To evaluate the role of autophagy and the molecular changes in the process of smooth muscle cells (SMCs) differentiation from human adipose-derived stem cells (hADSCs) and establish a protocol for inducing this differentiation.
The differentiation of hADSCs into SMCs was induced using the smooth muscle induction medium (SMIM) with a low serum level. Real-time PCR was used to examine the mRNA expression of smooth muscle marker genes. In addition, we used Western blot analysis and immumo fluorescence staining to evaluate changes in the protein level.
We observed an increase in the expression of smooth muscle marker genes, alpha- smooth muscle actin (α-SMA) and myosin heavy chain (MHC), in hADSCs exposed to SMIM for 6 weeks. The cellular complexity and granularity were increased in induced hADSCs, suggesting an increase in the content of intracellular organelles during the SMC differentiation process. The content of lysosomes, but not of mitochondria or endoplasmic reticulum, was significantly increased. The increased protein content of the lysosomal-associated membrane protein 1 confirmed the increase in the lysosomal content during SMC differentiation. By contrast, the conversion from LC3-I to LC3-II was increased during SMC differentiation, with a significant increase after 3 weeks of differentiation.
These results suggest that autophagy is significantly upregulated in the early stage of SMC differentiation. Autophagy might play a crucial role in SMC differentiation from hADSCs. Human ADSCs may be a potential biomaterial for urinary incontinence treatment and bladder reconstitution.
human adipose-derived stem cells; differentiation; smooth muscle cell; autophagy
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