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: bone marrow mesenchymal stem cells
Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (vol 40, pg 671, 2007)
OBJECTIVES: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential. MATERIALS AND METHODS: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis. RESULTS: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity. CONCLUSIONS: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.
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Culture of rabbit bone marrow mesenchymal stem cells and collagen scaffolds under the mimic microgravity environment in vitro
BACKGROUND:The method of differentiating from bone marrow mesenchymal stem cells(BMSCs) into chondrocytes included in vitro high-density micelle culture,in vitro monolayer cell culture,in vitro three dimensional stent induction,in vitro co-culture induction with chondrocytes and gene transfection.OBJECTIVE:To study adhesion,extension and proliferation of rabbit BMSCs in type Ⅰ and Ⅱ collagen scaffolds under the mimic microgravity environment in vitro.METHODS:BMSCs of rabbits were primarily cultured and subcultured in vitro,and then divided into two groups according to the difference of induction factors:experimental group receiving transforming growth factor(TGF)-β1 and insulin-like growth factor(IGF)-Ⅰ ;blank control group.After three weeks,the two groups were detected by methyl thiazolyl tetrazolium(MTT) assay,measurement of glycosaminoglycan(GAG) and immunohistochemistry.Chondrocytes in the experimental group were incubated in type Ⅰ and Ⅱ collagen scaffolds,and then divided into four groups:group 1:chondrocytes and type Ⅱ collagen scaffolds co-cultured statically;group 2:chondrocytes and type Ⅱ collagen scaffolds co-cultured under the mimic microgravity environment;group 3:chondrocytes and type Ⅰ collagen scaffolds co-cultured statically;group 4:chondrocytes and type Ⅰ collagen scaffolds co-cultured under the mimic microgravity environment.One week later,hematoxylin-eosin staining and toluidine blue staining were performed.RESULTS AND CONCLUSION:The results of the MTT assay(absorbance value) and the GAG content in experimental group were higher than in blank control group.Immunohistochemical detection of collagen Ⅱ was positive in experimental group.Results from hematoxylin-eosin staining and toluidine blue staining have demonstrated that composited cell number in type Ⅱ collagen scaffolds was evidently more than that of type Ⅰ collagen.Composited cell number under the mimic microgravity environment was evidently more than that of static culture in the same scaffold.Above-described results have confirmed that the mimic microgravity environment is conducive to adhesion and proliferation of high-density cells,and contributes to signal transmission among cells to provide suitable microenvironment for maintaining cell growth and metabolism.Collagen type Ⅱ scaffold can be used as a more satisfying scaffold material for chondrocytes than collagen type Ⅰ