Ibarra-Ibarra Blanca Rebeca, Franco Martha, Paez Araceli, López Elvira Varela, Massó Felipe
Laboratory of Translational Medicine, UNAM-INCar Research Unit, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico City, Mexico.
Department of Nephrology, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico City, Mexico.
Stem Cells Int. 2019 Apr 1;2019:8940365. doi: 10.1155/2019/8940365. eCollection 2019.
Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. 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 Ca 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 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.
基于细胞的疗法已成为治疗心血管疾病的一种手段；然而，仍有一些难题有待解决。心肌细胞生成可能是扩大临床应用选择的一种解决方案。先前关于脂肪组织来源的间充质干细胞（ASC）的研究中，心脏分化存在差异，且缺乏对心肌细胞分化效率的测量，这推动了本研究。在此，我们通过定向心肌细胞分化方案提高了ASC来源的类心肌细胞分化效率：BMP - 4 + VEGF（第0 - 4天），随后是含有细胞因子（IL - 6和IL - 3）的甲基纤维素培养基（第5 - 21天）。用定向心肌细胞分化方案处理的培养物从第7天起显示出类心脏细胞和“玫瑰花结样结构”。通过流式细胞术评估心肌肌钙蛋白T（cTnT）阳性细胞的百分比，以定量方式评估心肌细胞分化效率。用定向心肌细胞分化方案处理的ASC在第15天获得了高达44.03%（39.96%±3.78）的分化效率，且无需任何富集步骤。此外，在第21天，我们通过免疫荧光观察到，用定向心肌细胞分化方案处理的培养物中早期、晚期和心脏成熟分化标志物（Gata - 4、cTnT、心肌肌球蛋白重链（MyH）和肌浆/内质网Ca ATP酶（SERCa2））呈阳性表达。与其他方案不同，使用胚胎心肌发生的关键因子并结合含有先前报道的心脏发生细胞因子（IL - 6和IL - 3）的甲基纤维素培养基似乎有利于心肌细胞生成。这种新型高效培养方案使ASC来源的心脏分化更有效。需要进一步研究以鉴定用于心脏富集的ASC来源的心肌细胞表面标志物。
