Improved Efficiency of Cardiomyocyte-Like Cell Differentiation from Rat Adipose Tissue-Derived Mesenchymal Stem Cells with a Directed Differentiation Protocol

Abstract

Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and “rosette-like structures” from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

Stage specific transcriptome profiles at cardiac lineage commitment during cardiomyocyte differentiation from mouse and human pluripotent stem cells

“…These networks might have developed because PDGFRα + CLCs and KDR + PDGFRα + CPCs share some characteristics of other mesodermal lineage cells, such as immune cells and adipocytes. Although a few studies have reported that Il6 contributes to cardiomyocyte-like cell differentiation from mesenchymal stem cells (16,17), Il6 and TLR4 are primarily inflammatory, immune-related proteins, and are mainly associated with cardiovascular diseases such as heart failure and atherosclerosis (18,19). Similarly, Adipoq, Fabp4, Hcar2, and AHSG are associated with cardiometabolic diseases, including diabetes, obesity, heart failure, and coronary atherosclerosis (20)(21)(22)(23).…”

Role of miRNA-324-5p-Modified Adipose-Derived Stem Cells in Post-Myocardial Infarction Repair

“…Significantly, it was previously unmasked that miR-342-5p modified ADSCs potentiated endothelial cells to defense atherosclerosis ( 53 ). In addition to this, ADSCs limited the level of cTnT ( 54 ). Altogether, it might be quite certain that miR-342-5p modified ADSCs served as contributor to post-MI repair.…”

RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells

“…Yet, it was shown that cytokines like BMP4, IL and TGF improve cardiac development of human and non-human MSCs [57,77]. However, the cardiomyocyte-like cells derived from these programmed MSCs lack profound sarcomere formation, beating activity and physiological maturation [78,79]. This is in accordance to our data indicating that mRNA transfection could promote the expression of early cardiac proteins, while differentiation efficiency and elaboration of a terminal cardiac phenotype is profoundly limited when compared to PSC differentiation protocols [27,31].…”