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.