Microgravity Effect on Pancreatic Islets.

We previously demonstrated that boundary cap neural crest stem cells (BCs) induce the proliferation of beta-cells in vitro, increase survival of pancreatic islets (PIs) in vivo after transplantation, and themselves strongly increase their proliferation capacity after exposure to space conditions. Therefore, we asked if space conditions can induce the proliferation of beta-cells when PIs are alone or together with BCs in free-floating or 3D-printed form. During the MASER 15 sounding rocket experiment, half of the cells were exposed to 6 min of microgravity (µg), whereas another group of cells were kept in 1 g conditions in a centrifuge onboard. The proliferation marker EdU was added to the cells just before the rocket reached µg conditions. The morphological assessment revealed that PIs successfully survived and strongly proliferated, particularly in the free-floating condition, though the fusion of PIs hampered statistical analysis. Proliferation of beta-cells was displayed in 3D-printed islets two weeks after µg exposure, suggesting that the effects of µg may be delayed. Thus, PIs in 3D-printed scaffolds did not fuse, and this preparation is more suitable than free-floating specimens for morphological analysis in µg studies. PIs maintained their increased proliferation capacity for weeks after µg exposure, an effect that may not appear directly, but can emerge after a delay.

Effects of microgravity on neural crest stem cells.

Exposure to microgravity (µg) results in a range of systemic changes in the organism, but may also have beneficial cellular effects. In a previous study we detected increased proliferation capacity and upregulation of genes related to proliferation and survival in boundary cap neural crest stem cells (BC) after MASER14 sounding rocket flight compared to ground-based controls. However, whether these changes were due to µg or hypergravity was not clarified. In the current MASER15 experiment BCs were exposed simultaneously to µg and 1 g conditions provided by an onboard centrifuge. BCs exposed to µg displayed a markedly increased proliferation capacity compared to 1 g on board controls, and genetic analysis of BCs harvested 5 h after flight revealed an upregulation, specifically in µg-exposed BCs, of Zfp462 transcription factor, a key regulator of cell pluripotency and neuronal fate. This was associated with alterations in exosome microRNA content between µg and 1 g exposed MASER15 specimens. Since the specimens from MASER14 were obtained for analysis with 1 week's delay, we examined whether gene expression and exosome content were different compared to the current MASER15 experiments, in which specimens were harvested 5 h after flight. The overall pattern of gene expression was different and Zfp462 expression was down-regulated in MASER14 BC µg compared to directly harvested specimens (MASER15). MicroRNA exosome content was markedly altered in medium harvested with delay compared to directly collected samples. In conclusion, our analysis indicates that even short exposure to µg alters gene expression, leading to increased BC capacity for proliferation and survival, lasting for a long time after µg exposure. With delayed harvest of specimens, a situation which may occur due to special post-flight circumstances, the exosome microRNA content is modified compared to fast specimen harvest, and the direct effects from µg exposure may be partially attenuated, whereas other effects can last for a long time after return to ground conditions.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.