Ohtsu Yoshinori, Johkura Kohei, Ito Ken-ichi, Akashima Tomohiro, Asanuma Kazuhiko, Ogiwara Naoko, Oka Toru, Komuro Issei, Sasaki Katsunori, Amano Jun
Department of Surgery, Shinshu University School of Medicine, Nagano, Japan.
Curr Med Res Opin. 2005 May;21(5):795-803. doi: 10.1185/030079905X41499.
Injection of stem cells into ischaemic areas of the heart is expected to be an effective method for myocardial regeneration. The embryogenic carcinoma (EC) cell line P19CL6 is known to differentiate into cardiomyocytes when cultured with dimethyl sulfoxide (DMSO) and is expected to be a promising source for regenerative therapy in cardiac disease. To establish a high-yield method of cardiomyocyte differentiation, P19CL6 cells were double-stimulated with 5-azacytidine. Double stimulation-induced cardiomyocytes were also transplanted into ectopic sites in mice and their function evaluated.
To induce differentiation under adherent conditions, P19CL6 cells were incubated in growth medium with 10 microM 5-azacytidine for 24 h. After 5-azacytidine treatment, P19CL6 cells were incubated with 1% DMSO for nine days until they began to pulsate. Prior to transplantation, cells were treated again with 5-azacytidine. Differentiated cells were injected into the greater omentum, para-aorta region of the retroperitoneum and peri-femoral artery of adult BALB/c nude mice. Nine days after transplantation, irregularly pulsating tissues at a rate slower than the host heart were observed in the transplanted sites. Light microscopy showed formation of cardiac muscle tissues originating from P19CL6 cells. Differentiated cardiomyocytes were positive for cardiac troponin I, cadherin and alpha-smooth muscle actin, and the expressions of Csx/Nkx2.5 and GATA4 mRNAs were up-regulated. Electron microscopy demonstrated components specific to cardiomyocytes, such as Z-bands, desmosomes, fasciae adherens, myofibrils and mitochondria, which confirmed successful heterotopic cardiac muscle differentiation from P19CL6 cells.
This study demonstrated high-yield cardiac muscle differentiation of P19CL6 by 5-azacytidine and DMSO double stimulation and successful formation of cardiac muscle-like tissue by ectopic transplantation of cardiomyocytes derived from P19CL6 into the retroperitoneal area as well as into the peripheral vessel area.
将干细胞注入心脏缺血区域有望成为心肌再生的有效方法。胚胎癌（EC）细胞系P19CL6在与二甲基亚砜（DMSO）共培养时可分化为心肌细胞，有望成为心脏病再生治疗的一个有前景的细胞来源。为建立一种高效的心肌细胞分化方法，用5-氮杂胞苷对P19CL6细胞进行双重刺激。双重刺激诱导产生的心肌细胞也被移植到小鼠的异位部位，并对其功能进行评估。
为在贴壁条件下诱导分化，将P19CL6细胞在含10微摩尔5-氮杂胞苷的生长培养基中孵育24小时。5-氮杂胞苷处理后，将P19CL6细胞与1%DMSO共孵育9天，直至其开始搏动。移植前，细胞再次用5-氮杂胞苷处理。将分化的细胞注入成年BALB/c裸鼠的大网膜、腹膜后主动脉旁区域和股动脉周围。移植9天后，在移植部位观察到有不规则搏动的组织，其搏动速率比宿主心脏慢。光学显微镜显示形成了源自P19CL6细胞的心肌组织。分化的心肌细胞心肌肌钙蛋白I、钙黏着蛋白和α-平滑肌肌动蛋白呈阳性，并且Csx/Nkx2.5和GATA4 mRNA的表达上调。电子显微镜显示了心肌细胞特有的成分，如Z带、桥粒、黏合带、肌原纤维和线粒体，这证实了P19CL6细胞成功地异位分化为心肌。
本研究证明了通过5-氮杂胞苷和DMSO双重刺激可使P19CL6细胞高效分化为心肌，并且将源自P19CL6的心肌细胞异位移植到腹膜后区域以及外周血管区域可成功形成心肌样组织。
