**4. Combining stem cell transplantation and Angiopoietin-1 delivery**

Gene delivery strategy has developed over the years from direct plasmid injection to stem cell based ex-vivo delivery strategy. Stem cells are excellent carriers of therapeutic genes for delivery to the various body tissues and organs including the heart (Suzuki et al. 2001; Yau et al. 2007; Ye et al. 2007; Haider et al. 2008). Despite all the progress made, it remains to be defined whether cell based gene therapy can overcome several potential impediments such as poor transfection efficiency, unregulated transgene expression, low survival rate of transplanted cells into ischemic zones etc. On the same note, there are a number of parameters which require optimization including cell type, number of transfected cells to be transplanted, time of cell transplantation after infarction, and route of cell transplantation.

Angiopoietin-1 is one of the many angiogenic growth factors which have been extensively studied for pro-angiogenic activity in the ischemic tissues. We performed a comparative assessment of the methods to deliver angiopoietin-1 gene delivery for angiogenic repair of the infarcted heart using an experimental porcine heart model of chronic infarction (Ye et al. 2007). Our results showed that skeletal myoblast based delivery of angiopoietin-1 transgene was more effective as compared to the approach of direct injection of adenoviral vector encoding for angiopoietin-1. The genetically modified skeletal myoblasts carrying angiopoietin-1 transgene served as a reservoir of the transgene product and ensured localized release of angiopoietin-1 at the site of the cell graft without safety concerns associated with the use of direct injection of adenoviral vector (Ye et al. 2007). Besides we observed extensive survival of the transplanted skeletal myoblasts which underwent myogenic differentiation to repopulate the infarcted myocardium.

Given that the development of stable and functional blood vessels is regulated by a critical balance between several pro- and anti-angiogenic factors which also co-ordinate with various vasculogenic cells, we hypothesized that a single angiogenic factor may be insufficient to achieve the desired outcome. We therefore opted to combine angiopoietin-1 and Vegf for co-expression to achieve angiogenic synergism between the two growth factors (Ye et al. 2007). We developed a bicistronic adenoviral vector which encoded for human Vegf165 and angiopoietin-1 driven by the same promoter. The vector was used to genetically modify human skeletal myoblasts which were later transplanted in a porcine heart model of coronary artery ligation. We observed excellent survival of the transplanted skeletal myoblasts for up to 12 weeks using transient immunosuppression. Immunohistological studies showed myogenic differentiation of the skeletal myoblasts and increased blood vessel density in the infarct as well as peri-infarct regions with highest maturation index in the animal heart treated with skeletal myoblasts co-expressing Vegf and angiopoietin-1. Regional blood flow, measured with fluorescent microspheres, was significantly improved which revealed the functional competence of the newly formed blood vessels. These findings signified the feasibility of multimodal therapeutic approach based on simultaneous delivery of angiopoietin-1 and Vegf combined with cell transplantation.

Although skeletal myoblasts showed excellent ability as transgene carriers, one of the major drawbacks is their failure to develop gap junctions with the host cardiomyocytes and arrhythmogenicity (Fouts et al. 2006). We therefore hypothesized that the use of bone marrow derived mesenchymal stem cells might be a better option. Mesenchymal stem cells have been extensively studied for their cardiac reparability and regenerative potential (Chen et al. 2010; Kim et al 2010.; Labovsky et al. 2010; Haider et al. 2009) besides having superior transgene carrying capability (Chen et al. 2010; Huang et al. 2010; Tang et al. 2010; Haider et

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