Directional differentiation of human embryonic stem cells into cardiomyocytes by direct adherent culture

Abstract

Myocardial infarction is a serious and common disease in clinics, with a poor prognosis and a high mortality in the long term. The latest research progress in stem cell research, which has been at the frontier of medical research in recent years, gives hope to the patients with heart disease. Researchers have been trying to find an efficient and practical differentiation procedure to induce embryonic stem cells (ESCs) to differentiate into cardiomyocytes. This study used a direct adherent-culture method to induce the differentiation of human ESCs (hESCs) into cardiomyocytes in vitro and its differentiation efficiency was detected. The hESCs were induced into cardiomyocytes by adherence culture using the inducers activin A and BMP4. The time of appearance of beating cardiomyocytes, the percentage of beating colonies, and the beating frequency of cardiomyocytes under the microscope were observed and counted; the specific cardiomyocyte marker cTnT was started by immunofluorescence, and the electrophysiological function of cardiomyocytes was detected by patch clamp experiment. An apoptosis-Hoechst staining kit was used to detect the apoptosis ratio of beating of cardiomyocytes which had been treated by hypoxia for 24 hours. Widespread spontaneous beating cardiomyocytes was typically observed by day 13 after differentiation. The statistical result was that the average time of appearance of beating cardiomyocytes was 13·0±1·1 days, the percentage of beating colonies was 66·7%, and the beating frequency of cardiomyocytes 63·0±7·0 times/min; beating cardiomyocytes were positive to cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected, and the apoptosis ratio of beating cardiomyocytes which had been treated by hypoxia for 24 hours was 8·0±0·5%. The direct adherent-culture method was successfully used to induce the differentiation of hESCs into cardiomyocytes. The adherent method of hESC induced with activin A+BMP4 was determined to be more simple and effective than other methods. The differentiation efficiency reached 66·7%, and the differentiation time was about 13 days.