Experimental study on the preparation of endothelial cells by induced pluripotent stem cell technology for the construction of patient-specific tissue-engineered valve
10.3760/cma.j.issn.1009-6906.2015.05.004
- VernacularTitle:诱导多功能干细胞技术制备内皮细胞构建患者组织工程瓣膜的实验研究
- Author:
Bin LIN
1
;
Deguang FENG
1
;
Feng WANG
1
Author Information
1. 河南省人民医院心血管外科,郑州,450003
- Publication Type:Journal Article
- Keywords:
Human skin fibroblast;
Induced pluripotent stem cell;
Endothelial cell;
Tissue engineered heart valve
- From:
Chinese journal of nautical medicine and hyperbaric medicine
2015;22(5):352-355,363
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the feasibility of constructing tissue engineered heart valve (TEHV) in deeellularized porcine aortic valve scaffolds by seeding endothelial cells (EC) differentiated from human skin fibroblasts (HSF) derived from pluripotent stem cells (iPSc).Methods Primary HSF were isolated from the patient and infected by lentiviral supernatant harboring OCT4, SOX2, KLF4, c-MYC genes.Changes in cell morphology were observed after infection.Alkaline phosphatase (AP) staining,immunofluorescence staining were used to identify the derived HSF-iPSc.Then, HSF-iPSc were differentiated directly in induction medium.The markers of endothelial cells were detected by Western Blot during differentiation.CD31-positive cells were sorted by flow cytometry after differentiation.HSF-iPSc-EC markers were detected by immunofluorescence staining, and HSF-iPSc-EC function was analyzed by phagocytosis assay and tube formation.Decellularized porcine aortic valve scaffolds were prepared and observed by HE staining and scanning electron microscopy.TEHV was constructed by seeding HSF-iPSc-EC on the valve scaffolds and cultured by static-rotary-static model.Morphological characteristics of TEHV were observed by HE staining and scanning electron microscopy.oα-SMA expression levels were detected by immunofluorescence staining.Results Morphology of the reprogrammed HSF was identical to the embryonic stem cells.AP staining was positive, and pluripotency markers of Nanog, SSEA3, SSEA4, Tra-1-60 and Tra-1-81 were expressed positively.The endothelial markers of CD31, VE-cad, vWF, eNOS were up-regulated continually during the differentiation of HSF-iPSc into EC.CD31, VE-cad, vWF, and eNOS were expressed in sorted HSF-iPSc-EC.Meanwhile, HSF-iPSc-EC had obvious acetylated LDL phagocytosis and tube formation capacity.Cells in porcine aortic valve were stripped completely and the reticular structure of fibers remained intact.Endothelial-like cells displayed stratified growth on the surface of the constructed TEHV with good order and adhesion.In addition,the planted HSF-iPSc-EC had the expression of α-SMA with spatial specificity.Conclusions The obtained HSF-iPSc-EC could be used as ideal seed cells for the construction of TEHV, and the goal of reendothelialization in decellularized aortic valve in vitro was realized preliminarily.
