Transcriptional Abundance of Myosin Light Chain 2 Gene in Cardiac Differentiated Canine Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are promising cell source for cardiac tissue engineering and cell based therapies for heart repair as they can be expanded in vitro and differentiated into most cardiovascular cell types, including cardiomyocytes. During embryonic heart development, this differentiation occurs under the influence of internal and external stimuli that guide cells to go down the cardiac lineage. The aim of this study was to characterize the cardiac differentiation potential of a canine iPS cell. With the use of a standard embryoid body–based differentiation protocol for iPS cells were differentiated for 24 days. In vitro differentiations of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB’s were differentiated using 5-azacytidine (5-Aza). During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 to evaluate the expression of cardiomyocyte specific marker. Analyses on molecular, structural, and functional levels demonstrated that iPS cell– derived cardiomyocytes show typical features of ES cell– derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes. The differentiated cells expressed cardiac-specific gene myosin light chain 2 (MYL2) which started from day 8 of differentiation and highest expression was observed on day 16. Immunocytochemistry and relative expression of cardiac specific genes revealed that iPS cells differentiate into functional cardiomyocytes and allow to derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering

Generation of transgenic mesenchymal stem cells expressing green fluorescent protein as reporter gene using no viral vector in caprine.

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. While there are many ways to label and track cells each with strengths and weakness, the green fluorescent protein (GFP) is a reporter gene commonly employed. In the present study, caprine MSC were collected from bone marrow and cells were characterised with MSC specific markers. Passage 10 (P10) MSC cells were transfected using plasmid vector containing GFP as reporter gene with different concentrations of DNA and lipofectamine. Six different concentrations of DNA and lipofectamine as 1 µg DNA: 2 µL lipofectamine, 1 µg DNA: 2.5 µL lipofectamine, 1.2 µg DNA: 2.2 µL lipofectamine, 1.2 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 2.5 µL lipofectamine, 1.5 µg DNA: 3 µL lipofectamine were used. After 24 h and 48 h of transfection, caprine MSC were observed under florescent microscope. Highest transfection rate indicating green flourecscent MSC were found when the cells were transfected with 1.2 µg DNA: 2.2 µL lipofectamine and 1.5 µg DNA: 2.5 µL lipofectamine than other combinations. These cells have been propagated beyond 4th passage maintaining GFP expression. The results indicated that stable GFP positive MSC cells can be generated using the above protocol. These cells are being used for transplantation studies.