Mesenchymal stem cells (MSCs) were induced into a nucleus pulposus-like phenotype utilizing simulated microgravity in vitro in order to establish a new cell-based tissue engineering treatment for intervertebral disc degeneration. For induction of a nucleus pulposus-like phenotype, MSCs were cultured in simulated microgravity in a chemically defined medium supplemented with 0 (experimental group) and 10 ng/mL (positive control group) of transforming growth factor β1 (TGF-β1). MSCs cultured under conventional condition without TGF-β1 served as blank control group. On the day 3 of culture, cellular proliferation was determined by WST-8 assay. Differentiation markers were evaluated by histology and reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-β1 slightly promoted the proliferation of MSCs. The collagen and proteoglycans were detected in both groups after culture for 7 days. The accumulation of proteoglycans was markedly increased. The RT-PCR revealed that the gene expression of Sox-9, aggrecan and type II collagen, which were chondrocyte specific, was increased in MSCs cultured under simulated microgravity for 3 days. The ratio of proteoglycans/collagen in blank control group was 3.4-fold higher than positive control group, which denoted a nucleus pulposus-like phenotype differentiation. Independent, spontaneous differentiation of MSCs towards a nucleus pulposus-like phenotype in simulated microgravity occurred without addition of any external bioactive stimulators, namely factors from TGF-β family, which were previously considered necessary.

Mesenchymal stern cells (MSCs) were induced into a nucleus pulposus-like phenotype utilizing simulated microgravity in vitro in order to establish a new cell-based tissue engineering treatment for intervertebral disc degeneration.For induction of a nucleus pulposus-like phenotype,MSCs were cultured in simulated microgravity in a chemically defined medium supplemented with 0 (experimental group) and 10 ng/mL (positive control group) of transforming growth factor β1 (TGF-β1).MSCs cultured under conventional condition without TGF-β1 served as blank control group.On the day 3 of culture,cellular proliferation was determined by WST-8 assay.Differentiation markers were evaluated by histology and reverse transcriptase-polymerase chain reaction (RT-PCR).TGF-β1 slightly promoted the proliferation of MSCs.The collagen and proteoglycans were detected in both groups after culture for 7 days.The accumulation of proteoglycans was markedly increased.The RT-PCR revealed that the gene expression of Sox-9,aggrecan and type Ⅱ collagen,which were chondrocyte specific,was increased in MSCs cultured under simulated microgravity for 3 days.The ratio of proteoglycans/collagen in blank control group was 3.4-fold higher than positive control group,which denoted a nucleus pulposus-like phenotype differentiation.Independent,spontaneous differentiation of MSCs towards a nucleus pulposus-like phenotype in simulated microgravity occurred without addition of any external bioactive stimulators,namely factors from TGF-β family,which were previously considered necessary.

Background Mitochondrial dynamin-related protein 1 (DRP1) regulates mitochondrial division and plays an important role in maintaining hepatocyte function. However, the role of DRP1 in cadmium exposure-induced maternal liver damage in pregnant mice remains unclear. Objective To investigate the role and mechanism of DRP1 in maternal liver damage induced by cadmium exposure during pregnancy. Methods This study consisted of animal experiments and cell experiments. (1) Animal experiments. Mice at 14 days of gestation were randomly divided into three groups: a control group, a low-dose cadmium group (LCd group: 2.5 mg·kg⁻¹), and a high-dose cadmium group (HCd group: 5 mg·kg⁻¹). The pregnant mice were intraperitoneally injected with cadmium chloride (CdCl₂) for 6 and 24 h in the next morning. The weights of pregnant mice, uterus, maternal liver, and fetal mice were recorded after sacrifice. Serum and liver of pregnant mice were collected, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected, and liver tissues were stained with HE to observe changes in liver function and liver tissue structure. The expressions of oxidative phosphorylation-related proteins, hypoxia inducible factor-1α (HIF-1α) and DRP1 proteins in liver of pregnant mice were detected by Western blotting. (2) Cell experiments. AML12 cells were treated with CdCl₂ (10 μmol·L⁻¹) for 0, 2, 6, 12, and 24 h. The expressions of oxidative phosphorylation-related proteins, DRP1, and hypoxia inducible factor-1α (HIF-1α) proteins were detected. AML12 cells were pretreated with DRP1 inhibitor Mdivi-1 for 1 h and then CdCl₂ (10 μmol·L⁻¹) for 12 h to detect the expression of oxidative phosphorylation-related proteins and DRP1 protein. AML12 cells were treated with Hif-1α siRNA for 48 h and CdCl₂ (10 μmol·L⁻¹) for 6 h to detect the expression of HIF-1α and DRP1 proteins. Results The results of animal experiments showed that cadmium exposure in pregnant mice had no effects on maternal liver weight and liver coefficient. However, the histomorphological changes and necrosis in hepatocytes were observed. Compared with the control group, the serum ALT and AST levels of pregnant mice in the LCd group were significantly increased after 6 h (P＜0.05), and the levels in the HCd group were significantly increased after 6 and 24 h (P＜0.05). Cadmium exposure during pregnancy significantly up-regulated HIF-1α and DRP1 expressions and down-regulated the expressions of oxidative phosphorylation-related proteins in maternal livers. In vitro cell experiments showed that the expressions of oxidative phosphorylation-related proteins was significantly decreased and HIF-1α and DRP1 protein expressions were significantly increased in the AML12 cells treated with CdCl₂ for 6 h. Mdivi-1 pretreatment significantly antagonized the inhibitory effect of cadmium on the expressions of oxidative phosphorylation-related proteins in AML12 cells, while Hif-1α siRNA pretreatment significantly antagonized the up-regulative effect of cadmium on DRP1 expression in AML12 cells. Conclusion Cadmium exposure in pregnant mice may up-regulate DRP1 expression by activating HIF-1α signaling, then inhibit oxidative phosphorylation level of hepatic cells, and ultimately lead to maternal liver damage.