Stem cell research in low Earth orbit (LEO) has shown promise in understanding how microgravity alters stem cell properties and its potential for biomanufacturing applications. Studies in simulated microgravity have already revealed changes in differentiation capacity, viability, and proliferation in various stem cell types, including progenitor cells derived from pluripotent stem cells, cardiomyocytes, neural stem cells, mouse embryonic stem cells, and cardiac progenitor cells. While some pilot data suggest that microgravity might enhance the differentiation of specialized cell types from pluripotent stem cells, more research is needed to confirm this and explore the effects across various cell types. The influence of microgravity on fundamental stem cell properties, including self-renewal and the expression of pluripotency network components, via altered mechanotransduction remains a significant area of investigation. Prior research has indicated that microgravity might impact bone formation by inhibiting osteoblast function, and influence mesenchymal stem cell (MSC) differentiation, morphology, and migration. Epigenetic shifts and alterations in signaling pathways are also potential mechanisms by which microgravity exerts its effects. Human induced pluripotent stem cells (hiPSCs), with their ethical advantages and ability to differentiate into various cell types, are a promising tool for investigating these effects. However, the impact of spaceflight on hiPSC production, maintenance, and differentiation has been relatively unexplored. Efficient DNA plasmid delivery into cells for temporary expression without viral methods remains crucial for genome editing, cell fate direction, and somatic cell reprogramming. Traditional methods like lipofection can be inefficient, but altered fluid dynamics in LEO might potentially enhance transfection efficiency. The use of off-the-shelf hardware, like commercially available 96-well plates, offers a more cost-effective and reproducible approach for space-based research compared to custom-built systems, simplifying medium exchange and reducing costs. This study aimed to explore the feasibility of culturing and transfecting hiPSCs and their derived fibroblasts in commercially available 96-well plates during the Ax-2 mission to the ISS, advancing our understanding of stem cell behavior in microgravity and the potential for space-based biomanufacturing.

Previous research has demonstrated the potential of using spaceflight to understand stem cell behavior. Studies have shown altered differentiation capacity, viability, and proliferative potential of various stem cell types in simulated or real microgravity conditions. Specifically, research on cardiomyocytes, neural stem cells, and mouse embryonic stem cells revealed enhanced proliferative capacity in space. However, the impact of sustained microgravity on stem cell differentiation needs further investigation, particularly for hiPSCs, which have seen a rapid rise in prominence due to their ethical advantages and versatility in disease modeling and cell therapy. The challenge of producing sufficient cells for research on Earth highlights the potential of space-based biomanufacturing. Furthermore, while simulated microgravity studies have offered insights, conducting experiments in LEO is crucial for validating findings under true microgravity conditions. The impact of microgravity on fundamental stem cell properties such as self-renewal and pluripotency maintenance has been studied, with findings indicating potential alterations in signaling pathways and epigenetic modifications. The literature also highlights the potential of enhanced transfection efficiency in microgravity due to altered fluid dynamics. Finally, the transition from custom-made, low-throughput cell culture hardware to more readily available and cost-effective systems such as commercially available 96-well plates promises to improve the reproducibility and accessibility of space-based research.

This study utilized a SOX2-GFP hiPSC line and hiPSC-derived fibroblasts. These cells were expanded on Earth, frozen, and transported to the ISS aboard Axiom Mission 2 (Ax-2). Astronauts then thawed the cells and seeded them into commercially available 96-well plates containing Matrigel. A detailed experimental timeline condensed cell culture, imaging, and transfection into a five-day experiment. The astronauts were involved in all hands-on aspects, including thawing, seeding, media exchanges, imaging, and transfection. To assess cell growth and transfection efficiency, both hiPSCs and hiPSC-fibroblasts were cultured in the 96-well plates and subsequently transfected with an RFP-expressing plasmid. Imaging was performed using a Keyence microscope to capture phase-contrast, GFP, and RFP signals. Cell culture media remained within the wells of the 96-well plates due to surface tension, even in microgravity. Quantitative analysis was conducted to assess SOX2-GFP and RFP signal intensities, both per well and per cell, using automated workflows to minimize bias. Immunocytochemistry staining with OCT4 confirmed the pluripotency of hiPSCs. Statistical analysis utilized 2-way ANOVA and Tukey's multiple comparisons test to compare the signal intensities between ground controls and in-space samples. The study also included an Experimental Verification Test (EVT) conducted on Earth to ensure the safety and success of the space-based experiment, including a complete run-through using the same materials and protocols.

This study successfully demonstrated the feasibility of culturing and transfecting hiPSCs and hiPSC-derived fibroblasts in microgravity using commercially available 96-well plates. The surface tension of the media within the small wells of the plates prevented loss of media in microgravity, thereby overcoming a significant hurdle for microgravity cell culture. The hiPSCs spontaneously aggregated into spheroids on day 1 post-seeding in space, and subsequently adhered to the Matrigel substrate by day 2. This spheroid formation occurred naturally in microgravity, unlike Earth-based methods which require additional force vectors or shear stress. The SOX2-GFP reporter provided a robust and efficient way to monitor hiPSC health and pluripotency. Quantitative analysis of SOX2-GFP expression per single cell showed less variability in space, potentially indicating more homogenous expression of the SOX2 gene. Significant differences in SOX2-GFP signal intensity per well between space and Earth samples were observed, potentially related to cell detachment in space, cell health, or increased transfection plasmid uptake. Successful transfection with an RFP plasmid was demonstrated in both hiPSCs and hiPSC-fibroblasts in space. While a slight increase in transfection efficiency was observed in space for hiPSCs, this was not statistically significant. HiPSC-derived fibroblasts also exhibited both 2D and 3D growth patterns in space and were successfully transfected. Overall, this study shows that hiPSCs and hiPSC-fibroblasts can be successfully grown and transfected in commercially available hardware in space, demonstrating the practicality of space-based stem cell research.

This study addresses the research question of whether hiPSCs and their derivatives can be effectively cultured and transfected in microgravity using readily accessible materials. The results demonstrate that commercially available 96-well plates can be utilized successfully for these purposes, highlighting the potential for increased accessibility and reproducibility of space-based stem cell research. The spontaneous formation of hiPSC spheroids in microgravity is intriguing and suggests a novel approach to culturing these cells. The observed differences in SOX2-GFP expression and transfection efficiency between space and ground controls warrants further investigation. Future research should explore the underlying mechanisms driving these differences and examine long-term effects of microgravity on hiPSC behavior. This work lays the groundwork for future experiments involving more complex and prolonged hiPSC cultures in space, including hiPSC reprogramming and genome editing studies. The findings open new avenues for space-based biomanufacturing of stem cell-derived products.

This study successfully demonstrated the culture and transfection of hiPSCs and hiPSC-derived fibroblasts in space microgravity using commercially available 96-well plates. Surface tension proved crucial for maintaining cell culture media. The spontaneous formation of spheroids in microgravity was observed. Successful transfection of both cell types was also achieved, opening avenues for future studies involving reprogramming and genome editing in space. This research enhances the accessibility and cost-effectiveness of space-based stem cell studies.

This study was limited by the short duration (5 days) of the spaceflight mission. Longer-term experiments are needed to comprehensively assess the long-term effects of microgravity on hiPSC behavior and pluripotency. The relatively small sample size (n=6 wells per condition) may have limited statistical power. Further studies with larger sample sizes are needed to confirm the observed differences between in-space and ground-control groups. The inadvertent movement of the plate during the experiment may have influenced the adhesion of hiPSC spheroids to Matrigel. Future experiments should implement stricter controls to eliminate such variables. Lastly, the qualitative and quantitative analysis methods should be further refined to enhance accuracy and eliminate bias.