title: Stimulation of P19CL6 with multiple reagents induces pulsating particles in vivo.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Nagano, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohtsu
authors.forename: Kohei
authors.initials: K
authors.lastname: Johkura
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Ito
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Akashima
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Asanuma
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogiwara
authors.forename: Toru
authors.initials: T
authors.lastname: Oka
authors.forename: Issei
authors.initials: I
authors.lastname: Komuro
authors.forename: Katsunori
authors.initials: K
authors.lastname: Sasaki
authors.forename: Jun
authors.initials: J
authors.lastname: Amano
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D045744:Cell Line, Tumor; D019070:Cell Lineage; D018507:Gene Expression Regulation, Developmental; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009206:Myocardium / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000637:transplantation*; D033581:Stem Cell Transplantation; D013234:Stem Cells
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins