Reversal of Ischemic Cardiomyopathy with Sca-1+ Stem Cells Modified with Multiple Growth Factors

Background We hypothesized that bone marrow derived Sca-1 + stem cells (BM Sca-1 + ) transduced with multiple therapeutic cytokines with diverse effects will induce faster angiomyogenic differentiation in the infarcted myocardium. Methods and Results BM Sca-1 + were purified from transgenic male mice expressing GFP. Plasmids encoding for select quartet of growth factors, i.e., human IGF-1, VEGF, SDF-1α and HGF were prepared and used for genetic modification of Sca-1 + cells ( GF Sca-1 + ). Scramble transfected cells ( Sc Sca-1 + ) were used as a control. RT-PCR and western blotting showed significantly higher expression of the growth factors in GF Sca-1 + . Besides the quartet of the therapeutic growth factors, PCR based growth factor array showed upregulation of multiple angiogenic and prosurvival factors such as Ang-1, Ang-2, MMP9, Cx43, BMP2, BMP5, FGF2, and NGF in GF Sca-1 + ( p< 0.01 vs Sc Sca-1 + ). LDH and TUNEL assays showed enhanced survival of GF Sca-1 + under lethal anoxia ( p< 0.01 vs Sc Sca-1 + ). MTS assay showed significant increased cell proliferation in GF Sca-1 + ( p< 0.05 vs Sc Sca-1 + ). For in vivo study, female mice were grouped to receive the intramyocardial injection of 15 μl DMEM without cells (group-1) or containing 2.5×10 5 Sc Sca-1 + (group-2) or GF Sca-1 + (group-3) immediately after coronary artery ligation. As indicated by Sry gene, a higher survival of GF Sca-1 + in group-3 on day4 (2.3 fold higher vs group-2) was observed with massive mobilization of stem and progenitor cells (cKit + , Mdr1 + , Cxcr4 + cells). Heart tissue sections immunostained for actinin and Cx43 at 4 weeks post engraftment showed extensive myofiber formation and expression of gap junctions. Immunostaining for vWF showed increased blood vessel density in both peri-infarct and infarct regions in group-3. Infarct size was attenuated and the global heart function was improved in group-3 as compared to group-2. Conclusions Administration of BM Sca-1 + transduced with multiple genes is a novel approach to treat infarcted heart for its regeneration.