1. Isolation, culture and identification of hBMSCs.
1.1 Regular stem cell culture medium was used to inoculate and culture hBMSCs for 48 hours. Observed under an inverted microscope, hBMSCs showed a round shape, and most of them had adhered to the wall. After 4 days, the cells were spindle-shaped.At about 7 days, the cell colony gradually increased, and the confluence was 70-80%.The morphology of the hBMSCs was shown in Fig.1.
1.2 Identification of hBMSCs antigen expression by flow cytometry. Flow cytometry results showed positive rates of antigen expressions including CD44, CD90, CD105, CD45, and CD34 were 99.7%, 99.6%, 99.4%, 2.0%, and 1.5%, respectively, As shown in Fig. 1.
1.3 Detection of hBMSCs multi-diffenentiation potential tests. hBMSCs were cultured in osteogenic medium, adipogenic culture medium and chondrogenic medium, respectively. After 6 days, hBMSCs were collected and subjected to qRT-PCR to detect the mRNA expressions of RUNX2, PPARγ and SOX9, marker genes of bone differentiation, adipogenic differentiation and chondrogenic differentiation. They were compared with the control group (the hBMSCs were cultured in regular stem cell culture medium) see Fig.1.
2. Alkaline Phosphatase and Alizarin Red staining experiments observation of TGF-β1’s promotion effect on the bone differentiation of hBMSCs
2.1 Alkaline Phosphatase staining. In the osteogenic culture group, A small amount of cell cytoplasm was found to be stained, indicating that a small amount of osteoblasts were formed. The results of TGF-β1 group (addition of osteogenic medium and TGF-β1 final concentration 10ng/mL) showed that the cell cytoplasm staining darkened significantly, which denoted hBMSCs osteogenesis enhanced. See Fig. 2
2.2 Alizarin Red staining. After 14 days of culture in the osteogenic culture group, the hBMSCs were stained with Alizarin Red. After 14 days of culture in the TGF-β group, the staining of the cells in the osteogenic culture group was significantly increased and the color was darkened, suggesting that the hBMSCs in the TGF-β group osteogenesis enhanced. See Fig. 2.
3. Study on the epigenetic mechanism of TGF-β1’ promotion effect on the bone differentiation of hBMSCs.
3.1 The expressions of some methyltransferase and demethylase mRNA genes changed after the osteogenic differentiation of hBMSCs.
Compared with the hBMSCs cultured in regular culture medium, the demethylase KDM2B, KDM4A, KDM4B, KDM4C, KDM4D, KDM6A, KDMA6B and methyltransferase EZH1 mRNA genes were significantly increased in hBMSCs cultured by osteogenic medium (P<0.05). There was no statistical difference in the mRNA expression of the demethylase KDM2A and the methyltransferase EZH2 (P>0.05), which denoted that KDM2B, KDM4A, KDM4B, KDM4C, KDM4D, KDM6A, KDMA6B and EZH1 were related to the osteogenic differentiation of hBMSCs. The methyltransferase EZH2 and the demethylase KDM2A have no correlation with the osteogenic differentiation of hBMSCs. See Fig.3.
3.2 The expressions of methyltransferases and demethylases were partly regulated by TGF-β/Smad signaling pathway.
3.21 mRNA expression levels of methyltransferase and demethylase were regulated by TGF-β1. The hBMSCs were divided into the osteogenic culture group (osteogenic medium culture), the TGF-β1 group (osteogenic medium and TGF-β1 10ng/mL) and the SB group (osteogenic medium and TGF-β1 10ng/mL added with SB431542 1uM). SB431542 is a specific blocker of the TGF-β/Smad signaling pathway. After 72 hours of incubation in each group, qRT-PCR was used to detect the mRNA expression of the target genes in the hBMSCs of each group.
Compared with the osteogenic culture group, the mRNA expression of the demethylases KDM2B, KDM4B and KDM4C in the TGF-β1 group had no statistically significant difference (P>0.05), suggesting that expressions of the above three methylases were not associated with TGF-β1 during the osteogenic differentiation of hBMSCs. The relative mRNA expressions of KDM4A, KDM4D and EZH1 were significantly reduced (P<0.01), which denoted that the mRNA expressions of KDM4A, KDM4D and EZH1 were negatively regulated by TGF-β1. The relative expressions of KDM6A and KDM6B mRNA in the TGF-β1 group were significantly increased by 1.5 times (P<0.01), which showed that the mRNA expressions of KDM6A and KDM6B were both positively regulated by TGF-β1. See Fig. 4.
3.22 Protein expression levels of methyltransferase and demethylase were regulated by TGF-β1.
To confirm the regulatory effect of TGF-β1 on the protein expressions of methyltransferase and demethylase, hBMSCs were grouped into the regular culture group, the osteogenic culture group, the TGF-β1 group and the SB group. The grouping methods were the same as the contents of 3.1 and 3.21. A Western Blot was used to detect the protein expressions of methyltransferase EZH1 and demethylase KDM4A, KDM4D, KDM6A and KDM6B in the above 4 groups of hBMSCs. The results showed that during the osteogenic differentiation of hBMSCs, the protein expression trends of methyltransferase and demethylase were the same with mRNA expressions of these enzymes. See Fig. 4.
3.3 Osteogenic transcription factor RUNX2 was regulated by KDM6A and KDM6B, which elucidated the mechanism of TGF-β1 promoting the hBMSCs osteogenic differentiation.
The hBMSCs were divided into the regular culture group, the osteogenic culture group, the TGF-β1 group and the GSK-J4 group (osteogenic medium and TGF-β1 (10ng/mL) and GSK (10uM)). After 72 hours of incubation in each group, qRT-PCR was performed to detect the mRNA expressions of KDM6A, KDM6B and RUNX2. A Western Blot was used to detect the protein levels of KDM6A, KDM6B and RUNX2 in hBMSCs. See Fig. 5.
Comparing the osteogenic culture group with the regular culture group, the relative expressions of mRNA and proteins of KDM6A, KDM6B and RUNX2 in the osteogenic culture group were significantly increased (P<0.01), suggesting that the expressions of KDM6A and KDM6B were significantly related to the osteogenic differentiation of hBMSCs.
Compared with the osteogenic culture group, the relative expressions of mRNA and proteins of KDM6A, KDM6B, and RUNX2 in the TGF-β1 group were furtherly increased (P<0.01), which denoted TGF-β1 had a positive regulatory effect on the expression of KDM6A, KDM6B and RUNX2.
Compoared with the TGF-β1 group, it showed that the relative expressions of mRNA and proteins of KDM6A and KDM6B in the GSK-J4 group did not change significantly (P>0.05) while the expressions of RUNX2 reduced significantly (P<0.01), which indicated that osteogenic transcription factor RUNX2 was regulatd by the demethylases KDM6A and KDM6B. GSK-J4 was the specific inhibitor of KDM6A /6B activity. See Fig.5.