Effects of simulated microgravity on the expression profiles of RNA during osteogenic differentiation of human bone marrow mesenchymal stem cells

Abstract Objectives Exposure to microgravity induces many adaptive and pathological changes in human bone marrow mesenchymal stem cells (hBMSCs). However, the underlying mechanisms of these changes are poorly understood. We revealed the gene expression patterns of hBMSCs under normal ground (NG) and simulated microgravity (SMG), which showed an interpretation for these changes by gene regulation and signal pathways analysis. Materials and methods In this study, hBMSCs were osteogenically induced for 0, 2, 7 and 14 days under normal ground gravity and simulated microgravity, followed by analysis of the differences in transcriptome expression during osteogenic differentiation by RNA sequencing and some experimental verification for these results. Results The results indicated that 837, 399 and 894 differentially expressed genes (DEGs) were identified in 2, 7 and 14 days samples, respectively, out of which 13 genes were selected for qRT‐PCR analysis to confirm the RNA‐sequencing results. After analysis, we found that proliferation was inhibited in the early stage of induction. In the middle stage, osteogenic differentiation was inhibited, whereas adipogenic differentiation benefited from SMG. Moreover, SMG resulted in the up‐regulation of genes specific for tumorigenesis in the later stage. Conclusion Our data revealed that SMG inhibits the proliferation and inhibits the differentiation towards osteoblasts but promotes adipogenesis. SMG also selects highly tumorigenic cells for survival under prolonged SMG.


| INTRODUC TI ON
Previous studies have shown that microgravity induces significant bone loss in weight-bearing bones at a rate of approximately 1%-2% per month. 1 Bone loss under microgravity may be due to the inhibition of osteoblast development and the increase in osteoclastic activity. 2 Exposure to microgravity for a few days has shown a marked suppression of differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into osteoblasts detected by a decrease in ALPL, COL1A1, SPARC and RunX2. 3 Under microgravity, osteoblasts show shorter and wavier microtubules, smaller and fewer focal adhesions, and thinner cortical actin and stress fibres. Simultaneously, microgravity causes an increase in bone resorption by osteoclasts and a decrease in osteoblast cellular integrity. 4 Microgravity also has a significant effect on cell proliferation. Mouse embryonic stem (ES) cells cultured under the SMG condition had a significantly reduced total cell number compared to cells cultured under normal ground gravity (NG). 5 Another study found that the proliferation of murine bone marrow stromal cells was also inhibited in space flight. 6 It has been reported that cells under SMG are more flattened and reached confluence at a lower cell density. 7 Random positioning machine (RPM) is a kind of 3D clinostat that provides continuous random changes in orientation relative to the gravity vector, which can be used to simulate microgravity. 8 In this study, we used an RPM to simulate microgravity and evaluated the effects of SMG on whole gene expression during osteogenesis of hBMSCs. We performed an RNA-seq analysis of hBMSCs induced osteogenically under SMG and NG, and clustered differentially expressed genes (DEGs) into several functional groups to analyse the impacts of SMG on the physiology of hBMSCs.

Whole bone marrow samples were collected at the First Affiliate
Hospital, and hBMSCs were isolated, cultured and passaged as previously described. 9 Cells of 3rd passage were used for the experiments, and the cells examined for the expression profile of special surface antigens expressed CD19 − , CD34 − , CD14 − , CD45 − , HLA − , DR − , CD105 + , CD90 + , CD73 + and CD29 + ( Figure S1). In addition, these cells had osteogenic and adipogenic potentials indicating that the cells prepared from human bone marrow can be accepted as hBMSCs ( Figure S2). 10,11

| Microgravity simulation and induction with random position machine
The SM-31 random position machine (RPM) (Chinese Academy of Science, Beijing, China) was used to simulate microgravity condition. It was operated in random modes of speed (0.1-10 rpm) inside a CO 2 incubator (5% CO 2 , 37°C and 100% humidity). After the cells of 3rd passage reached 60%-70% confluency, the medium was replaced with osteogenic medium 12 (L-Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Shanghai, China) supplemented with 10% FBS, 50 μg/mL l-ascorbic acid (Sigma, Shanghai, China), 10 mmol/L β-glycerophosphate (Sigma), 0.1 μmol/L dexamethasone (Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin. The flasks were fixed on a cell culture vessel for RPM exposure. Cells grown in NG condition were also filled with osteogenic medium and were statically placed in the same CO 2 incubator. The medium was changed every other day. Cells induced for 0, 2, 7 and 14 days were used to study the effects of SMG.
The subsequent results follow these time-points unless otherwise specified.

| Validation of gene expression by qRT-PCR
Reverse transcription of 1 μg of total RNA was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Shanghai, China). Primers (Table S1) were designed using Primer

| Immunohistochemistry assays
The analysis of ALP activity was performed using cells osteogenically induced for 2, 7 and 14 days. ALP activity was analysed using the ALP Quantitative Analysis Kit (Nanjing Jiancheng Institute, China) according to the manufacturers' instructions. The ALP activity was determined at 405 nm using ARM-100 Microplate Reader (Allsheng, Hangzhou, China). In addition, cells induced for 2, 7 and 14 days were also stained with Oil Red O as previously described. 15 For immunolabelling of cytoskeleton, Filamentous actin (F-actin) was stained with phalloidin-TRITC (P1951; Sigma), diluted with 1:300 and nuclei with 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 10 minute. Images were obtained using ZEISS LSM 710 NLO Multiphoton microscope (Shanghai, China).

| Statistical analysis
All values are expressed as the mean ± standard deviation (SD).

Statistical analysis was performed using a two-tailed unpaired
Student's t test or analysis of variance (ANOVA) for multiple comparisons. P < 0.05 was considered statistically significant and P < 0.01 as highly significant.

| Sequencing results and quality control
Paired-end RNA-Seq reads of 150 bp were generated by using the accounted for an average of 91.09%, 6.87% and 2.04%, respectively.
The base quality distribution of all samples is shown in Table S2.
As shown in Figure 1A, 61 DEGs were common in NG group and 93 DEGs in SMG group when the four induction points were compared. In total, 837 DEGs were detected in NG2 vs SMG2 samples and SMG14 clustered together. Similarity between groups of day 2 and day 7 was more compared to those of day 14. In comparison with NG and SMG, we found that most genes in cells under SMG2 and SMG7 had a down-regulated expression pattern (more blue bands) compared with those in cells under NG2 and NG7, respectively. In NG14 vs SMG14, there was no obvious change in the number of up-and down-regulated genes. However, the genes upor down-regulated were different between cells under NG14 and SMG14. Eight main clusters were plotted with expression patterns of genes ( Figure 1D). We selected the DEGs in each cluster and classified them based on UniProtKB keywords (Table S3). The first four clusters were in the NG group and the last four clusters were in the SMG group. Cluster 1 represents genes whose expression level decreased from day 0 to day 2 and then increased from day  Moreover, we also found that there were two GO terms related to tubulin and cytoskeleton in molecular function (Figure 2A,B). To study the middle and later stages of osteogenesis, days 7 and 14 were chosen to perform GO and KEGG analysis. In SMG7, 10 of the most enriched GO terms of three ontologies were selected ( Figure 4A and Figure S4). GO analysis indicated that most of the enriched DEGs were related to multicellular organismal process. The pathway results showed that PPAR signing pathway and calcium signalling pathway were the most enriched pathways ( Figure 4B). In the PPAR signalling pathway, two-third of the genes were up-regulated ( Figure 4C); some amongst them, such as PPARγ and SCD (2.06-fold) were important for adipose differentiation.

| Gene ontology and Kyoto encyclopaedia of genes and genomes enrichment analysis of differentially expressed genes
In the calcium signalling pathway, 80% of the DEGs were downregulated ( Figure 4C). In NG14 vs SMG14 group, multicellular organismal process, receptor binding and extracellular region were the three most enriched GO terms of the three ontologies studied ( Figure 5A and Figure S5). Analysis of the top 20 statistics of   Figure S6A). Furthermore, with ALP assay, no significant difference was found between the NG and SMG groups on days 2, but a significant difference in the activity of ALP was detected on days 7 and 14. However, there was no significant change in ALP activity at the three time-points within the SMG group ( Figure S6B). Cell proliferation assays on days 7 and 14 were also performed to study the effect of prolonged SMG. Interestingly, enhanced proliferation of cells in SMG14 group was found ( Figure S6C). However, there was no significant difference in SMG7 and NG7.

| Validation of DEGs by qRT-PCR
Quantitative real-time PCR (qRT-PCR) was performed to validate the differentially expressed genes involved in the RNA-seq data ( Figure S7). Thirteen genes were selected from the differentially expressed genes of different time periods. Of these genes, five were involved in cell cycle, four were associated with osteogenesis were significantly down-regulated, while the genes specific for osteogenic differentiation, RunX2, ALPL, BMP2 and COL1A1 were down-regulated. Of four genes specific for adipogenic differentiation, PPARγ, CEBPA, CEBPB and CFD were up-regulated. In 7 days, although the genes specific for cell cycle were up-regulated, no significant difference between NG and SMG groups were observed.
The genes specific for osteogenic differentiation were significantly down-regulated, and all the genes specific for adipogenic differentiation were significantly up-regulated. In 14 days, we found the same trend as 7 days in the genes specific for differentiation, but the cell cycle-related genes MCM5, CCNB1 and CDC20 were significant up-regulated. The results showed that the expression patterns of these thirteen genes were highly in agreement with the RNA-seq results.

| D ISCUSS I ON
In the present study, whole transcriptome analysis revealed that SMG affected many biological processes of hBMSCs. Not only tissue-specific genes but also genes related to proliferation and differentiation were affected. On day 2, hBMSCs cultured under SMG exhibited down-regulation of the genes related to cell cycle, such as MCM5, CCNA2, CCNB1, CDK1, E2F1, CDC25B and CDC25C. [16][17][18][19][20][21][22] It has been reported that microgravity changes the distribution of the cell cycle phase in many mammalian cells, such as primitive human haematopoietic progenitor cells 23 and endothelial cells. 24 In addition, several studies have shown that microgravity has a major effect on the cytoskeleton, 25,26 and changes in cytoskeleton may showing that the cell cycle was arrested in G0/G1 phase. However, our results were consistent with another study showing that modelled μ-g induced alterations in cell cycle kinetics characterized by prolonged S phase and reduced cyclin A expression. 23 The previous study showed that continuous inhibition of genes cyclin A and CDC2 inhibits the cell exit from S phase. 29 In our results, genes of CCNA2 Gene ontology (GO) and KEGG pathway analysis showed that the differentiation of hBMSCs was mainly affected by SMG in the middle stage of induction (day 7). Many studies have reported that microgravity inhibited osteogenic differentiation and enhanced adipogenic differentiation. 32,33 In agreement with these reports, KEGG pathway enrichment in our study showed that the PPAR signalling pathway and calcium signalling pathway were the most enriched pathways with two-thirds of the DEGs in PPAR signalling pathway being up-regulated, while 80% of the DEGs in calcium signalling pathway being down-regulated. From previous reports, we know that PPAR-γ, a well-known adipogenic marker, powerfully induces adipogenesis at the morphological and molecular levels in response to a number of PPAR-γ activators. 34 CEBPA (2.52-fold) has also been showed to have roles in differentiation and lipogenesis. 35 PPAR-γ is essential for the differentiation of adipocytes in vivo and in vitro, and CEBPA has been shown to induce adipocyte differentiation through transactivation of PPAR-γ. 36 Up-regulation of these genes promotes adipogenic differentiation of hBMSCs under SMG. Calcium signalling pathway plays an important role in osteogenic differentiation. 37,38 The expression of osteogenic genes in hBMSCs was highly increased with high extracellular Ca 2+ concentration in the in vitro environment, 39 and Ca 2+ influx through the transient receptor potential melastatin type 7 (TRPM7) further triggered Ca 2+ release from the inositol trisphosphate receptor type 2 on the endoplasmic reticulum and promoted osteogenesis. 40 In addition to inhibiting the osteogenesis-specific genes, inhibition of calcium signalling pathway is also responsible for SMG inhibition of osteogenic differentiation. In the later stages of induction (day 14), GO enrichment analysis showed that SMG mainly affected the multicellular organism process and developmental process. Through pathway enrichment, we found that cytokine-cytokine receptor interaction and pathways in cancer were the most enriched pathways. From these DEGs, we found that many genes associated with growth factors, cytokines, tumorigenesis and angiogenesis were up-regulated, while the genes associated with apoptosis, inflammation and signal transduction were down-regulated. Previous reports have suggested that prolonged spaceflight has a negative impact on the immune system resulting in hypoplasia of lymphoid organs and alterations in mitogen-induced blastogenesis. 41 Under SMG, a significant loss of antigen-specific cytotoxic T lymphocyte activity was observed, suggesting the negative effect of SMG on cell-mediated immunity. 42 Moreover, microgravity has adverse effects on tumour cells. Although cancer stem cells are committed to selective differentiation when cultured in microgravity, a study has provided evidence of significant apoptosis under this condition. 43 Another study reported that some tumours seem to be much less aggressive in the microgravity environment of space compared to Earth. 44 Nevertheless, SMG may have altered the tumour cell characteristics and enhanced the invasive property. Therefore, it is possible that the microgravity analogue culture environment may have selected highly tumorigenic cells for survival. 45 Our RNA-seq results are more inclined to support this conclusion.
To confirm the effect of simulated microgravity on osteogenic differentiation and adipogenic differentiation derived from RNA-seq data, we performed assays of ALP activity and Oil Red O staining.
ALP activity, as a marker of osteogenic differentiation, reflects bone formation and/or mineralization. 11 We found that ALP activity at 7 and 14 days was significantly increased compared to that at 2 days in NG group, but no obvious change in SMG group. These results indicated that SMG inhibited osteogenic differentiation of hBMSCs.
For analysis of adipogenic potential of hBMSCs under osteogenic induction, we found the significantly positive staining of Oil Red O in SMG group compared to those in NG group, especially at 14 days, which shows a tendency of adipogenic differentiation even under osteogenic induction. These results are consistent with the conclusion of RNA-seq data.
In conclusion, SMG inhibits the proliferation of cells in the early stage of osteogenesis of hBMSCs while in the middle stage, it inhibits the differentiation towards osteoblasts and promotes adipogenesis. It may select highly tumorigenic cells for survival under prolonged SMG. We believe that the data provided in this study may clarify some of the physiological changes, such as bone loss and immune system dysfunction occurring during spaceflight, and in favour of a better understanding of the mechanism of these changes.

CO N FLI C T O F I NTE R E S T S
Authors indicate no potential conflict of interests.