Research in Medicine

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Background and Aim: Adipose-derived stem cells (ADSCs) represent a promising source of cells for cardiac tissue engineering and repair of the injured heart. However, better understanding of the factors affecting the cardiac differentiation of ADSCs is required before clinical application of these cells. Current study was designed to investigate the role of bFGF and BMP4 in cardiac differentiation of human ADSCs.
Materials and Methods: ADSCs were isolated from human abdominal subcutaneous adipose tissue and cultured. For cardiac differentiation, ADSCs were treated with 10 ng/ml bFGF and 20, 50 or 100 ng/ml BMP4 in a medium containing 10% FBS or 0.5% B27 for four days. Then the induction factors were completely omitted, and the cells were maintained in 10% FBS-containing medium for up to three weeks. At the end of differentiation period, the expression of some cardiac markers was assessed by RT-PCR, qPCR and immunocytochemistry.
Results: The differentiated ADSCs expressed cardiac-specific genes. Based on qPCR analysis, the maximum expression level of ANF and MLC2A mRNAs was detected in the cells treated with 10 ng/ml bFGF and 20 ng/ml BMP4 in the FBS-containing medium. Moreover, FBS supplementation of induction medium was more effective than the B27-containing medium for cardiac differentiation of ADSCs by bFGF and BMP4. The cells treated with 10 ng/ml bFGF and 20 ng/ml BMP4 in FBS-containing medium expressed cardiac troponin I and α-actinin proteins.
Conclusion: It seems that a combination of bFGF and BMP4 improves cardiac differentiation of ADSCs. Moreover, bFGF and BMP4 are more effective for cardiac differentiation when the induction medium is supplemented with FBS than B27. This may be due to the presence of insulin in B27 supplement.  

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Background and Aim: The miR-302/367 cluster plays a critical role in reprogramming of somatic cells into pluripotent stem cells and in maintaining pluripotency. Until now, several studies have been conducted on the mechanism of miR-302/367 cluster in reprogramming, while little research has been done specifically on the effect of this cluster on mTOR signaling pathway. The mTOR pathway not only controls various cellular processes, such as proliferation, differentiation and autophagy, but also plays a significant role in the reprogramming process. Therefore, the present study was designed to identify the effect of miR-302/367 cluster on the mTOR pathway in adipose tissue-derived stem cells (ADSCs).
Methods: In this experimental study, the third to fifth- passaged ADSCs were transfected with TDH101PA- GP vector expressing mir-302/367 cluster and the mock vector using a Neon Transfection Kit and Neon Transfection System. One week after transfection, the expression of some mTOR signaling molecules in the ADSCs was assessed by comparative Real- Time PCR. Relative gene expression between the miR-302/367 and mock groups (4 replicates) was calculated by REST 2009 software based on Pair Wise Reallocation Randomization Test. Also, the expression of some mTOR pathway proteins was assessed by western blot analysis.
Results: The overexpression of miR-302/367 cluster significantly reduced the expression of AKT, MTOR, RAPTOR, and RICTOR genes; the expression of these genes in the miR-302/367 transfection group compared to the mock group decreased to 0.44, 0.51, 0.56 and 0.6, respectively (P< 0.05). The expression of genes in the mock group was assumed as one. Also, as revealed by western blot analysis, the expression of phosphorylated AKT and phosphorylated MTOR proteins decreased in the ADSCs transfected with miR- 302/367 cluster compared to the mock group.
Conclusion: Overexpression of the miR-302/367 cluster appears to inhibit the activity of the mTOR signaling pathway in ADSCs and affect the cell reprogramming through this mechanism.