in MSCs, we employed a method of stress-induced senescence utilizing mitomycin C (MmC). Subsequent experiments involved the interaction of ECs with MSCs in a coculture or the treatment of ECs with the secretome of senescent MSCs. After 48 h, we assessed the EC state. Our findings revealed that direct interaction led to a decrease in EC proliferation and migratory activity of the coculture. Furthermore, there was an increase in the activity of the lysosomal compartment, as well as an upregulation of the genes P21, IL6, IL8, ITGA1, and ITGB1. Treatment of ECs with the "senescent" secretome resulted in less pronounced effects, although a decrease in proliferation and an increase in ICAM-1 expression were observed. The maintenance of high levels of typical "senescent" cytokines and growth factors after 48 h suggests that the addition of the "senescent" secretome may have a prolonged effect on the cells. It is noteworthy that in samples treated with the "senescent" secretome, the level of PDGF-AA was higher, which may explain some of the pro-regenerative effects of senescent cells. Therefore, the detected changes may underlie both the negative and positive effects of senescence. The findings provide insight into the effects of cell senescence in vitro, where many of the organism's regulatory mechanisms are absent.

Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells.

The process of aging is intimately linked to alterations at the tissue and cellular levels. Currently, the role of senescent cells in the tissue microenvironment is still being investigated. Despite common characteristics, different cell populations undergo distinctive morphofunctional changes during senescence. Mesenchymal stem cells (MSCs) play a pivotal role in maintaining tissue homeostasis. A multitude of studies have examined alterations in the cytokine profile that determine their regulatory function. The extracellular matrix (ECM) of MSCs is a less studied aspect of their biology. It has been shown to modulate the activity of neighboring cells. Therefore, investigating age-related changes in the MSC matrisome is crucial for understanding the mechanisms of tissue niche ageing. This study conducted a broad proteomic analysis of the matrisome of separated fractions of senescent MSCs, including the ECM, conditioned medium (CM), and cell lysate. This is the first time such an analysis has been conducted. It has been established that there is a shift in production towards regulatory molecules and a significant downregulation of the main structural and adhesion proteins of the ECM, particularly collagens, fibulins, and fibrilins. Additionally, a decrease in the levels of cathepsins, galectins, S100 proteins, and other proteins with cytoprotective, anti-inflammatory, and antifibrotic properties has been observed. However, the level of inflammatory proteins and regulators of profibrotic pathways increases. Additionally, there is an upregulation of proteins that can directly cause prosenescent effects on microenvironmental cells (SERPINE1, THBS1, and GDF15). These changes confirm that senescent MSCs can have a negative impact on other cells in the tissue niche, not only through cytokine signals but also through the remodeled ECM.

The Decellularized Cell-Derived Extracellular Matrix Enhances the Paracrine Function of Human Mesenchymal Stromal/Stem Cells.

The mesenchymal stromal/stem cells (MSCs) are known to secrete pleiotropic paracrine factors, contributing to tissue regeneration. This unique ability makes MSCs promising therapeutic tools for many diseases, including even those that were previously untreatable. Thus, the development of preconditioning approaches aimed at enhancing the paracrine function of MSCs attracts great interest. In the present work, we studied how the extracellular matrix, the essential part of the native tissue microenvironment, affects the secretory capacity of MSCs of various origins. The MSC-derived decellularized extracellular matrix (dECM), used as the cell culture substrate, triggered strong upregulation of FGF-2, MMP-1, HGF, GRO-α, GRO-ß, CXCL-5, CXCL-6, IL-6, IL-8, G-CSF and MCP-1. Functional in vitro tests revealed that conditioned media derived from MSCs cultured on dECM significantly improved 3T3 fibroblast and HaCaT keratinocyte scratch wound healing, stimulated THP-1 monocyte migration and promoted capillary-like HUVEC-based tube formation compared to conditioned media from MSCs grown on plastic. In addition, we found that FAK inhibition promoted dECM-induced upregulation of paracrine factors, suggesting that this kinase participates in the MSCs' paracrine response to dECM. Together, these findings demonstrate that dECM provides cues that considerably enhance the secretory function of MSCs. Thus, dECM usage as a cell culture substrate alone or in combination with a FAK inhibitor may be viewed as a novel MSC preconditioning technique.

Microgravity Effects and Aging Physiology: Similar Changes or Common Mechanisms?

Despite the use of countermeasures (including intense physical activity), cosmonauts and astronauts develop muscle atony and atrophy, cardiovascular system failure, osteopenia, etc. All these changes, reminiscent of age-related physiological changes, occur in a healthy person in microgravity quite quickly - within a few months. Adaptation to the lost of gravity leads to the symptoms of aging, which are compensated after returning to Earth. The prospect of interplanetary flights raises the question of gravity thresholds, below which the main physiological systems will decrease their functional potential, similar to aging, and affect life expectancy. An important role in the aging process belongs to the body's cellular reserve - progenitor cells, which are involved in physiological remodeling and regenerative/reparative processes of all physiological systems. With age, progenitor cell count and their regenerative potential decreases. Moreover, their paracrine profile becomes pro-inflammatory during replicative senescence, disrupting tissue homeostasis. Mesenchymal stem/stromal cells (MSCs) are mechanosensitive, and therefore deprivation of gravitational stimulus causes serious changes in their functional status. The review compares the cellular effects of microgravity and changes developing in senescent cells, including stromal precursors.

Hypoxic Extracellular Matrix Preserves Its Competence after Expansion of Human MSCs under Physiological Hypoxia In Vitro.

Tissue-relevant O2 levels are considered as an important tool for the preconditioning of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine needs. The present study investigated the quality and functions of the extracellular matrix (ECM) of MSCs under low O2 levels. Human adipose tissue-derived MSCs were continuously expanded under normoxia (20% O2, N) or "physiological" hypoxia (5% O2, Hyp). Decellularized ECM (dcECM) was prepared. The structure of the dcECM was analyzed using confocal laser and scanning electron microscopy. Collagen, dcECM-N, and dcECM-Hyp were recellularized with MSC-N and further cultured at normoxia. The efficacy of adhesion, spreading, growth, osteogenic potential, and paracrine activity of recellularized MSC-N were evaluated. At low O2, the dcECM showed an increased alignment of fibrillar structures and provided accelerated spreading of MSC-N, indicating increased dcECM-Hyp stiffness. We described O2-dependent "ECM-education" of MSC-N when cultured on dcECM-Hyp. This was manifested as attenuated spontaneous osteo-commitment, increased susceptibility to osteo-induction, and a shift in the paracrine profile. It has been suggested that the ECM after physiological hypoxia is able to ensure the maintenance of a low-commitment state of MSCs. DcECM, which preserves the competence of the natural microenvironment of cells and is capable of "educating" others, appears to be a prospective tool for guiding cell modifications for cell therapy and tissue engineering.

Heterotypic Cell Culture from Mouse Bone Marrow under Simulated Microgravity: Lessons for Stromal Lineage Functions.

Muscle and skeleton structures are considered most susceptible to negative factors of spaceflights, namely microgravity. Three-dimensional clinorotation is a ground-based simulation of microgravity. It provides an opportunity to elucidate the effects of microgravity at the cellular level. The extracellular matrix (ECM) content, transcriptional profiles of genes encoding ECM and remodelling molecules, and secretory profiles were investigated in a heterotypic primary culture of bone marrow cells after 14 days of 3D clinorotation. Simulated microgravity negatively affected stromal lineage cells, responsible for bone tissue formation. This was evidenced by the reduced ECM volume and stromal cell numbers, including multipotent mesenchymal stromal cells (MSCs). ECM genes encoding proteins responsible for matrix stiffness and cell-ECM contacts were downregulated. In a heterotypic population of bone marrow cells, the upregulation of genes encoding ECM degrading molecules and the formation of a paracrine profile that can stimulate ECM degradation, may be mechanisms of osteodegenerative events that develop in real spaceflight.

Stromal Lineage Precursors from Rodent Femur and Tibia Bone Marrows after Hindlimb Unloading: Functional Ex Vivo Analysis.

Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.

Real and Simulated Microgravity: Focus on Mammalian Extracellular Matrix.

The lack of gravitational loading is a pivotal risk factor during space flights. Biomedical studies indicate that because of the prolonged effect of microgravity, humans experience bone mass loss, muscle atrophy, cardiovascular insufficiency, and sensory motor coordination disorders. These findings demonstrate the essential role of gravity in human health quality. The physiological and pathophysiological mechanisms of an acute response to microgravity at various levels (molecular, cellular, tissue, and physiological) and subsequent adaptation are intensively studied. Under the permanent gravity of the Earth, multicellular organisms have developed a multi-component tissue mechanosensitive system which includes cellular (nucleo- and cytoskeleton) and extracellular (extracellular matrix, ECM) "mechanosensory" elements. These compartments are coordinated due to specialized integrin-based protein complexes, forming a distinctive mechanosensitive unit. Under the lack of continuous gravitational loading, this unit becomes a substrate for adaptation processes, acting as a gravisensitive unit. Since the space flight conditions limit large-scale research in space, simulation models on Earth are of particular importance for elucidating the mechanisms that provide a response to microgravity. This review describes current state of art concerning mammalian ECM as a gravisensitive unit component under real and simulated microgravity and discusses the directions of further research in this field.

Short-Term Reloading After Prolonged Unloading Ensures Restoration of Stromal but Not Hematopoietic Precursor Activity in Tibia Bone Marrow of C57Bl/6N Mice.

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sµg). To evaluate the effect of sµg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.

Microgravity Effects on the Matrisome.

Gravity is fundamental factor determining all processes of development and vital activity on Earth. During evolution, a complex mechanism of response to gravity alterations was formed in multicellular organisms. It includes the "gravisensors" in extracellular and intracellular spaces. Inside the cells, the cytoskeleton molecules are the principal gravity-sensitive structures, and outside the cells these are extracellular matrix (ECM) components. The cooperation between the intracellular and extracellular compartments is implemented through specialized protein structures, integrins. The gravity-sensitive complex is a kind of molecular hub that coordinates the functions of various tissues and organs in the gravitational environment. The functioning of this system is of particular importance under extremal conditions, such as spaceflight microgravity. This review covers the current understanding of ECM and associated molecules as the matrisome, the features of the above components in connective tissues, and the role of the latter in the cell and tissue responses to the gravity alterations. Special attention is paid to contemporary methodological approaches to the matrisome composition analysis under real space flights and ground-based simulation of its effects on Earth.

Simulated Microgravity Remodels Extracellular Matrix of Osteocommitted Mesenchymal Stromal Cells.

The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight.

Magnetic levitational bioassembly of 3D tissue construct in space.

Magnetic levitational bioassembly of three-dimensional (3D) tissue constructs represents a rapidly emerging scaffold- and label-free approach and alternative conceptual advance in tissue engineering. The magnetic bioassembler has been designed, developed, and certified for life space research. To the best of our knowledge, 3D tissue constructs have been biofabricated for the first time in space under microgravity from tissue spheroids consisting of human chondrocytes. Bioassembly and sequential tissue spheroid fusion presented a good agreement with developed predictive mathematical models and computer simulations. Tissue constructs demonstrated good viability and advanced stages of tissue spheroid fusion process. Thus, our data strongly suggest that scaffold-free formative biofabrication using magnetic fields is a feasible alternative to traditional scaffold-based approaches, hinting a new perspective avenue of research that could significantly advance tissue engineering. Magnetic levitational bioassembly in space can also advance space life science and space regenerative medicine.

Secretome of Senescent Adipose-Derived Mesenchymal Stem Cells Negatively Regulates Angiogenesis.

Nowadays, paracrine regulation is considered as a major tool of mesenchymal stem cell (MSC) involvement in tissue repair and renewal in adults. Aging results in alteration of tissue homeostasis including neovascularization. In this study, we examined the influence of replicative senescence on the angiogenic potential of adipose-derived MSCs (ASCs). Angiogenic activity of conditioned medium (CM) from senescent and "young" ASCs was evaluated in chorioallantoic membrane (CAM) assay in ovo using Japanese quail embryos. Also, the formation of capillary-like tubes by human umbilical vein endothelial cells (HUVECs) in 3D basement membrane matrix ''Matrigel'' and HUVEC migration capacity were analyzed. Multiplex, dot-blot and gene expression analysis were performed to characterize transcription and production of about 100 angiogenesis-associated proteins. The results point to decreased angiogenic potential of senescent ASC secretome in ovo. A number of angiogenesis-associated proteins demonstrated elevation in CM after long-term cultivation. Meanwhile, VEGF (key positive regulator of angiogenesis) did not change transcription level and concentration in CM. Increasing both pro- (FGF-2, uPA, IL-6, IL-8 etc.) and antiangiogenic (IL-4, IP-10, PF4, Activin A, DPPIV etc.) factors was observed. Some proangiogenic genes were downregulated (IGF1, MMP1, TGFB3, PDGFRB, PGF). Senescence-associated secretory phenotype (SASP) modifications after long-term cultivation lead to attenuation of angiogenic potential of ASC.

Adipose tissue-derived stromal cells retain immunosuppressive and angiogenic activity after coculture with cord blood hematopoietic precursors.

Adipose-tissue derived stromal cells (ASCs) are currently considered as a full value alternative source of bone marrow MSCs for prevention of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation due to their immunosuppressive potential. Besides, ASCs are known to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ex vivo expansion enables to amplify significantly the number of HSPCs of different commitment. Mononuclear cells (MNCs) from cord blood (cb) contain HSPCs and are easily assessed. The rarity of those HSPCs is a serious limitation of its application in cell therapy. Here we expanded cbMNCs in stroma-dependent setting to generate heterocellular associates consisting of ASCs and undifferentiated and low committed hematopoietic cbHSPCs. A part of cbHSPCs in associates demonstrated a primitive phenotype confirmed by formation of "cobblestone areas". ASCs associated with cbHSPCs demonstrated up-regulation of immunosuppressive indoleamine 2,3-dioxygenase (IDO), leukemia inhibitory factor (LIF), cyclooxygenase-2 (PTGS2) genes. ASC-cbHSPCs as well as ASCs provoked the suppression of HLA-DR activation and apoptosis of mitogen-stimulated T cells. VEGF transcription and secretion were elevated providing stimulation of blood vessel formation in ovo. Thus, ASCs retain immunosuppressive and proangiogenic capacities evidencing "third party" potential along with the effective support of ex vivo expansion of cbHSPCs. Above functions expand the relevance of ASCs for needs of regenerative medicine.

Low-dose photodynamic therapy promotes angiogenic potential and increases immunogenicity of human mesenchymal stromal cells.

Photodynamic therapy (PDT) is a non-invasive FDA and EMA-approved anticancer treatment modality. Initially developed for elimination of malignant cells, PDT affects all cells in the tumor bed including stromal cells. Stroma represents not only an important component of tumor microenvironment, but has a significant impact on tumor susceptibility to PDT and other anticancer therapies. However, the effects of PDT on stromal cells are poorly investigated. During PDT the tumor stroma can receive low-dose irradiation as a result of chosen regimen or limited depth of light penetration. Here, we characterized response of human mesenchymal stromal cells (MSCs) to low-dose PDT. In an in vitro model we demonstrated that low-dose PDT resulted in activation of Erk1/2 and inhibition of GSK-3 signaling in MSCs. PDT-mediated induction of intracellular reactive oxygen species (ROS) resulted in reorganization of MSC cytoskeleton and decreased cell motility. More importantly, low-dose PDT dramatically upregulated secretion of various proangiogenic factors (VEGF-A, IL-8, PAI-1, MMP-9, etc.) by MSCs and improved MSC ability to promote angiogenesis suggesting an increase in the pro-tumorigenic potential of MSCs. In contrast, co-cultivation of PDT-treated MSCs with lymphocytes resulted in significant decrease of MSC viability and potential increase in MSC immunogenicity, which may lead to increased anti-tumor immunity. Low-dose PDT in MSCs significantly inhibited secretion of CCL2 (MCP-1) potentially limiting infiltration of pro-tumorigenic macrophages. Altogether, our findings demonstrate that low-dose PDT significantly modifies functional properties of MSCs improving their pro-tumorigenic potential while simultaneously increasing potential immune stimulation suggesting possible mechanisms of stromal cell contribution to PDT efficacy.

Simulated microgravity modulates the mesenchymal stromal cell response to inflammatory stimulation.

The duration and distance of manned space flights emphasizes the importance of advanced elucidation of space flight factors and their effects on human beings. The exposure to inflammatory mediators under microgravity may contribute to the activity of different cells, perivascular stromal cells (MSCs) in particular. Inflammatory activation is now considered as a principal cue of MSC engagement in reparative remodeling. In the present paper, the effect of simulated microgravity (sµg) on TNFα-mediated priming of adipose tissue-derived MSC (ASCs) was examined. Sµg per se did not induce inflammatory-related changes, such as elevation of ICAM-1 and HLA-ABC expression, soluble mediator production, or shifting of the transcription profile in ASCs. Moreover, the attenuated ASC response to TNFα priming under sµg was manifested in decreased production of TNFα-dependent pleiotropic cytokines (IL-8 and MCP-1), matrix remodeling proteases, and downregulation of some genes encoding growth factors and cytokines. Time-dependent analysis detected the first signs of priming attenuation after 48 hours of 3D-clinorotation. A reduced response of MSCs to priming under sµg can be a negative factor in terms of MSC involvement in tissue remodeling processes.

Reciprocal modulation of cell functions upon direct interaction of adipose mesenchymal stromal and activated immune cells.

The interaction of adipose mesenchymal stromal cells (ASCs) and allogeneic peripheral blood mononuclear cells (PBMCs) is regulated either through direct or paracrine mechanisms. Here, we examined the impact of direct contact in reciprocal regulation of ASC-PBMC functions. Activated PBMCs in vitro induced ASC immunomodulatory activity, while direct and paracrine intercellular interactions regulated PBMCs themselves: the functional state of the organelles was altered, and activation decreased. Direct contact with immune cells affected the activity of ASC intracellular compartments, in particular, reactive oxygen species (ROS) production, and decreased the growth rate. Some ASC properties, including motility, intercellular adhesion molecule-1 (ICAM-1), and major histocompatibility complex class I and II antigens (HLA-ABC and HLA-DR, respectively) expression, did not depend on contact with PBMCs and were only regulated by paracrine means. Direct ASC and PBMC contact favoured an angiogenesis-supportive microenvironment, possibly due to the greater production of VEGF by ASCs; this microenvironment also contained a higher leukemia inhibitory factor (LIF) level. Thus, a change in the functional activity of ASCs and PBMCs upon interaction promoted the formation of an immunosuppressive, anti-inflammatory, and proangiogenic microenvironment. This environment could help resolve inflammation and further restore damaged tissue. SIGNIFICANCE OF THE STUDY: Numerous studies have demonstrated the beneficial effects of transplanted mesenchymal stromal cells, particularly ASCs, for the treatment of a number of autoimmune diseases as well as various tissue injuries. To improve the efficiency of these methods, it is necessary to understand the principal events that occur when ASCs are introduced, primarily the molecular mechanisms of interaction between ASCs and the recipient immune system. We demonstrated that an anti-inflammatory, immunosuppressive, and angiostimulatory shift in the paracrine profile upon the interaction of activated PBMCs and ASCs changes the functional activity of both cell types, a phenomenon that is potentiated by direct cell-cell contact.

Hematopoiesis-supportive function of growth-arrested human adipose-tissue stromal cells under physiological hypoxia.

Ex vivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of ex vivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the ex vivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p < 0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration in vivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.

Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45+MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

Angiogenic Activity of Human Adipose-Derived Mesenchymal Stem Cells Under Simulated Microgravity.

Multipotent mesenchymal stem/stromal cells (MSCs) are strongly involved in tissue homeostasis mainly through paracrine regulation. In this study, we examined the influence of simulated microgravity on the angiogenic potential of adipose-derived MSCs (ASCs). The conditioned medium (CM) from random positioning machine (RPM)-exposed ASCs stimulated the formation of vessel network in ovo, endothelial cell (EC) capillary-like network, and nondirected EC migration in vitro. These effects were driven by alteration of both angiogenesis-related gene and protein expression. The elevation of angiogenic regulators Serpin E1, Serpin F1, IGFBP, VEGF, and IL-8 was detected in ASC-CM after 3D-clinorotation. In addition, transcription of genes encoding growth factors with proangiogenic activity were upregulated including VEGF-c and VEGF-a. These data evidenced that besides direct effect on ECs, microgravity could provoke MSC-mediating specific microenvironment for ECs supporting their functions, that is, proliferation and migration via increased production of IL-8 and VEGF as well as other paracrine factors involved in angiogenesis regulation.