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.