Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, 100029, People's Republic of China.
Department of Ultrasound, The Third Medical Center of PLA General Hospital, Beijing, 100039, People's Republic of China.
In Vitro Cell Dev Biol Anim. 2023 Apr;59(4):256-263. doi: 10.1007/s11626-023-00764-4. Epub 2023 Apr 17.
To promote the differentiation of human-induced pluripotent stem cells (hiPSCs) into myocardium through a standard chemically defined and small-molecule-based induction protocol (CDM3), and preliminarily prepare myocardial patches that provide experimental data and theoretical support for further maturation through other in vitro experiments and safety studies in vivo. After resuscitation, culture, and identification of hiPSCs, they were inoculated onto Matrigel-coated polycaprolactone (PCL). After 24 h, cell growth was observed by DAPI under a fluorescence microscope and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, scanning electron microscopy was performed to observe the morphology of cells on the patch surface. On days 1, 3, 5, and 7 of culture, cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and a curve was drawn to observe cell growth and proliferation. After co-culture with Matrigel-covered PCL for 24 h, hiPSCs were divided into control and CDM3 groups, and cultured for an additional 6 d. On the eighth day, cell growth was observed by DAPI under a fluorescence microscope, hiPSC stemness was identified by OCT4 fluorescence, and cardiomyocytes were identified by cardiac troponin T (cTnT) and α-actin expression. hiPSCs co-cultured with Matrigel-covered PCL for 24 h emitted green fluorescence indicating OCT4, showing that hiPSCs maintained their stemness on Matrigel-covered PCL scaffolds. DAPI emitted blue fluorescence, indicating that cells grew clonally with uniform cell morphology. Scanning electron microscopy showed that hiPSCs adhered and grew on PCL covered with Matrigel, with clearly visible cell outlines indicating normal morphology. Assessment of cell viability by the CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds covered with Matrigel. After 6 d of culture, immunofluorescence showed that control group hiPSCs highly expressed the stem cell marker OCT4 but not myocardial markers cTnT or α-actin. In contrast, notable expression of myocardial markers cTnT and α-actin but not OCT4 occurred in the CDM3 group. hiPSCs can proliferate and grow on PCL scaffolds covered with Matrigel. Under the influence of CDM3, hiPSCs differentiated into cardiomyocyte-like cells, allowing the preliminary preparation of myocardial patches that can provide a better method for clinical treatment of myocardial infarction.
通过标准化的化学定义和基于小分子的诱导方案（CDM3）促进人诱导多能干细胞（hiPSC）向心肌分化，并初步制备心肌贴片，通过其他体外实验和体内安全性研究为进一步成熟提供实验数据和理论支持。复苏、培养和鉴定 hiPSC 后，将其接种到涂有 Matrigel 的聚己内酯（PCL）上。24 小时后，通过荧光显微镜观察 DAPI 下细胞的生长情况，并通过 OCT4 荧光鉴定 hiPSC 的干性。固定后，通过扫描电子显微镜观察贴片表面细胞的形态。培养第 1、3、5 和 7 天，通过细胞计数试剂盒-8（CCK-8）测定细胞活力并绘制曲线观察细胞生长和增殖情况。与涂有 Matrigel 的 PCL 共培养 24 小时后，将 hiPSC 分为对照组和 CDM3 组，再培养 6 天。第 8 天，通过荧光显微镜观察 DAPI 下细胞生长情况，通过 OCT4 荧光鉴定 hiPSC 干性，通过心肌肌钙蛋白 T（cTnT）和α-肌动蛋白表达鉴定心肌细胞。与涂有 Matrigel 的 PCL 共培养 24 小时的 hiPSC 发出绿色荧光，表明 OCT4，表明 hiPSC 在涂有 Matrigel 的 PCL 支架上保持其干性。DAPI 发出蓝色荧光，表明细胞以均匀的细胞形态克隆生长。扫描电子显微镜显示，hiPSC 附着并在涂有 Matrigel 的 PCL 上生长，细胞轮廓清晰可见，表明形态正常。CCK-8 法评估细胞活力显示，hiPSC 在涂有 Matrigel 的 PCL 支架上增殖和生长。培养 6 天后，免疫荧光显示对照组 hiPSC 高度表达干细胞标志物 OCT4，但不表达心肌标志物 cTnT 或α-actin。相比之下，CDM3 组明显表达心肌标志物 cTnT 和α-actin，但不表达 OCT4。hiPSC 可在涂有 Matrigel 的 PCL 支架上增殖和生长。在 CDM3 的影响下，hiPSC 分化为心肌样细胞，为心肌贴片的初步制备提供了可能，为心肌梗死的临床治疗提供了更好的方法。
