Differentiation of rat embryonic stem cells into functional cardiomyocytes

Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental studies.The recent breakthrough in the generation of rat embryonic stem cells(rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation of rESCs into derivatives of three germ lines will serve as a powerful tool and resource for the investigation of mammalian development,cell function, tissue repair,and drug discovery.However, the distinct culture condition and signal inhibitor-depended maintenance of rESCs stand as a considerable challenge for its in vitro differentiation.To address it,we investigated whether rESCs are capable of forming terminal differentiated cardiomyocytes. We found that the embryoid bodies(EBs)-based method used in mouse ESC(mESC) differentiation failed to work in the cultivation of rESCs.We then modified the differentiation protocol and successfully developed an in vitro differentiation system to differentiate rESCs into three embryonic germ layers.By using this method,the rESCs form spontaneous beating cardiomyocytes with the properties similar to those derived from fetal rat hearts and mESCs.This unique cellular system will provide a new approach to study the early development and cardiac function as well as to perform pharmacological test and cell therapy study(Grants:the State Major Research Program of China(2009ZX09503-024,2010CB945603) and CAS(XDA01030000).

Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Cells and Expression of VEGF After Gene Transfection

Objectives To induce the differentiation of rabbit bone marrow mesenchymal stem cells （MSCs） to myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor （VEGF） gene in MSCs transfected by AdTrackCMV-VEGF165. Methods MSCs were isolated and purified from rabbit bone marrow by percoll （11）73 g/ml） and then cultured in low glucose DMEM with 10% FBS. AdTrackCMV-VEGF165 eukaryotic expression vector was constructed and transfected into the MSCs. After being incubated with 5-azacytidine （5- Aza）, the expression of troponin I in MSCs was assayed by immunohistochemistry and the expression of VEGF gene was identified by northern blot and western blot. The concentration of VEGF in the supernatant was measured by ELASA. Results MSCs were isolated and cultured successfully from rabbit bone marrow. The positive cTnI stain of some MSCs after the induction of 5-aza indicated that the cells were differentiated to myogenic cells. Northern blot and western blot showed that the expression of VEGF 165 mRNA was much higher in the hVEGF165 gene transfected cells than that of the control. The concentration of VEGF in the supernatant got to the peak 3-5 days after hVEGF165 gene transfection （1011- 1027 pg/ml） and decreased gradually thereafter, but still higher than that of control group or pAdTrackCMV group （349 pg/mLvs 116 pg/mL or 125pg/ml respectively, MSCs could be induced P〈 0.01）. Conclusions to differentiate to myogenic cells by 5-aza in vitro and could express VEGF by VEGF gene transfection.