Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells

Abstract

Objectives: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential.

Materials and methods: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis.

Results: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity.

Conclusions: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.

Figure 1

Effects of different erythrocyte lysis buffers. Bone marrow erythrocytes were treated with erythrocyte lysis buffers. Group A: 0.15 mol/L NH₄CL, 10 mmol/L KHCO₃, 0.01 mmol/L EDTA; group B: 4% acaetic acid; and group C: 0.01 mmol/L PBS. After centrifugation, haemoglobin (Hb) concentration in the supernatant was determined by spectrophotometry. Values were obtained from three experiments and represent the mean ± SE. *P < 0.05, groups B and C versus group A.

Figure 2

Primary culture of rBMSCs from marrow treated with different lysis buffers. (a) Treated with NH₄CL, resulting in more cells being adherent; (b) treated with 4% acaetic acid, and almost no cells were adherent; and (c) treated with PBS. The images of a, b and c were taken on the 6th day after seeding. d was part of the rBMSC clone from group A after 2 weeks of culture (×4).

Figure 3

Characteristics of rBMSCs. Passage 4 rBMSCs were stained for surface markers, CD44 (a), CD106 (b) and CD34 (c), using 3,3′‐diaminobenzidine as the final reaction product. The brown deposit (a and b) suggests positive results, but no deposit is seen in c. These results imply that the isolated rBMSCs expressed CD44 and CD106 but not CD34.

Figure 4

Growth curves of 2nd, 4th and 6th passage rBMSCs and the percentage of 3rd passage cells in each phase of the cell cycle. rBMSCs were plated on coverslips for the methylene blue proliferation assay from day 1 to day 7 (a), or cells were placed into flasks for cell cycle analysis based on propidium iodide staining and FACScan flow cytometer on the 3rd day (b). Growth curves and the percentages of cells in each phase of the cell cycle represent the results of two separate experiments.

Figure 6

rBMSCs proliferation under simulated microgravity. rBMSCs were plated on glass coverslips or placed in flasks, and were cultured in normal medium under simulated microgravity for 1–4 days. Cell proliferation was detected by methylene blue staining (a), Am presents the optical density value and S is the area of the measured coverslips. Cell cycle participation was assessed by flow cytometry (b) and represented the same tendency in three separated experiments. *P < 0.05, SMG versus CN, n = 3. CN, normal gravity; SMG, simulated microgravity.

Figure 5

rBMSC differentiation into osteogenic (a) and adipogenic cells (b). rBMSC differentiation into osteogenic cells was confirmed by von Kossa staining after 18 days of culture in osteogenic media; differentiation into adipogenic cells was confirmed by oil red O staining after 7 days of culture, in adipogenic medium. Cells in normal media served as controls. The images represent the results of three experiments.

Figure 7

Disruption of actin stress fibre formation under simulated microgravity. rBMSCs were plated on coverslips and were cultured in normal medium for 3 days under normal gravity (CN) or simulated microgravity (SMG). At the end of the study, cells were labelled with Texas red isothiocyanate‐conjugated phalloidin, to visualize filamentous actin. Images were acquired using a fluorescence microscope (Leica Microsystems GmbH Wetzlar, Germany) using a ×40 objective. Images represent the results of three separate experiments.

Figure 8

Reduction of Akt and ERK1/2 phosphorylation levels and Cbfa1 expression under simulated microgravity. rBMSCs were cultured for 3 days in the clinostat either using normal medium (for phosphorylation detection) or using osteogenic induction medium (for Cbfa1 detection). Total protein was extracted, quantified and separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Immunoblots were probed using specific antibodies to phosphorylated Akt (a), phosphorylated Erk1/2 (b) and Cbfa1 (c). Images represent the results of three separate experiments. CN, normal gravity (1 g); SMG, simulated microgravity.

Figure 9

Simulated microgravity affected the stimulation of rBMSCs by growth factors. Cell proliferation effects were detected by methylene blue staining. IGF‐I at concentrations ranging from 0 to 20 ng/mL significantly stimulated rBMSCs growth from day 1 to day 4 (a). rBMSCs were cultured for 3 days under simulated microgravity (SMG) with or without growth factors (EGF 20 ng/mL, bFGF 2 ng/mL, IGF‐I 20 ng/mL) and then were assessed once more using methylene blue staining (bFGF and control, n = 3; EGF and IGF‐I, n = 5). Compared to normal gravity (CN), all treated cells presented a significant difference in picture A. SMG markedly inhibited the effect of the growth factors (b) ##P < 0.01 growth factor treated versus control; **P < 0.01 CN (1 g control) versus SMG.