One of the biggest problems for human beings to space exploration and future space colonization is microgravity which often leads to unusual phenomena and behaviors 3. For example, human bone density and muscle mass can decrease significantly 4,5. However, the research on the adverse effects of microgravity on human beings is very limited. One of the core bottlenecks is the lack of a real microgravity environment; thus simulated microgravity conditions were created to study how microgravity affects cell proliferation and differentiation, as well as organismal development and homeostasis. Our self-developed RFC, which is also called Random Positioning Machine (RPM) in other labs, is one of the microgravity-simulated machines that is designed to rotate samples, creating a pseudo-weightless environment randomly 6. It offers the flexibility to adjust the rotation speed, allowing researchers to replicate various levels of microgravity 7. The RPM has been extensively utilized in various biomedical and biological studies 7,8. It has contributed to research fields such as cell biology, tissue engineering, bone health, muscle physiology, plant biology, and drug development 9. Thus, RPM is a useful tool to detect the effect of simulated microgravity on cell fate and also able to explore the related molecular mechanisms.
The liver is a central organ for numerous physiological processes such as body energy metabolism, detoxification, and immunology 10. Therefore, stem/progenitor cells in the developing liver have been carefully studied to explore how they can be differentiated into functional hepatocytes. Hepatic differentiation is a complex process that involves four distinct stages for stem cells (stemness maintenance (STEM), definitive endodermal lineage (DE), precursor hepatocytes (Pre-H), and hepatocyte-like cells (M-H)), each characterized by specific cellular and molecular changes 11. The key markers associated with the stem stage include OCT-4 and NANOG; at the DE stage, the two DE biomarkers SOX17 and CXCR4 were sound expression; the critical markers associated with Pre-H stage are AFP and SOX9; at the M-H stage, the two hepatic biomarkers ALB and CK18 were uniformly distributed inside the cells and presented over the entire cells 11,12. Because Human hepatic stem cells are relatively difficult to obtain and their growth and differentiation rates are much slower in cell culture conditions, other cell lines were often alternatively used to study the differentiation process of human hepatic stem cells. Among them, the HepaRG cell line is a kind of cell model that exhibits many of the same characteristics as primary human developing hepatocytes. It can differentiate into both well-differentiated hepatocyte-like cells (HLCs) that resemble primary human hepatocytes (PHH), and biliary epithelial cells (cells lining the bile ducts) under defined culture conditions 13. Therefore, HepaRG cells have been extensively used as an in vitro model for studying drug metabolism, cell differentiation, and liver diseases 14. Although physical forces such as substrate stiffness and shear stress have been shown to play a critical role in stem cell differentiation. However, whether microgravity affects hepatic stem/progenitor cell differentiation is still largely unknown.
Cells can adapt to the changes caused by physical forces by activating diversity of mechanical signaling pathways, thereafter affecting cells’ morphological appearance and biological functions 15. Mechanoreceptors on cell membranes are capable of detecting and transmitting mechanical signals like pressure, stretch, and vibration into biochemical signals for further processing and interpretation 16, finally regulating a wide range of cellular processes, including cell proliferation, differentiation, migration, and gene expression 15. Until now, hundreds of mechanical sensors have been identified, and they play an essential role in various physiological processes, such as touch sensation, balance and proprioception, cardiovascular regulation, and pain perception 17. For example, mechanosensitive ion channels, such as Piezo and TRP channels, are open in response to mechanical stimuli, allowing ions to flow across the cell membrane to initiate downstream signaling cascades18; mechanical forces can promote cytoskeleton remodeling and regulate gene expression by activating the adhesion protein (focal adhesion kinase, FAK) 19. Thus, understanding the properties and roles of mechanoreceptors is essential for elucidating how cells sense and adapt to their mechanical environment and developing new approaches for diagnosing and treating mechanical-related diseases.
MCOLN1/TRPML1 is one of the transient receptor potential (TRP) channel family members, primarily localized to the endosomal and lysosomal compartments of cells, and plays a crucial role in controlling membrane trafficking, lysosomal biogenesis, autophagy, etc. 20. It is a large protein consisting of 580 amino acids and possesses 6 transmembrane domains, an intracellular N-terminal domain, and a C-terminal domain 21. The activity of TRPML1 is stimulated by PI(3, 5)P2 that is synthesized depending on Vac14 and PIKfyve 22. TRPML1 is a nonselective cation channel that conducts calcium ions across the lysosomal membranes to facilitate membrane trafficking between endolysosomal compartments and plasma membranes 23. In addition, it also promotes the fusion and maturation of endosomes with lysosomes, enabling the degradation of internalized macromolecules and the recycling of membrane proteins 24. Moreover, as a calcium-permeable channel, TRPML1 modulates various calcium signaling pathways, including those involved in cell growth, proliferation, differentiation, and apoptosis 21,25. However, the research on the physiological functions of TRPML1 under simulated microgravity conditions is still totally unknown.
In the article, we found that the differentiation rate of HepaRG cells could be significantly enhanced under simulated microgravity effects created by using RFC. Mechanically, simulated microgravity increases the content of PI (3, 5) P2 through remodeling the cytoskeleton, which in turn activates TRPML1 to increase the calcium flux of lysosomal, finally promoting Wnt/β-catenin pathway dependent cell differentiation. Our results disclosed the mechanical-biological coupling regulation mechanism that how simulated microgravity affects hepatic differentiation of HepaRG cells, which is expected to provide scientific foundations for embryonic liver development under simulated microgravity conditions.