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Cellular repressor of E1A-stimulated genes (CREG) is widely expressed in mature tissues and cells. Previous research has confirmed that for the in vitro smooth muscle cells (SMCs), CREG can maintain their differentiation phenotype and inhibit their transformation to synthetic phenotype, but it has not been reported about whether CREG has similar effects on differentiation of SMC. This study aims to investigate the role of CREG in the differentiation of mouse embryonic stem cells (ESCs) to SMCs.
Pluripotency of ESCs were verified by alkaline phosphatase (ALP) staining, SSEA-1 staining and in vivo tumor formation experiments. ESCs were firstly cultivated to form embryonic body (EB) and induced by type IV collagen to establish SMCs differentiation model in vitro. ESCs with over-expression of CREG or knout-out of CREG were prepared by genetic modification. EB at indicated time points was collected and markers of SMC including SMα-actin, Calponin, Desmin and SM-MHC were detected by Western blot, Real-time PCR, or immunofluorescence staining. Cells with positive SMα-actin fluorescence were counted at 14d after attached differentiation by flow cytometry. Contractility of differentiated SMCs was assessed by vasotonic reagents. Calcium current was evaluated by Fluo-3AM treatment.
We firstly confirmed that ESCs we used were pluripotent. Then a positive correlation was found between CREG expression level and SMCs differentiation. Real-time PCR showed that in CREG-knockout group, the transcription levels of SMα-actin, Calponin Desmin and SM-MHC were significantly lower than those in the control group, while transcription levels of these markers in CREG-overexpression group were obviously higher than those in the control group. Western blot showed the similar results. Immunofluorescence staining results showed that compared with CREG-knockout group, there were more expressions of SMC markers in the normal group and CREG-overexpression group; in addition to the large quantity, CREG-overexpression group also showed dense lamellar intercellular connections, which might be related to its function. Flow cytometry showed that among 14d EB, CREG-overexpression group had the most cells with positive SM-αactin, while CREG-knockout group had the least. After stimulation of AngII on 14d EB, calcium ion concentration was detected. The reactivity of CREG-overexpression group was closer to the primary smooth muscle cells isolated from adult mice aorta. After stimulation of carbachol on 14d EB, area changes of SMC were detected, and the changes were more apparent in CREG-overexpression group compared with the other two groups.
CREG promotes differentiation and maturation of SMCs derived from mouse ESCs.