Effect of CDM3 on co-culture of human-induced pluripotent stem cells with Matrigel-covered polycaprolactone to prepare cardiac patches

Abstract

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.

Keywords: CDM3; Cardiac patch; Human-induced pluripotent stem cells; Polycaprolactone.