Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

Abstract

Growing stem cells on Earth is very challenging and limited to a few population doublings. The standard two-dimensional (2D) culture environment is an unnatural condition for cell growth. Therefore, culturing stem cells aboard the International Space Station (ISS) under a microgravity environment may provide a more natural three-dimensional environment for stem cell expansion and organ development. In this study, human-derived mesenchymal stem cells (MSCs) grown in space were evaluated to determine their potential use for future clinical applications on Earth and during long-term spaceflight. MSCs were flown in Plate Habitats for transportation to the ISS. The MSCs were imaged every 24-48 h and harvested at 7 and 14 days. Conditioned media samples were frozen at -80 °C and cells were either cryopreserved in 5% dimethyl sulfoxide, RNAprotect, or paraformaldehyde. After return to Earth, MSCs were characterized to establish their identity and cell cycle status. In addition, cell proliferation, differentiation, cytokines, and growth factors' secretion were assessed. To evaluate the risk of malignant transformation, the space-grown MSCs were subjected to chromosomal, DNA damage, and tumorigenicity assays. We found that microgravity had significant impact on the MSC capacity to secrete cytokines and growth factors. They appeared to be more potent in terms of immunosuppressive capacity compared to their identical ground control. Chromosomal, DNA damage, and tumorigenicity assays showed no evidence of malignant transformation. Therefore, it is feasible and potentially safe to grow MSCs aboard the ISS for potential future clinical applications.

Keywords: Preclinical research; Stem-cell research; Translational research.

Fig. 1. Cell culture and real-time imaging.

MSCs were seeded and cultured in BioCells cassettes (a) and transported in Plate Habitats (PHABS) (b). Real-time images were taken from ground control and ISS microgravity groups. Imaging started 3 days before launching (L − 3) until 13 days after launching (L + 13) (c). Scale bar: 400 µm.

Fig. 2. Establishing MSC identity.

MSCs thawed from frozen BioCells after 1 week (a–d) and 2 weeks in culture at ISS were tripled stained with CD73, CD90, and CD105 antibodies and then analyzed using flow cytometry. a, c, e, g were from ground control samples. b, d, f, h were from ISS microgravity samples. R7 gate was used to select double positive for CD90 and CD73. R2 gate was applied out of R7 gate to demonstrate triple-positive cells for CD73, CD90, and CD105 (n = 3).

Fig. 3. Post-flight MSC proliferation analyses.

a MSCs were cultured in triplicates after 1- or 2-week expansion in the ISS environment and on Earth in our laboratory. Cells were seeded into a 96-well plate and stained with IncuCyte Nuclight red to track cell proliferation by nucleus count. Real-time images were acquired by IncuCyte S3 at 2-h interval up to 48 h. Post flight, the ISS-expanded cells proliferated at comparable rate with ground control cells. Statistics determined by one-way ANOVA (n = 3). Error bars represent standard deviation (SD) (n = 3, P > 0.05). b Similarly, assessment of population doubling in longer-term cultures of 1-week ISS-expanded MSCs and the corresponding ground control showed no statistically significant difference in growth rate (two-sample Wilcoxon’s rank-sum (Mann–Whitney) test, P = 0.68). Error bars represent standard deviation (SD).

Fig. 4. Impact of microgravity on expression of cell cycle checkpoints associated genes.

MSCs were culture for 1 and 2 weeks. Cells were harvested and saved in RNAprotect. RNA was later isolated and quantitative reverse transcription PCR (RT-qPCR) analysis was performed as described in the “Methods” section. Expression of cell cycle checkpoint-associated genes: a cyclin-dependent kinase inhibitor 2A (CDKN2A), b transcription factor (E2F1), and c serine/threonine-protein kinase, known as polo-like kinase 1 (PLK1) were evaluated and compared to ground control gMSCs. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n = 3, *P < 0.05), error bars represent standard deviation (SD).

Fig. 5. Influence of microgravity on osteogenic and adipogenic differentiation.

sMSC and gMSC cultures were stained with Alizarin Red and Oil Red O (a). Each condition was seeded and measure in triplicates. Representative pictures (×100) are shown from post-flight MSC culture in a growth medium or differentiation medium after 2-week culture expansion at ISS or ground (Earth). Quantitative real-time PCR analysis of osteogenic (COL1A1, RunX2) and adipogenic (CEBPB) genes was done after 21 days in differentiation medium (b). RNA from three 1-week and 2-week MSC cultures were used for quantitative PCR analysis. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n = 3). Error bars represent standard deviation (SD). Scale bar: 100 µm.

Fig. 6. Conditioned media from sMSC and gMSC cultures were evaluated for cytokine and growth factor secretion profile.

PDGF-AA (a) was increased and sCD40L (i) was decreased significantly after 1 week in microgravity. IL-10 (d), MCP-3 (f), and SDF-1α/β (h) were decreased and IP-10 (l) was increased significantly in microgravity environment after 2 weeks in microgravity environment relative to ground controls. Statistics determined by Student’s t test (n = 3, *P < 0.05 **P < 0.01). Error bars represent standard deviation (SD).

Fig. 7. Immunomodulation potency assay comparing the immunosuppressant capacity of gMSC and sMSC towards peripheral blood mononuclear cell (PBMC).

MSCs were incubated for 72 h with phytohemagglutinin (PHA) stimulated PBMCs at 1:3 ratio of MSCs to PBMCs. Luminescence is proportional to the amount of ATP present, and the amount of ATP is proportional to the number of metabolically active cells present. Luminescence fold change is relative to the positive control (PBMCs with PHA). Each condition was seeded and measured in quintuplicate. Statistics determined by one-way ANOVA (n = 5, *P < 0.05, **P < 0.005). Error bars represent standard deviation (SD).

Fig. 8. Genomic integrity analysis of the safety of growing MSC in space.

a Bone marrow-derived mesenchymal stem cells (5000 cells/well) previously grown for 1 week or 2 weeks in space, along with ground control cells were seeded to ibidi µ-Slide 8 Well. Cells were stimulated with 100 μM etoposide as indicated, fixed, and subjected to immunofluorescence analysis to visualize pS139-H2A.X. Scale bars indicate 10 μm. b shows quantitation of the fluorescence intensity of pS139-H2A.X (n = 40 cells). Analysis was conducted using the Image J software. Fluorescence intensities were normalized to the etoposide-treated group for each experimental condition. **** indicates statistical significance (<0.0001) determined by one-way ANOVA as compared to cells treated with 100 μM etoposide. Error bars represent standard deviation (SD).

Fig. 9. Chromosomal studies were performed using confluent cultures of sMSCs and gMSCs.

Twenty cells were analyzed from sMSCs (a) and gMSCs (b) and two of these were karyotyped using standard G banding method. No chromosomal abnormalities were detected.

Fig. 10. Tumorigenicity assay.

Cells were seeded on a soft agar to evaluate their tumorigenic potential. Five thousand cells/well of positive control cell line HT-1080 (a) and 67,000 cells/well of negative control cell line W138 (b) were used as control to validate the procedure. Twelve cells/well of MSCs were used for this assay. (c) Ground control and (d) microgravity were cells that were culture expanded for 1 week and (e) ground control and (f) microgravity were cells cultured for 2 weeks on Earth and in microgravity, respectively.