Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells☆

Yihua Zhang 0 Yuankui Chu 0 Wenzheng Shen 0 Zhongying Dou 0 0 Shaanxi Branch of National Stem Cell Engineering and Technology Centre, College of Veterinary Medicine, Northwest A & F University , Yangling 712100, Shaanxi, China The aim of this study is to investigate effects of 5-azacytidine (5-aza) induction duration on differentiation of bone marrow mesenchymal stem cells (MSCs) from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. hfMSCs were stimulated with 10 mmolyl 5-aza for 24 h (group A), 48 h (group B) and 21 days (group C), respectively. During the induction, 30-40% of the cells gradually enlarged, elongated, connected with adjoining cells and formed myotube-like structures, branches and string-bead-like nuclei. Some of the cells congregated into cell clusters or strips. After the induction, numerous myofilaments in the cytoplasm and conjunction of intercalated disclike structure between adjoining cells were observed. The induced cells expressed messenger ribonucleic acids (mRNAs) and proteins of myocardium-specific a-actin, sarcomeric b-myocin heavy chain and troponin-T. The positive cell percentages for the three antigens in group C were each significantly higher than those antigens in group A and B (P-0.01) and the cell population doubling time (PDT) of group C was longer than those of group A and B (P-0.01). These indicate that 21-d induction with 10 mmolyl 5-aza slows down proliferation speed of hfMSCs but increases differentiation rate of hfMSCs into cardiomyocyte-like cells if compared with 24-48 h induction. 2009 Published by European Association for Cardio-Thoracic Surgery. All rights reserved. 1. Introduction Previous researches show that bone marrow cell transplantation at the time of acute myocardial infarction can improve cardiac function w1, 2x. Further researches substantiate that (1) hematopoietic stem cells improve cardiac function in the section of myocardial infarction through inducing neoangiogenesis w3x, not through regenerating cardiomyocytes w4x, (2) the beneficial effects of mesenchymal stem cells (MSCs) directly injected into the infarcted myocardium most likely result from the trophic effects of MSCreleased substances on native cardiac and vascular cells, not from trans-differentiation into cardiomyocytes w5x, and there is the risk for unwanted differentiation toward calcifications andyor ossifications after direct transplantation w6, 7x, and (3) MSCs undergoing directed induction in vitro can improve cardiac function through regenerating cardiomyocytes after injections into the infarcted myocardium w8, 9x. Consequently, the search for novel strategies of inducing the cells toward a cardiac lineage in vitro has been recommended w10x. 5-azacytidine (5-aza) is a DNA This research was supported by grants from the Key Program of National Ministry of Education (03160), the National Natural Science Foundation of China (30671067) and the Key Program of Shaanxi Province (2006Kz05-G1). *Corresponding author. Tel./fax: q86-029-8708-0068. E-mail address: (Z. Dou). 2009 Published by European Association for Cardio-Thoracic Surgery demethylating chemical compound and previous studies have proved that 5-aza induces uncontrolled myogenic specification by random demethylation w11x and the optimal concentration of 5-aza to induce cardiomyogenic differentiation is 10 mmolyl w1x. However, almost in all reports relative to differentiation of MSCs induced with 5-aza into cardiomyocyte-like cells in vitro, the induction time of 5-aza is 24 h and no paper to focus on selecting the induction time of 5-aza was seen. This study was conducted to investigate the effects of 5-aza induction duration on proliferation and differentiation of bone marrow MSCs from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. 2. Material and methods 2.1. Preparation of hfMSCs Human first trimester abortuses (age of 1012 weeks) were obtained from a local hospital with permission from the patients, hospital and the Ethics Committee of Northwest A & F University. hfMSCs were isolated from the aborted fetuses by scissoring their long bones lengthwise, followed by rinsing and culturing of the whole marrow cells in Minimum Essential Medium alpha (a-MEM, Gibco, Billings, Montana, USA), containing 20% fetal calf serum (FCS, Y. Zhang et al. / Interactive CardioVascular and Thoracic Surgery 9 (2009) 943946 Stemcell Technologies Inc, Vancouver, Canada), 100 IUyml penicillin, 100 mgyml streptomycin and 0.1 mmolyl b-mercaptoethanol (Sigma, Loveland, CO, USA). At 80% confluence of the cultured hfMSCs were harvested with 2.5 gyl trypsin (Gibco) containing 0.4 gyl edetic acid (Invitrogen, Carlsbad, California, USA) and passaged in a ratio of 1:3, with the medium changes every other day. Before differentiation the cells of passage 3 were detected by flow cytometer (Beckman Coulter Inc, Fullerton, California, USA) for antigen markers of the putative hfMSCs, CD29, CD44, CD71, CD105 and CD166, and those of hemopoietic stem cells, CD11a, CD14, CD34 and CD45 (all from Beckman Coulter Inc) as described previously w12x. 2.2. Differentiation of cardiomyocyte-like cells hfMSCs of the fourth passage were cultured in 12-well plates at 4050% confluence and induced in three groups. The cells in group A and B were cultured respectively for 24 h and 48 h in a complete medium (RPMI1640, Gibco; 10% FCS, Stemcell; 100 IUyml Penicillin and 100 mgyml Streptomycin) plus 10 mmolyl 5-aza (Sigma-Aldrich Co, St Louis, USA), and then both switched to the complete medium without 5-aza. Those in group C were always cultured in the complete medium plus 10 mmolyl 5-aza. And cells cultured in the complete medium were used as uninduced stem cell control. All treatments were terminated at day 21 of total culture, with the medium changes every three days. 2.3. Transmission electron microscopy The induced and uninduced cells were scraped and centrifugalized to cell clusters. The cell pellets were pre-fixed 4% glutaraldehyde in phosphate buffer solution (PBS) for 2 h, washed in PBS, post-fixed in 2% osmium tetroxide, dehydrated, stained in uranyl acetate, embedded in epoxy resin and made into ultrathin sections (60 nm). And the ultrastructure of cells in all groups was viewed and analysed under a LIBRA 200FE transmission electron microscope (SII NanoTechnology Inc, Japan). 2.4. Cell population doubling time (PDT) To evaluate cell growth rate before, during and after the induction, cells were randomly collected and counted from five wells in every group. The PDT of the cells was calculated according to the formula PDTs(TT0) lg2y(lgNt lgN0). In this formula, PDT represents population doubling time, T0 and T separately represent starting time and ending time of cell culture, and N0 and Nt separately represent the cell number at the start and the end of each culture. 2.5. Immunocytochemical analysis Induced and uninduced cells were fixed with paraformaldehyde in PBS for 1 (...truncated)