| Experimental cell research studying the influence of microgravity on cell growth and tissue regeneration in space is currently a hot topic in space life science and gravitional biology.Although a variety of Earth-bound equipment has been used to study the effect of microgravity on proliferation and differentiation of ESCs,simulated microgravity remains a controversial substitute for real microgravity.Recently,NASA reported a space based stem cell research that they performed an experiment using embryoid bodies(EBs)as a model to test the impacts of space microgravity on early lineage commitment of mouse embryonic stem cells(ESCs).They demonstrated that microgravity inhibited EB differentiation and maintained sternness of EBs by gene expression analysis and post-microgravity cellular differentiation and viability assays when the samples returned to Earth.However,there was no system available that allowed long-term culture of stem cells or real-time imaging in space microgravity.Due to resource limitation and an unmanned spacecraft,there is currently no efficient method or very difficult to real-time investigate the properties of stem cells under microgravity environment.Thus,it is necessary to futher investigate the effect of space microgravity on the stem cell character of proliferation and differentiation with real-time observation in orbit.In this study,OCT4-GFP reporter mouse ESCs line,OCT4-GFP mouse ESCs-derived EB and Brachyury-GFP mouse ESCs-derived EB were used as experiment samples in the TZ-1 spaceflight mission.First,a novel suspension culture method of serum-free and feeder-free with chemical defined medium for maintaining the sternness of pluripotent stem cells was exploitation.ESCs formed three dimensional(3-D)aggregates resembling the neurospheres and sustained long-term proliferation,their undifferentiated state and pluripotency in the suspension culture that comparable to conventional ESCs cultured method.Addition,we have developed an automated culture system to culture ESCs and EB in C02 independent conditions.Our results demonstrated that mouse ESCs cultured in matrigel-coated chambers grew well and propagated in this automated culture system,which could be used to culture mouse ESCs and EBs for the subsequent spaceflight experiment.Finally,we utilized real microgravity environment in the TZ-1 cargo spacecraft,an automatic cell culture equipment,and live cell imaging techniques to examine the effects of microgravity on the morphology,proliferation,and differentiation of mESCs.The image data obtained from spaceflight(?g),in comparison with the results from ground control(1g),showed that mESCs can be cultured for long-term survival,and enhancement of sternness marker in space microgravity.Additionally,we observed that stem cell colonies grew from original flat layers to 3-D aggregates during spaceflight,suggesting the advantage of 3-D growth in microgravity.For EB differentiation,mouse EBs cultured in attachment dishes were capable of developing outgrowth on the ECM substrate in microgravity conditions,expressing a lower level of OCT4-GFP.Interestingly,we found that although EB could differentiate into the mesendoderm lineage in the space environment,these differentiated cells maintained high expression of Brachyury,even after 15 days of culture.In conclusion,our results demonstrate that the space microgravity environment might play a potential role in supporting 3-D growth of cells and maintenance of sternness in embryonic stem cells,while spaceflight may have a negative effect on the terminal differentiation of ESCs.Our results possibly lead to a new understanding of the effect of space microgravity on the proliferation of pluripotent stem cells and initiation of their early differentiation.These results will also allow us to offer a novel strategy for tissue engineering in regenerative medicine,and it should provide useful information for stem cells expansion under microgravity condition. |
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