Human cranial bone-derived mesenchymal stem cells cultured under simulated microgravity can improve cerebral infarction in rats.

The efficacy of transplanting human cranial bone-derived mesenchymal stem cells (hcMSCs) cultured under simulated microgravity (sMG) conditions has been previously reported; however, their effect on cerebral infarction remains unknown. Here, we examined the efficacy of transplanting hcMSCs cultured in an sMG environment into rat models of cerebral infarction. For evaluating neurological function, hcMSCs cultured in either a normal gravity (1G) or an sMG environment were transplanted in rats 1 day after inducing cerebral infarction. The expression of endogenous neurotrophic, axonal, neuronal, synaptogenic, angiogenic, and apoptosis-related factors in infarcted rat brain tissue was examined using real-time polymerase chain reaction and western blotting 35 days after stroke induction. The RNAs of hcMSCs cultured under 1G or sMG environments were sequenced. The results showed that neurological function was significantly improved after transplantation of hcMSCs from the sMG group compared with that from the 1G group. mRNA expressions of nerve growth factor, fibroblast growth factor 2, and synaptophysin were significantly higher in the sMG group than in the 1G group, whereas sortilin 1 expression was significantly lower. RNA sequencing analysis revealed that genes related to cell proliferation, angiogenesis, neurotrophy, neural and synaptic organization, and inhibition of cell differentiation were significantly upregulated in the sMG group. In contrast, genes promoting microtubule and extracellular matrix formation and cell adhesion, signaling, and differentiation were downregulated. These results demonstrate that hcMSCs cultured in the sMG environment may be a useful source of stem cells for the recovery of neurological function after cerebral infarction.

Simulated microgravity-induced dysregulation of cerebrospinal fluid immune homeostasis by disrupting the blood-cerebrospinal fluid barrier.

BACKGROUND: The blood-cerebrospinal fluid barrier (BCSFB) comprises the choroid plexus epithelia. It is important for brain development, maintenance, function, and especially for maintaining immune homeostasis in the cerebrospinal fluid (CSF). Although previous studies have shown that the peripheral immune function of the body is impaired upon exposure to microgravity, no studies have reported changes in immune cells and cytokines in the CSF that reflect neuroimmune status. The purpose of this study is to investigate the alterations in cerebrospinal fluid (CSF) immune homeostasis induced by microgravity and its mechanisms. This research is expected to provide basic data for brain protection of astronauts during spaceflight. METHODS: The proportions of immune cells in the CSF and peripheral blood (PB) of SMG rats were analyzed using flow cytometry. Immune function was evaluated by measuring cytokine concentrations using the Luminex method. The histomorphology and ultrastructure of the choroid plexus epithelia were determined. The concentrations of intercellular junction proteins in choroid plexus epithelial cells, including vascular endothelial-cadherin (VE-cadherin), zonula occludens 1 (ZO-1), Claudin-1 and occludin, were detected using western blotting and immunofluorescence staining to characterize BCSFB injury. RESULTS: We found that SMG caused significant changes in the proportion of CD4 and CD8 T cells in the CSF and a significant increase in the levels of cytokines (GRO/KC, IL-18, MCP-1, and RANTES). In the PB, there was a significant decrease in the proportion of T cells and NKT cells and a significant increase in cytokine levels (GRO/KC, IL-18, MCP-1, and TNF-α). Additionally, we observed that the trends in immune markers in the PB and CSF were synchronized within specific SMG durations, suggesting that longer SMG periods (≥21 days) have a more pronounced impact on immune markers. Furthermore, 21d-SMG resulted in ultrastructural disruption and downregulated expression of intercellular junction proteins in rat choroid plexus epithelial cells. CONCLUSIONS: We found that SMG disrupts the BCSFB and affects the CSF immune homeostasis. This study provides new insights into the health protection of astronauts during spaceflight.

[Effect of Collagen Peptides on Function of Mouse T Lymphocytes under Simulated Microgravity].

OBJECTIVE: To understand the effect of collagen peptides on the function of mouse lymphocytes under simulated microgravity. METHODS: The splenocytes of mice were isolated, and the rotary cell culture system was used to simulate the microgravity. The T lymphocytes were stimulated with mitotic agents, concanavalin A (ConA), and the cells were treated with different concentrations of collagen peptides. The proliferation of lymphocytes and the levels of cytokines in the supernatant were detected. RESULTS: Simulated microgravity could inhibit the proliferation of spleen T lymphocytes and decrease the level of cytokines in the supernatant. Collagen peptides could promote the lymphocyte proliferation and cytokine production in cells cultured under simulated microgravity. CONCLUSION: Collagen peptides may attenuate the inhibitory effect of simulated microgravity on T lymphocytes by regulating the cell proliferation and the secretion of cytokines.

Can Simulated Microgravity and Darkness Conditions Influence the Phytochemical Content and Bioactivity of the Sprouts?-A Preliminary Study on Selected Fabaceae Species.

Sprouts' consumption has become popular due to their wide availability, easy cultivation process, and proven biological activity. Moreover, stress factors, such as limited access to light or disturbed gravity during growth, may contribute to the increased activity and the synthesis of bioactive compounds. In this study, for the first time, the examination of the impact of darkness and simulated microgravity conditions on the white clover sprouts from the Fabaceae family was conducted. Among several species, used in the preliminary attempts, only white clover was satisfactory sprouting in the disturbed gravity conditions, and thus was chosen for further examination. A random positioning machine setup was used during the cultivation process to simulate microgravity conditions. Additionally, the sprouts were cultivated in total darkness. Simulated microgravity and/or darkness during the first few days of the sprouts' growth caused biomass reduction, the increased synthesis of bioactive compounds (isoflavones and phenolics), and changes in the level of abscisic acid and phenylalanine ammonia-lyase. Moreover, it increased the antioxidant properties of the sprouts, while the enhancement of their cytotoxic impact was observed only for androgen-dependent prostate cancer LNCaP cells. To conclude, the presented results are promising in searching for novel functional food candidates and further studies are necessary, directed at other plant families.

Simulated microgravity increases CD226+ Lin- CD117- Sca1+ mesenchymal stem cells in mice.

Microgravity is one of the most common causes counting for the bone loss. Mesenchymal stem cells (MSCs) contribute greatly to the differentiation and function of bone related cells. The development of novel MSCs biomarkers is critical for implementing effective therapies for microgravity induced bone loss. We aimed to find the new molecules involved in the differentiation and function of MSCs in mouse simulated microgravity model. We found CD226 was preferentially expressed on a subset of MSCs. Simulation of microgravity treatment significantly increased the proportion of CD226+ Lin- CD117- Sca1+ MSCs. The CD226+ MSCs produced higher IL-6, M-CSF, RANKL and lower CD200 expression, and promoted osteoclast differentiation. This study provides pivotal information to understand the role of CD226 in MSCs, and inspires new ideas for prevention of bone loss related diseases.

Simulated microgravity altered the gene expression profiles and inhibited the proliferation of Kupffer cells in the early phase by downregulating LMO2 and EZH2.

Microgravity is a primary challenge that need to overcome, when human travel to space. Our study provided evidence that Kupffer cells (KCs) are sensitive to simulated microgravity (SMG), and no similar research report has been found in the literature. Using transcriptome sequencing technology, it was showed that 631 genes were upregulated and 801 genes were downregulated in KCs after treatment under SMG for 3 days. The GO analysis indicated that the proliferation of KCs was affected when exposed to SMG for 3 days. CCK-8 assay confirmed that the proliferation of KCs was inhibited in the third day under the environment of SMG. Furthermore, we identified 8 key genes that affect the proliferation of KCs and predicted 2 transcription factors (TFs) that regulate the 8 key genes. Significantly, we found that microgravity could affect the expression of LMO2 and EZH2 to reduce the transcription of Racgap1, Ccna2, Nek2, Aurka, Plk1, Haus4, Cdc20, Bub1b, which resulting in the reduction in KCs proliferation. These finding suggested that the inhibition of KCs proliferation under microgravity may influence the homeostasis of liver, and LMO2 and EZH2 can be the targets in management of KCs' disturbance in the future practice of space medicine.

Fluid and Bubble Flow Detach Adherent Cancer Cells to Form Spheroids on a Random Positioning Machine.

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask's rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth's gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research.

The Critical Role of The Piezo1/ß-catenin/ATF4 Axis on The Stemness of Gli1+ BMSCs During Simulated Microgravity-Induced Bone Loss.

Disuse osteoporosis is characterized by decreased bone mass caused by abnormal mechanical stimulation of bone. Piezo1 is a major mechanosensitive ion channel in bone homeostasis. However, whether intervening in the action of Piezo1 can rescue disuse osteoporosis remains unresolved. In this study, a commonly-used hindlimb-unloading model is employed to simulate microgravity. By single-cell RNA sequencing, bone marrow-derived mesenchymal stem cells (BMSCs) are the most downregulated cell cluster, and coincidentally, Piezo1 expression is mostly enriched in those cells, and is substantially downregulated by unloading. Importantly, activation of Piezo1 by systemically-introducing yoda1 mimics the effects of mechanical stimulation and thus ameliorates bone loss under simulated microgravity. Mechanistically, Piezo1 activation promotes the proliferation and osteogenic differentiation of Gli1+ BMSCs by activating the ß-catenin and its target gene activating transcription factor 4 (ATF4). Inhibiting ß-catenin expression substantially attenuates the effect of yoda1 on bone loss, possibly due to inhibited proliferation and osteogenic differentiation capability of Gli1+ BMSCs mediated by ATF4. Lastly, Piezo1 activation also slightly alleviates the osteoporosis of OVX and aged mice. In conclusion, impaired function of Piezo1 in BMSCs leads to insufficient bone formation especially caused by abnormal mechanical stimuli, and is thus a potential therapeutic target for osteoporosis.

Simulated Microgravity Exposure Induces Antioxidant Barrier Deregulation and Mitochondria Enlargement in TCam-2 Cell Spheroids.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell-cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

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.

Frizzled-9 triggers actin polymerization and activates mechano-transducer YAP to rescue simulated microgravity-induced osteoblast dysfunction.

Long-term spaceflight can result in bone loss and osteoblast dysfunction. Frizzled-9 (Fzd9) is a Wnt receptor of the frizzled family that is vital for osteoblast differentiation and bone formation. In the present study, we elucidated whether Fzd9 plays a role in osteoblast dysfunction induced by simulated microgravity (SMG). After 1-7 days of SMG, osteogenic markers such as alkaline phosphatase (ALP), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2) were decreased, accompanied by a decrease in Fzd9 expression. Furthermore, Fzd9 expression decreased in the rat femur after 3 weeks of hindlimb unloading. In contrast, Fzd9 overexpression counteracted the decrease in ALP, OPN, and RUNX2 induced by SMG in osteoblasts. Moreover, SMG regulated phosphorylated glycogen synthase kinase-3ß (pGSK3ß) and ß-catenin expression or sublocalization. However, Fzd9 overexpression did not affect pGSK3ß and ß-catenin expression or sublocalization induced by SMG. In addition, Fzd9 overexpression regulated protein kinase B also known as Akt and extracellular signal-regulated kinase (ERK) phosphorylation and induced F-actin polymerization to form the actin cap, press the nuclei, and increase nuclear pore size, thereby promoting the nuclear translocation of Yes-associated protein (YAP). Our study findings provide mechanistic insights into the role of Fzd9 in triggering actin polymerization and activating YAP to rescue SMG-induced osteoblast dysfunction and suggest that Fzd9 is a potential target to restore osteoblast function in individuals with bone diseases and after spaceflight.

Combined effect of simulated microgravity and low-dose ionizing radiation on structure and antibiotic resistance of a synthetic community model of bacteria isolated from spacecraft assembly room.

Understanding the structural and antibiotic resistance changes of microbial communities in space environments is critical for identifying potential pathogens that may pose health risks to astronauts and for preventing and controlling microbial contamination. The research to date on microbes under simulated space factors has primarily been carried out on single bacterial species under the individual effects of microgravity or low-dose radiation. However, microgravity (MG) and low-dose ionizing radiation (LDIR) coexist in the actual spacecraft environment, and microorganisms coexist as communities in the spacecraft environment. Thus, the microbial response to the real changes present during space habitation has not been adequately explored. To address this knowledge gap, we compared the dynamics of community composition and antibiotic resistance of synthetic bacterial communities under simulated microgravit, low-dose ionizing radiation, and the conditions combined, as it occurs in spacecraft. To ensure representative bacteria were selected, we co-cultured of 12 bacterial strains isolated from spacecraft cleanrooms. We found that the weakened competition between communities increased the possibility of species coexistence, community diversity, and homogeneity. The number of Bacilli increased significantly, while different species under the combined conditions showed various changes in abundance compared to those under the individual conditions. The resistance of the synthetic community to penicillins increased significantly under low doses of ionizing radiation but did not change significantly under simulated microgravity or the combined conditions. The results of functional predictions revealed that antibiotic biosynthesis and resistance increased dramatically in the community under space environmental stress, which confirmed the results of the drug sensitivity assays. Our results show that combined space environmental factors exert different effects on the microbial community structure and antibiotic resistance, which provides new insights into our understanding of the mechanisms of evolution of microorganisms in spacecraft, and is relevant to effective microbial pollution prevention and control strategies.

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1ß, Il6, and Tgfß1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Results: Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Discussion: In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Influence of Simulated Microgravity on Mammary Epithelial Cells Grown as 2D and 3D Cultures.

During space travel, astronauts will experience a unique environment that includes continuous exposure to microgravity and stressful living conditions. Physiological adaptation to this is a challenge and the effect of microgravity on organ development, architecture, and function is not well understood. How microgravity may impact the growth and development of an organ is an important issue, especially as space flight becomes more commonplace. In this work, we sought to address fundamental questions regarding microgravity using mouse mammary epithelial cells in 2D and 3D tissue cultures exposed to simulated microgravity. Mouse mammary HC11 cells contain a higher proportion of stem cells and were also used to investigate how simulated microgravity may impact mammary stem cell populations. In these studies, we exposed mouse mammary epithelial cells to simulated microgravity in 2D and then assayed for changes in cellular characteristics and damage levels. The microgravity treated cells were also cultured in 3D to form acini structures to define if simulated microgravity affects the cells' ability to organize correctly, a quality that is of key importance for mammary organ development. These studies identify changes occurring during exposure to microgravity that impact cellular characteristics such as cell size, cell cycle profiles, and levels of DNA damage. In addition, changes in the percentage of cells revealing various stem cell profiles were observed following simulated microgravity exposure. In summary, this work suggests microgravity may cause aberrant changes in mammary epithelial cells that lead to an increase in cancer risk.

Long-term simulated microgravity alters gut microbiota and metabolome in mice.

Spaceflight and microgravity has a significant impact on the immune, central nervous, bone, and muscle support and cardiovascular systems. However, limited studies are available on the adverse effects of long-term microgravity on the intestinal microbiota, metabolism, and its relationships. In this study, a ground-based simulated microgravity (SMG) mouse model was established to evaluate the impact of long-term microgravity on gut microbiota and metabolome. After 8 weeks of SMG, alterations of the intestinal microbiota and metabolites were detected using 16S rRNA sequencing and untargeted metabolomics. Compared to the control, no significant differences in α-diversity were observed at weeks 2, 4 and 8. Nevertheless, there were clear differences in community structures at different time points. The phylum Verrucomicrobia significantly declined from 2 to 8 weeks of SMG, yet the relative abundance of Actinobacteria and Deferribacteres expanded remarkably at weeks 8. SMG decreased the genus of Allobaculum and increased Bacteroides significantly throughout the period of 8 weeks. Besides, Genus Akkermansia, Gracilibacter, Prevotella, Odoribacter, Rothia, Sporosarcina, Gracilibacter, Clostridium, and Mucispirillum were identified as biomarkers for SMG group. Desulfovibrio_c21_c20, Akkermansia_muciniphila, and Ruminococcus_gnavus dropped at week 2, which tend to recover at week 4, except for Akkermansia_muciniphila. Bacteroides_uniformis and Faecalibacterium_prausnitzii declined significantly, while Ruminococcus_flavefaciens and Mucispirillum_schaedleri elevated at week 8. Furthermore, intestinal metabolome analysis showed that 129 were upregulated and 146 metabolites were downregulated in SMG. Long-term SMG most affected steroid hormone biosynthesis, tryptophan, cysteine, methionine, arginine, proline metabolism, and histidine metabolism. Correlated analysis suggested that the potential beneficial taxa Allobaculum, Akkermansia, and Faecalibacterium were negatively associated with tryptophan, histidine, arginine, and proline metabolism, but positively with steroid hormone biosynthesis. Yet Bacteroides, Lachnospiraceae_Clostridium, Rothia, Bilophila, and Coprococcus were positively correlated with arginine, proline, tryptophan, and histidine metabolism, while negatively associated with steroid hormone biosynthesis. These results suggest that Long-term SMG altered the community of intestinal microbiota, and then further disturbed intestinal metabolites and metabolic pathways, which have great potential to help understand and provide clues for revealing the mechanisms of long-term SMG involved diseases.

Simulated Microgravity Alters P-Glycoprotein Efflux Function and Expression via the Wnt/ß-Catenin Signaling Pathway in Rat Intestine and Brain.

The drug efflux transporter permeability glycoprotein (P-gp) plays an important role in oral drug absorption and distribution. Under microgravity (MG), the changes in P-gp efflux function may alter the efficacy of oral drugs or lead to unexpected effects. Oral drugs are currently used to protect and treat multisystem physiological damage caused by MG; whether P-gp efflux function changes under MG remains unclear. This study aimed to investigate the alteration of P-gp efflux function, expression, and potential signaling pathway in rats and cells under different simulated MG (SMG) duration. The altered P-gp efflux function was verified by the in vivo intestinal perfusion and the brain distribution of P-gp substrate drugs. Results showed that the efflux function of P-gp was inhibited in the 7 and 21 day SMG-treated rat intestine and brain and 72 h SMG-treated human colon adenocarcinoma cells and human cerebral microvascular endothelial cells. P-gp protein and gene expression levels were continually down-regulated in rat intestine and up-regulated in rat brain by SMG. P-gp expression was regulated by the Wnt/ß-catenin signaling pathway under SMG, verified by a pathway-specific agonist and inhibitor. The elevated intestinal absorption and brain distribution of acetaminophen levels also confirmed the inhibited P-gp efflux function in rat intestine and brain under SMG. This study revealed that SMG alters the efflux function of P-gp and regulates the Wnt/ß-catenin signaling pathway in the intestine and the brain. These findings may be helpful in guiding the use of P-gp substrate drugs during spaceflight.

The Effects of Combined Exposure to Simulated Microgravity, Ionizing Radiation, and Cortisol on the In Vitro Wound Healing Process.

Human spaceflight is associated with several health-related issues as a result of long-term exposure to microgravity, ionizing radiation, and higher levels of psychological stress. Frequent reported skin problems in space include rashes, itches, and a delayed wound healing. Access to space is restricted by financial and logistical issues; as a consequence, experimental sample sizes are often small, which limits the generalization of the results. Earth-based simulation models can be used to investigate cellular responses as a result of exposure to certain spaceflight stressors. Here, we describe the development of an in vitro model of the simulated spaceflight environment, which we used to investigate the combined effect of simulated microgravity using the random positioning machine (RPM), ionizing radiation, and stress hormones on the wound-healing capacity of human dermal fibroblasts. Fibroblasts were exposed to cortisol, after which they were irradiated with different radiation qualities (including X-rays, protons, carbon ions, and iron ions) followed by exposure to simulated microgravity using a random positioning machine (RPM). Data related to the inflammatory, proliferation, and remodeling phase of wound healing has been collected. Results show that spaceflight stressors can interfere with the wound healing process at any phase. Moreover, several interactions between the different spaceflight stressors were found. This highlights the complexity that needs to be taken into account when studying the effect of spaceflight stressors on certain biological processes and for the aim of countermeasures development.

Simulated microgravity affects stroma-dependent ex vivo myelopoiesis.

Microgravity is known negatively affect physiology of living beings, including hematopoiesis. Dysregulation of hematopoietic cells and supporting stroma relationships in bone marrow niche may be in charge. We compared the efficacy of ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) in presence of native or osteocommitted MSCs under simulated microgravity (Smg) using Random Positioning Machine (RPM). In comparison with 1 g, a decrease of MSC-associated HSPCs and an increase of floating HSPCs was observed after 7 days of Smg exposure. Among floating HSPCs, primitive progenitors were presented by late CD34+/133-. Total CFUs as well as erythroid (BFU-E) and granulocytic (CFU-G) numbers were lower. MSC-associated primitive HSPCs demonstrated increased proportion of late CD34+/133- in expense of early CD34-/133+. Osteo-MSCs preferentially supported late primitive CD34+ and more committed HSPCs as followed from increase of CFUs, and CD235a+ erythroid progenitors. Under Smg, an increased VEGF, eotaxin, and GRO-a levels, and a decrease in RANTES were found in the osteo-MSC-HSPC co-cultures. IL-6,-8, -13, G-CSF, GRO-a, MCP-3, MIP-1b, VEGF increased in co-culture with osteo-MSCs vs intact MSCs. Based on the findings, the misbalance between primitive/committed HSPCs and a decrease in hematopoiesis-supportive activity of osteocommitted cells are supposed to underline hematopoietic disorders during space flights.

Long-term osteogenic differentiation of human bone marrow stromal cells in simulated microgravity: novel proteins sighted.

Microgravity-induced bone loss is a major concern for space travelers. Ground-based microgravity simulators are crucial to study the effect of microgravity exposure on biological systems and to address the limitations posed by restricted access to real space. In this work, for the first time, we adopt a multidisciplinary approach to characterize the morphological, biochemical, and molecular changes underlying the response of human bone marrow stromal cells to long-term simulated microgravity exposure during osteogenic differentiation. Our results show that osteogenic differentiation is reduced while energy metabolism is promoted. We found novel proteins were dysregulated under simulated microgravity, including CSC1-like protein, involved in the mechanotransduction of pressure signals, and PTPN11, SLC44A1 and MME which are involved in osteoblast differentiation pathways and which may become the focus of future translational projects. The investigation of cell proteome highlighted how simulated microgravity affects a relatively low number of proteins compared to time and/or osteogenic factors and has allowed us to reconstruct a hypothetical pipeline for cell response to simulated microgravity. Further investigation focused on the application of nanomaterials may help to increase understanding of how to treat or minimize the effects of microgravity.

Latest knowledge about changes in the proteome in microgravity.

INTRODUCTION: A long-term stay of humans in space causes health problems and changes in protists and plants. Deep space exploration will increase the time humans or rodents will spend in microgravity (µg). Moreover, they are exposed to cosmic radiation, hypodynamia, and isolation. OMICS investigations will increase our knowledge of the underlying mechanisms of µg-induced alterations in vivo and in vitro. AREAS COVERED: We summarize the findings over the recent 3 years on µg-induced changes in the proteome of protists, plants, rodent, and human cells. Considering the thematic orientation of microgravity-related publications in that time frame, we focus on medicine-associated findings, such as the µg-induced antibiotic resistance of bacteria, the myocardial consequences of µg-induced calpain activation, and the role of MMP13 in osteoarthritis. All these point to the fact that µg is an extreme stressor that could not be evolutionarily addressed on Earth. EXPERT OPINION: In conclusion, when interpreting µg-experiments, the direct, mostly unspecific stress response, must be distinguished from specific µg-effects. For this reason, recent studies often do not consider single protein findings but place them in the context of protein-protein interactions. This enables an estimation of functional relationships, especially if these are supported by epigenetic and transcriptional data (multi-omics).