The SERiSM project: preliminary data on human stem cell reprogramming in microgravity

• ¹ Università degli Studi di Roma Tor Vergata, Department of Experimental Medicine and Surgery, NAST Centre, Tor Vergata University of Rome, Italy, Italy
• ² Università degli Studi di Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, Italy
• ³ Agenzia Spaziale Italiana, Italy
• ⁴ Università Campus Bio-Medico, Department of Medicine; European Center for Brain Research, IRCCS Santa Lucia Foundation, Rome, Italy, Italy

Bone loss is a major thread to astronauts’ health during Space missions, and thus the identification of novel biomarkers to be exploited in bone regeneration is a main focus of Space research. The “SERiSM” (Role of the Endocannabinoid System in Reprogramming Human Pluripotent Stem Cells under Microgravity) project has been selected by the Italian Space Agency (ASI) following the 2012 Life Science Research Announcement (ASI DC-MIC-2012-024), in order to evaluate the osteogenic differentiation under real microgravity and to investigate the involvement of the endocannabinoid system in this process. SERiSM was approved by the National Aeronautics and Space Administration (NASA) and was launched to the International Space Station (ISS) on board the Falcon rocket from Cape Canaveral, Florida (USA), on August 14th, 2017, in the frame of the VITA mission of ASI. Accumulated evidence has recently demonstrated that endocannabinoid (eCB) signaling is modulated by weightlessness (Chouker et al., 2010; Strewe et al., 2012). eCBs are lipid mediators with manifold pathophysiological roles both in the central nervous system (Chiurchiù et al., 2018), where they control also neuronal cell programming (Maccarrone et al., 2014), and at the periphery of our body (Maccarrone et al., 2015). Of note, eCB signaling seems to be a key for bone homeostasis (Bab and Zimmer, 2008; Deis et al., 2018). The SERiSM project aimed at interrogating alterations of eCBs that could be possibly linked to bone loss in Space. To this end, human Blood-Derived Stem Cells (hBDSCs) were used as an innovative and easily accessible stem cell model derived from peripheral blood (Marfe et al., 2012). Indeed, the hBDSC model is autologous and possesses a remarkable proliferative and differentiative capacity, thus leading to different cell types with a remarkable therapeutic potential (Marfe et al., 2012a and 2012b; Alaimo et al., 2013). Moreover, hBDSCs start osteogenic differentiation when exposed to a single agent (rapamycin), in the presence of the bone scaffold BioOss (Carpentieri et al., 2016; 2017). The SERiSM rationale and preliminary data showing eCB system expression and osteogenic induction during the on ground experiments and the early phases of differentiation in the preparatory Experiment Sequence Test (EST) and aboard the ISS, are presented here. hBDSCs were isolated from whole blood, withdrawn from a human healthy donor, and were purified by repetitive centrifugation in Phosphate Buffer Saline (PBS) containing macrophage colony-stimulating factor (M-CSF, 50 nM) and gentamicin sulfate (5 μM), and were incubated for 72 h at 37°C. One day before launch, cells were resuspended in DMEM/F-12 medium containing L-glutamine (300 mg/L), gentamicin sulfate (50 mg/L), HEPES (40 mM), penicillin/streptomycin (10 mL/L), fetal bovine serum (100 mg/L) and BioOss granulate, and were used for simulate microgravity experiments or loaded into a specific hardware developed by Kayser Italia for EST and ISS experiments. The latter was composed of 8 experimental units (EUs), each fitting into a KIC-SL container. Once aboard the ISS, the SERiSM experiment was installed into the KUBIK facility, pre-conditioned at 37°C, and was activated in the Columbus module. During inflight operations, hBDSCs were activated automatically for different times (0, 48 and 72 hrs), by adding rapamycin (1 µM) to each culture chamber (Carpentieri et al., 2017). Then, cells were fixed with RNAlater (900 µl/culture chamber; Ambion, Austin, TX, USA), and were immediately moved by the astronauts into the MELFI (minus 80°C laboratory freezer for ISS) facility, where they were stored until download with the Space Capsule Dragon. In the on-ground experiments, the viability of hBDSCs stained with Hoechst was tested every day by flow cytometry, to verify cell survival during the same time-window as the in-flight experiment. Our results demonstrated that cell viability remained > 90% of the controls for up to 10 days, which is the maximum planned time for SERiSM experiment in Space; then, viability decreased to ~80% on day 11, and down to ~30% on day 14 (Fig. 1a). Furthermore, osteogenic differentiation from hBDSCs was documented by the formation of calcium deposits stained by Alizarin Red, showing that metabolic and phenotypic changes towards bone formation took place already within 72 h (Fig. 1b). Therefore, all subsequent experiments on ground and on the ISS were carried out at t0 (control) and t72 (i.e., after 72h of osteogenic differentiation). The expression of the main eCB-binding type-1 and type-2 cannabinoid receptors (CB1 and CB2) was analyzed by Western blot, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping control (Fig. 2). CB1 and CB2 proteins were expressed at both t0 and t72, and showed opposite changes along the differentiation processes: CB1 increased, whereas CB2 decreased (Fig. 2). The samples recovered from the EUs at the end of the EST, and those flown aboard the ISS, showed an increased amount of cells at t72 than at t0 (Fig. 3a), suggesting a proper cell growth in the EUs. Moreover, electrophoretic analysis performed on the same samples, in order to evaluate the protein pattern of hBDSCs after addition of RNAlater and subsequent freezing, showed variations in the low molecular weight proteins that are typical of transcription factors (Fig. 3b and c). In order to ascertain the integrity of the samples and to standardize the experimental procedure for further analysis of ISS samples, Western blot was performed on t0 and t72 samples to measure lactate dehydrogenase (LDH) A and B (Fig 3d and e). Of note, LDH A is highly expressed in cells with a glycolytic metabolism that is typical of stem cells, whereas LDH B is more active in cells with oxidative metabolism that is typical of differentiated cells. The samples recovered from the EUs flown aboard the ISS showed a more marked reduction of LDH A than of LDH B at t72 compared to t0 and t72 ground experiments (Fig 3f and g), supporting the concept that stem cells were less abundant and that commitment towards differentiation took place (Shyh-Chang et al., 2013). Taken together, our preliminary data on endocannabinoid system, cellular proliferation and metabolic switch provide the basis for the ongoing experiments aimed at disclosing the potential of endocannabinoid signalling in bone remodeling and osteogenesis modulation under authentic microgravity conditions. Figure legends Figure 1. (a) hBDSCs viability during 15 days after isolation. (b) Staining with Alizarin Red to highlight bone formation after rapamycin treatment and incubation with BioOss scaffold. Figure 2. Expression of CB1 and CB2 receptors in hBDSCs at t0 and t72. Figure 3. Ground control (EST) and microgravity (ISS) experiment samples. (a) Sample pellets; (b, c) Gel Coomassie staining; (d, e) LDH A and LDH B protein expression in hBDSCs at t0 and t72; (f, g) densitometric analysis of LDH A/LDH B expression.