Abstract 1116: Preconditioning And Re-programming Of Bone Marrow Derived Sca-1+Cells By Insulin Like Growth Factor-1 Treatment For Cytoprotection And Cardiomyogenic Differentiation
Background: We hypothesized that preconditioning (PC) of stem cells with insulin like growth factor-1 (IGF-1) before transplantation is cytoprotective and re-programs Sca-1+cells for cardiomyogenic differentiation.
Methods: Sca-1+cells were purified from male C57/Black 6 mice bone marrow. The cells were preconditioned with 100 nM IGF-1 for 30 minutes at 37oC followed by 8 hours of oxygen glucose deprivation (OGD) to assess cytoprotective effects. LDH assay, cytochrome-c release, and mitochondrial membrane potential were used cell viability assessment. Expression of cardiac specific markers was analyzed by real-time PCR and Western blot. For in vivo study, acute myocardial infarction model was developed in female rats. Animals were grouped (n=8/group) to inject intramyocardially 50μl DMEM without cells (group 1), or containing 1x106 non-preconditioned (group 2) or IGF-1 preconditioned (group 3) male Sca-1+cells labeled with PKH26. Survival of the transplanted cells was assessed by real time PCR for sry-gene. Six weeks later, heart function was assessed by echocardiography after which the animals were sacrificed for histological and immunohistological studies.
Results: PC significantly improved donor cell survival (p<0.05) under OGD as compared with non-preconditioned cells. PC induced PI3K/Akt dependent caspase-3 downregulation for improved cytoprotection. Moreover, IGF-1 PC significantly upregulated connexin 43, GATA4 mRNA levels (3 and 3.5 folds respectively) and connexin 43 protein level (3.2 fold). Survival of the Sca-1+cells was 5.5 fold higher in group-3 as compared with group 2 at 7 days after transplantation. Confocal imaging after immunostaining for actinin and connexin 43 showed extensive engraftment and myogenic differentiation of the transplanted cells. Blood vessel density was increased in group-2 (21.4 ± 1.2 per 400x field, p=0.02 vs group 2). Fibrosis was greatly reduced in group-3 (21.5 ± 3.5%, p=0.01). Heart function indices including Ejection fraction (66.2 ± 3.5, p=0.03) and Fractional shortening (30.4 ± 2.0, p=0.03) were significantly improved in group 3.
Conclusions: PC with IGF-1 reprograms Sca-1+cells for cardiomyogenic differentiation besides activating pro-survival signaling under ischemic stress.