Microgravity produces a variety of physical, chemical, and biological cues leading to an intricate and largely unresolved network of mechanosensitive molecules, transduction pathways, oxidative stress-related responses, and adaptations. The bone loss observed in astronauts and animal models after spaceflight is attributable to alterations in the bone tissue formation that depends on the continuous remodelling through the activities of bone-resorbing osteoclasts of hematopoietic lineage and bone-forming osteoblast of mesenchymal origin. Focusing on osteogenic differentiation, we present the results of the ”SCD - Stem Cells Differentiation” experiment, aiming to determine how human bone marrow stem cells (hBMSCs) react to a prolonged (approx. 2 weeks) exposure to microgravity in terms of growth, and differentiation when treated with a physiological osteo-inducer as 1,25-dihydroxy vitamin D (Vit D3). The experiment was selected by the European Space Agency and transferred to ISS with the Soyuz-TMA- 16M (ISS 42S). It was carefully prepared because experiments performed on ISS remain a uniquely exceptional means of clarifying the microgravitational effects on osteogenesis, often only partially activated and detectable under simulated conditions. Because of the substantial reduction in calcification observed (about 50% inflight vs. on-ground control), we looked at significantly affected pathways in hBMSCs grown in microgravity vs. on-ground controls. Genome-wide expression changes were assessed via microarray and next generation sequencing (NGS) and integrated with exosomal mi-RNA measurements. Multi-scale pathway analysis of the omics datasets revealed evidence of cell cycle arrest, occurring with a number of osteogenic gene markers, but without indications of adipogenesis, senescence and/or apoptosis.

Due to spaceflight, astronauts experience serious, weightlessness-induced bone loss because of an unbalanced process of bone remodeling that involves bone marrow mesenchymal stem cells (BMSCs), as well as osteoblasts, osteocytes, 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. Previous 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 operative 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 partially 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 performed on ground to adapt the project to the spaceflight constraints in terms of culture conditions, fixation and storage of human BMSCs in space aiming at satisfying the biological requirements mandatory to retrieve suitable samples 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.