Investigation On The Molecular Mechanism By Which Hippo-YAP Signaling Pathway Regulates Proliferation And Cell Lineage Determination Of Adipose-derived Stem Cells

Objective This study was undertaken to isolate adipose-derived stem cells (ASCs) from SD rats, test the multi-differentiation ability and cell surface CD marker expressions of ASCs, in order to provide a seed cell with a clear background for the subsequent investigations.Methods1. Adipose tissue was isolated from SD rats, and ASCs were isolated from the adipose tissue with enzyme digestion methods.2. ASCs were tested for their multi-differentiation ability including osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation.3. Cell surface CD marker expression profiles of ASCs were tested with flow cytometry.Results1. Isolated ASCs showed a homogenous morphology, most of which were flat or fibroblast-like spindle shape.2. Osteogenic differentiation test showed that ASCs were positively stained by Alizarin Red. Adipogenic differentiation test showed that ASCs were positively stained by Oil Red O. Chondrogenic differentiation test showed that the cartilage formed by pellet culture of ASCs was positively stained by Alcian blue and type II collagen immunohistochemistry staining were also positive.3. The flow cytometry revealed that isolated ASCs positively expressed CD29and CD44, negatively expressed CD34, CD45and CD31.Conclusion ASCs were isolated and purified with enzyme digestion methods in this study, which could provide a seed cell for the subsequent investigations. Objective This study tried to investigate the upstream regulatory mechanism which regulates the subcellular localization and expression of YAP in the ASCs.Methods1. YAP subcellular localization of the ASCs was investigated under different culture conditions including both low density and high density culture.2. YAP subcellular location of the ASCs was investigated before and after the cell cytoskeleton was destroyed with Latrunculin B.3. YAP expression induced by serum starve (SS) or lysophosphatidic acid (LPA) was investigated, the relationship between LPA induced YAP expression and cytoskeleton was also strictly investigated.Results1. Confocal scanning laser microscopy revealed that YAP was mainly distributed in the nucleus when ASCs were cultured with a low density, YAP nuclear distribution decreased when cell culture density increased, but YAP was still mainly distributed in the nucleus.2. YAP was evenly distributed in both the nucleus and cytoplasm of ASCs when cytoskeleton of ASCs was destroyed with Latrunculin B.3. SS decreased YAP expression of ASCs, destruction of ASCs cytoskeleton further decreased YAP expression. LPA promoted YAP expression of ASCs, and this effect was inhibited when ASCs cytoskeleton was destroyed with Latrunculin B.Conclusion Cell culture density and actin cytoskeleton regulate YAP subcellular localization partly by modulating the strain of ASCs. LPA could promote YAP expression of ASCs, and this effect is related to the actin cytoskeleton. Objective To explore the molecular mechanism by which Hippo-YAP signaling pathway regulates proliferation and cell lineage determination of adipose-derived stem cells.Methods1. Short hairpin RNA(shRNA) was constructed to inhibit the protein and mRNA expression profiles of YAP.2. CTGF, Ankrdl, YAP and PCNA gene expressions of the ASCs+LPA group, ASC+shYAP+LPA group, ASCs+SIP group and ASC+shYAP+S1P group were respectively evaluated with RT-PCR.3. Proliferation of ASCs in the ASCs+LPA group, ASC+shYAP+LPA group, ASCs+S1P group and ASC+shYAP+S1P group were studied with the CCK8methods.4. Cell cycle distribution of the ASCs+LPA group、ASC+shYAP+LPA group、 ASCs+S1P group and ASC+shYAP+S1P group were studied with flow cytometry using PI staining.5. YAP protein and mRNA expression of ASCs during the osteogenic differentiation process and adipogenic differentiation process were evaluated with RT-PCR and Western Blot analysis.6. Osteogenic differentiation and adipogenic differentiation potential of ASCs were respectively studied after shYAP transfection.Results1. Short hairpin RNA of YAP (shYAP) transfection potently inhibited the protein and mRNA expression of YAP in the ASCs.2. Results of the RT-PCR revealed that LPA and SIP could increase the CTGF, Ankrdl, YAP and PCNA gene expressions of ASCs, while shYAP transfection could remarkably inhibit the stimulatory effect of both LPA and SIP.3. Results of the CCK8methods revealed that LPA and S1P could increase the proliferation of ASCs, while shYAP transfection potently inhibited this stimulatory effect caused by LPA and S1P.4. Flow cytometry with PI staining revealed that LPA and S1P could significantly increase the S and G2/M phases of ASCs, while shYAP transfection significantly inhibit the S and G2/M phases of ASCs stimulated by LPA and S1P.5. YAP protein expression of ASCs during the osteogenic differentiation process increased compared with control group, while YAP mRNA decreased when compared with contrl group. On the contrary, YAP protein expression of ASCs during the adipogenic differentiation process decreased compared with control group, while YAP mRNA increased when compared with contrl group.6. Alkaline phosphatase staining and Runx2gene expression of the Osteo+shYAP group were significantly higher than that in the Osteo or Osteo+shControl group. While the Oil Red O staining and PPARy gene expressions of the Adipo+shYAP group were significantly lower than that in the Adipo or Adipo+shControl group.Conclusion YAP could promote the proliferation of ASCs, knockdown of YAP with shYAP increased the osteogenic differentiation ability of ASCs and decreased the adipogenic differentiation ability of ASCs.