Effect of CDM3 on the co-culture of human induced pluripotent stem cells with matrigel-coated polycaprolactone to make cardiac patch
- VernacularTitle:CDM3对人诱导多能干细胞与覆有基质胶的聚己内酯共培养制作心肌补片的影响
- Author:
Yue DAI
1
;
Fan ZHOU
2
;
Jianwei ZHENG
3
;
Junsheng MU
1
;
Ping BO
1
;
Bin YOU
1
Author Information
1. Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, 100029, P. R. China
2. Department of Ultrasound, The Third Medical Center of PLA General Hospital, Beijing, 100039, P. R. China
3. Heart Center of Sunshine Fusion Hospital, Weifang, 261205, Shandong, P. R. China
- Publication Type:Journal Article
- Keywords:
Human induced pluripotent stem cells;
polycaprolactone;
CDM3;
cardiac patch
- From:
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery
2023;30(05):738-745
- CountryChina
- Language:Chinese
-
Abstract:
Objective To provide experimental data and theoretical support for further studying the maturity of cardiac patches in other in vitro experiments and the safety in other in vivo animal experiments, through standard chemically defined and small molecule-based induction protocol (CDM3) for promoting the differentiation of human induced pluripotent stem cells (hiPSCs) into myocardium, and preliminarily preparing cardiac patches. Methods After resuscitation, culture and identification of hiPSCs, they were inoculated on the matrigel-coated polycaprolactone (PCL). After 24 hours, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, electron microscope scanning was performed to observe the cell morphology on the surface of the patch. On the 1st, 3rd, 5th, and 7th days of culture, the cell viability was determined by CCK-8 method, and the growth curve was drawn to observe the cell growth and proliferation. After co-cultured with matrigel-coated PCL for 24 hours, hiPSCs were divided into a control group and a CDM3 group, and continued to culture for 6 days. On the 8th day, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and hiPSCs stemness was identified by OCT4 fluorescence, and cTnT and α-actin for cardiomyocyte marker identification. Results Immunofluorescence of hiPSCs co-cultured with matrigel-coated PCL for 24 hours showed that OCT4 emitted green fluorescence, and hiPSCs remained stemness on matrigel-coated PCL scaffolds. DAPI emitted blue fluorescence: cells grew clonally with uniform cell morphology. Scanning electron microscope showed that hiPSCs adhered and grew on matrigel-coated PCL, the cell outline was clearly visible, and the morphology was normal. The cell viability assay by CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds coated with matrigel. After 6 days of culture in the control group and the CDM3 group, immunofluorescence showed that the hiPSCs in the control group highly expressed the stem cell stemness marker OCT4, but did not express the cardiac markers cTnT and α-actin. The CDM3 group obviously expressed the cardiac markers cTnT and α-actin, but did not express the stem cell stemness marker OCT4. Conclusion hiPSCs can proliferate and grow on matrigel-coated PCL. Under the influence of CDM3, hiPSCs can be differentiated into cardiomyocyte-like cells, and the preliminary preparation of cardiac patch can provide a better treatment method for further clinical treatment of cardiac infarction.
