| Bone marrow mesenchymal stem cells has huge application potential intissue engineering and regenerative medicine. However there are manyproblems yet to be solved, such as stem cell differentiation efficiency, thenumber of seed cell and so on. For the differentiation of stem cells, the maininduction method is employed by gene recoding or chemical special inducer.although these methods can be successfully achieved, but the tumorigenicity andexcessive differentiation problems have been the subject of research. At present,there is no specific way to improve their differentiation potential.In the past40years, the space industry development makes people awareof the effects of microgravity on biological life, including cerebrospinal fluidflow change, body fluid electrolyte loss, muscle atrophy, and immune systemfunction decline. Similarly, microgravity alters some properties of cells,including cell morphology and function and the cellular response to the environment. Our study found that after microgravity intervention MSCs cellmorphology changed obviously, from spindle to round." With God, God formwith", shape change will inevitably lead to changes in their function. Under theguidance of the traditional Chinese medicine―Circumference philosophical‖, wethink the cells with round change is a primitive state. The round bone marrowmesenchymal stem cells have greater differentiation potential. This study usesground rotary cultivating device (2D-clinostat) simulation of cell in space toground forces, normal1g gravity (Normal gravity, NG) as a control, withsimulated microgravity (simulated microgravity, SMG) intervention of bonemarrow mesenchymal stem cells to observe the differentiate into neural cellsand endothelial cells. At the same time, cytoskeleton as well as the key moleculeof RhoA activity are observed, thereby directing the possible differentiationmechanism of stem cells. This study for the first time puts forward therelationship of cell morphology and stem cell induction efficiency based on thetraditional Chinese concept under SMG stimulation, which is expected to realizehigh efficiency and safety of stem cells differentiation.Part1The simulated microgravity enhances the differentiationof mesenchymal stem cells into neuronsObjective:to demonstrate that simulated microgravity can enhance thedifferentiation of mesenchymal stem cells into neurons, which might be a newstrategy for the treatment of central nervous system diseases.Method: After isolation and culture of MSCs， cells were identified by flow cytometry(FCM).The3passage cells were used in this study. Thestimulated weightlessness was mimicked by2D-clinostat. FCM was used toanalyze cell apoptosis. Confocal microscopy and real-time PCR analysis wereused to analyze the special markers of neurons.ELISA and real-time PCRdetected the expression of nervous relatve factors. action potentials weredetected by current-clamp whole-cell confguration.Results:rMSCs (rat mesenchymal stem cells) present spread, spindleshape when cultured in normal gravity (NG) while in simulated microgravity(SMG) they become unspread, round shape. rMSCs were cultured respectivelyin normal gravity and in a clinostat to simulate microgravity, followed withneuronal differentiated medium. The neuronal cells derived from rMSCs inSMG express higher microtubule-associated protein-2(MAP-2), tyrosinehydroxylase (TH) and choline acetyltransferase (CHAT). Furthermore, asrMSCs are subjected to SMG, they excrete more neurotrophins like nervegrowth factor (NGF), brain derived neurophic factor (BDNF) and ciliaryneurotrophic factor (CNTF). Neuronal cells from SMG group generated moremature action potentials and displayed repetitive action potentials bycomparison to cells from NG group.Conclusion: simulated microgravity can enhance the differentiation ofmesenchymal stem cells into neurons.Part2The simulated microgravity enhances the differentiation ofmesenchymal stem cells into endothelial cells Objective: to demonstrate that simulated microgravity can enhance thedifferentiation of mesenchymal stem cells into endothelial cells.Methods: primary rat bone marrow mesenchymal stem cells cultured andwere randomly divided into SMG culture group and NG control group;immunofluorescence and reverse transcription PCR detection were used todetect the expression of vWF, Flk-1of induced cells. DiI-Ac-LDL phagocytosisassays and in vitro angioplasty experiment kit for the detection of two groupsafter the induction of endothelial cell were used to analyze the function.Results:①the immune fluorescence detection: in microgravityintervention group, endothelial like cells expressed vWF and Flk-1significantlyhigher than that in normal gravity group. MSCs showed no expression of vWFand Flk-1.②The reverse transcription PCR: endothelial like cells in SMG groupexpressed the vWF level2.8times higher than normal gravity group (P <0.05);the expression of Flk-1level was1.6times higher than normal gravity group (P<0.05).③The DiI-Ac-LDL phagocytic experiment: no obvious differencebetween the two groups was observed after the differentiation of endothelialcells with respect to lipid uptake ability.④The in vitro angioplasty experiment:software Image J analysis software analysis of tube area, the pipe length.Differentiation6days later in SMG group, cells formed ring area and lengthsignificantly higher than that in normal gravity group (P <0.05).Conclusion: simulated microgravity can enhance the differentiation ofmesenchymal stem cells into endothelial cells.Part3cytoskeleton, and RhoA regulate the effects ofmicrogravity on MSCs differentiation Objective: To observe the effect of different time of microgravity oncytoskeleton and the changes of the activity of RhoA, and to investigate theeffect of SMG on regulating stem Cell lineage commitment.Methods:Cells were randomly divided into short time SMG72h, long timeintervention group SMG10d and NG control group; laser confocal detect thecytoskeletal alpha actin, Microtubules changes. MSCs in different groups weredifferentiated into neural, endothelial, adipogenic, osteogenic directiondirection.reverse transcription PCR detected the differentiation efficiency. RhoGTPase pull-down Assay was used to detect RhoA activity changes in differentgroups. The activity of RhoA Exoenzyme C3Transferase blockers was used todetect the changes of MSCs differentiation ability.Results:①phalloidin was as the cytoskeletal alpha actin markers. Shorttime microgravity made cytoskeletal tension decreased, and reduced expression.Microfilament expression was restored and cytoskeletal tension rised inSMG10d group. The beta tubulin was as MSCs cytoskeletal microtubulesprotein marker. Short time microgravity made microtubules disintegrate,obscure, reduce expression. Microtubules has gathered, and tubulin expressionhas been restored in SMG10d group.②The endothelial induction: vWF levelincreased in SMG72h group,1.6times higher than in normal gravity (P <0.05),but with the microgravity processing time for10days, induced cells expressedvWF levels significantly decreased.③The neural induction: induced cellexpression of MAP-2level increased in SMG72h2times higher than in normalgravity(P<0.05). In SMG10days group, MAP-2levels were significantlydecreased(P <0.05).④The osteogenic induction: In SMG72h group,ALPlevels decreased,0.6times less than in the normal gravity group (P <0.05), while in SMG10d group, induced cells expressed ALP levels significantlyincreased(P<0.05).⑤The adipogenic induction: In group SMG72h, PPARgamma2expression level was increased,1.6times higher than in the normalgravity group (P <0.05), but SMG10days,the expression of PPAR gamma2levels were decreased significantly, compared with the SMG72h group (P <0.05).⑥The total amount of RhoA expression in the intervention groups didnot change significantly, but the activation of RhoA expression in SMG72hgroup decreased significantly compared with the normal gravity, fell about80%(P <0.05); while the SMG10days after the RhoA activity picked upsignificantly(P <0.05).⑦With RhoA blockers intervention, the effects ofSMG10d on differentiation was reversed, osteogenic differentiation capacitydecreased, and the expression of ALP reduced80%(P<0.05); in contrast,adipogenic differentiation ability increased, and PPAR gamma2expressionlevels increased about3times (P <0.05).Conclusion: the effects of different time of microgravity on cytoskeletaltension and microtubules clutch are made possible through RhoA regulation.When intervention of MSCs with the short time, it reduces the activity ofRhoA,causing tension loss, promoting the differentiation to the stressunsensitive cells. When intervention with long time SMG, it increases RhoAactivity, causing cell tension rise, promoting the differentiation to the stresssensitivity cells. |
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