Due to spaceflight, astronauts experience serious, weightlessness-induced bone loss because of an unbalanced process of bone remodeling that involves bone marrow mes- enchymal stem cells (BMSCs), as well as osteoblasts, osteo- cytes, and osteoclasts. The effects of microgravity on osteo- cells have been extensively studied, but it is only recently that consideration has been given to the role of BMSCs. Pre- vious researches indicated that human BMSCs cultured in simulated microgravity (sim-μg) alter their proliferation and differentiation. The spaceflight opportunities for biomedical experiments are rare and suffer from a number of opera- tive constraints that could bias the validity of the experiment itself, but remain a unique opportunity to confirm and explain the effects due to microgravity, that are only par- tially activated/detectable in simulated conditions. For this reason, we carefully prepared the SCD – STEM CELLS DIFFERENTIATION experiment, selected by the European Space Agency (ESA) and now on the International Space Station (ISS). Here we present the preparatory studies per- formed on ground to adapt the project to the spaceflight constraints in terms of culture conditions, fixation and stor- age of human BMSCs in space aiming at satisfying the biological requirements mandatory to retrieve suitable sam- ples for post-flight analyses. We expect to understand better the molecular mechanisms governing human BMSC growth and differentiation hoping to outline new countermeasures against astronaut bone loss.
Research Containing: Gene Expression
Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET gamma radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET gamma radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFbeta gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.
Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.
AIM: The goal of the study was to evaluate changes in lung status due to spaceflight stressors that include radiation above levels found on Earth.;MATERIALS AND METHODS: Within hours after return from a 13-day mission in space onboard the Space Shuttle Atlantis, C57BL/6 mice (FLT group) were euthanized; mice housed on the ground in similar animal enclosure modules served as controls (AEM group). Lung tissue was collected to evaluate the expression of genes related to extracellular matrix (ECM)/adhesion and stem cell signaling. Pathway analysis was also performed. In addition, immunohistochemistry for stem cell antigen-1 (SCA-1), the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis, and staining for histological characteristics were performed.;RESULTS: There were 18/168 genes significantly modulated in lungs from the FLT group (p<0.05 vs. AEM); 17 of these were up-regulated and one was down-regulated. The greatest effect, namely a 5.14-fold increase, was observed on Spock1 (also known as Spark/osteonectin), encoding a multi-functional protein that has anti-adhesive effects, inhibits cell proliferation and regulates activity of certain growth factors. Additional genes with increased expression were cadherin 3 (Cdh3), collagen, type V, alpha 1 (Col5a1), integrin alpha 5 (Itga5), laminin, gamma 1 (Lamc1), matrix metallopeptidase 14 (Mmp14), neural cell adhesion molecule 1 (Ncam1), transforming growth factor, beta induced (Tgfbi), thrombospondin 1 (Thbs1), Thbs2, versican (Vcan), fibroblast growth factor receptor 1 (Fgfr1), frizzled homolog 6 (Fzd6), nicastrin (Ncstn), nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 (Nfatc4), notch gene homolog 4 (Notch4) and vang-like 2 (Vangl2). The down-regulated gene was Mmp13. Staining for SCA-1 protein showed strong signal intensity in bronchiolar epithelial cells of FLT mice (p<0.05 vs. AEM). TUNEL positivity was also significantly higher in the FLT mice (p<0.05 vs. AEM), but no consistent histological differences were noted. CONCLUSION: The results demonstrate that spaceflight-related stress had a significant impact on lung integrity, indicative of tissue injury and remodeling.
Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth’s gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth’s gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth.
Bone Proteomics experiment (BOP): the first proteomic analysis of mammalian cells cultured in weightlessness conditions
Purpose. Bone mass loss is a major consequence of extended periods of weightlessness. Many studies performed on astronauts and animals have shown that impaired maturation of osteoblast cells as well as a decrease of their bone-synthesising activity play key roles in microgravity-dependent bone mass loss. Several experiments on single cells and tissues showed that weightlessness can also influence cells cultured in vitro. Many molecular mechanisms are affected, among which the cytoskeleton, signal transduction cascades and gene expression. However, the underlying mechanisms of these changes and their molecular consequences are far from being fully understood. In contrast to weightlessness, dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production. A growing body of evidence points to extracellular nucleotides (i.e. ATP and UTP) as soluble factors that are released by several cell types in response to mechanical stimulation and that eventually trigger an intracellular signal. We have recently demonstrated that ATP and UTP, as well as mechanical stimulation, can activate two fundamental transcription factors, Runx2 and Egr-1, in the human HOBIT osteoblast cell line [Pines A. et al.,Biochem J. 2003; Costessi A. et al., Bone, 2005]. The purpose of the present study was to investigate the possible role(s) of extracellular nucleotides in the molecular response of osteoblast cells to weightlessness conditions. We focused on two aspects: 1. whether administration of ATP could stimulate osteoblast cells in weightlessness, possibly balancing or overcoming its known negative effects; 2. An analysis of the proteome of osteoblast cells exposed to weightlessness by means of two dimension electrophoresis (2-DE) coupled to mass spectrometry, to identify new molecular targets. Methodology – The BOP experiment. BOP was selected in the Success 2002 Student Contest organized by ESA and awarded to A.C. We developed and produced a new and low-cost dedicated hardware to support the experiment. We decided to use the human osteoblast cell line MG-63, since they had been studied in previous space experiments. The cells were grown in space for about five days in three chambers (control cells and cells treated with ATP for 20’ or 3 hours) and eventually lysed. A parallel ground experiment was performed. BOP flew during the Italian Soyuz Taxi flight in April 2005. Results and conclusions. We developed in less than nine months a new hardware that provided 70 cm2 per chamber and a total of more than 200 cm2. Preliminary analysis indicate that administration of ATP to MG-63 cells cultured in weightlessness conditions is able to increase ERK phosphorylation. Analysis of 2D gels revealed several differentially regulated proteins in response to ATP treatment. The identification of these proteins is in progress. To the best of our knowledge, BOP is the first proteomic study on mammalian cells cultured in space. The conclusion of the analysis will reveal new aspects of osteoblast biology and provide new insights into the molecular responses of human cells to weightlessness.
Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis
Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.
Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?
Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.
Spaceflight Effects and Molecular Responses in the Mouse Eye: Preliminary Observations After Shuttle Mission STS-133
Spaceflight exploration presents environmental stressors including microgravity-induced cephalad fluid shift and radiation exposure. Ocular changes leading to visual impairment in astronauts are of occupational health relevance. The effect of this complex environment on ocular morphology and function is poorly understood. Female 10-12 week-old BALB/cJ mice were assigned to a flight (FLT) group flown on shuttle mission STS-133, Animal Enclosure Module ground control group (AEM), or vivarium-housed (VIV) ground controls. Eyes were collected at 1, 5, and 7 days after landing and were fixed for histological sectioning. The contralateral eye was used for gene expression profiling by RT-qPCR. Sections were visualized by hematoxylin/eosin stain and processed for 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3, and glial fibrillary acidic protein (GFAP) and β-amyloid double-staining. 8-OHdG and caspase-3 immunoreactivity was increased in the retina in FLT samples at return from flight (R+1) compared to ground controls, and decreased at day 7 (R+7). Β-amyloid was seen in the nerve fibers at the post-laminar region of the optic nerve in the flight samples (R+7). Expression of oxidative and cellular stress response genes was upregulated in the retina of FLT samples upon landing, followed by lower levels by R+7. These results suggest that reversible molecular damage occurs in the retina of mice exposed to spaceflight and that protective cellular pathways are induced in the retina and optic nerve in response to these changes.
Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq
A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.