FoxO proteins are known tumor suppressors, cause cell cycle arrest and induce apoptosis besides their vital involvement in maintenance of cellular homeostasis. We observed that MSC with Akt and Ang-1 co-expression ( AAMSC) phosphorylated forkhead box O1 (FoxO1) transcription factor to abrogate its activity in the cells. 7, 8 The present study was designed to determine the downstream signaling involved during coordinated interaction between Akt and Ang-1 transgenes in MSC and their effect on cell proliferation. 5, 6 We have already shown that simultaneous overexpression of Akt and angiopoietin-1 (Ang-1) in bone marrow-derived mesenchymal stem cells (MSC) led to coordinated interaction between the two transgenes and gave better stem cell survival, promoted their angiomyogenic differentiation and stably improved global heart function. Moreover, additive actions associated with genetic manipulation of stem cells may also include their angiomyogenic differentiation and altered paracrine activity in the heart. Given that stem cells are excellent carriers of transgenes, the strategy of genetic reprogramming of stem cells can be exploited to improve their post-transplantation characteristics, including survival and proliferation, for effective participation in myocardial repair process. 4 We hypothesized that manipulation of donor stem cells for sustenance of their inherent property of self-renewal during the acute phase after transplantation would compensate for the massive cell loss. As a part of the latter strategy, up to 1 billion cells have been transplanted in the experimental animal models, and dose-escalating clinical studies have shown that increasing the number of donor cells supported better therapeutic outcome. With regards to the former approach, we have previously reported that preconditioning of stem cells by pharmacological manipulation, 1 growth factor treatment 2 or by exposure to intermittent repeated cycles of anoxia/reperfusion 3 effectively promoted donor stem cell survival in the ischemic heart. The problem is generally addressed by either priming of the cells by activation of survival signaling for improved survival or by increasing the number of donor cells for transplantation to compensate for the cell loss in the cytokine-rich microenvironment of the ischemic heart. Stem cell therapy for the infarcted heart is confronted with the problem of massive death of the donor stem cells post-transplantation, which results in poor prognosis. In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling. EmpMSC) 7 d after engraftment (n = 4 animals/group). During in vivo studies, male GFP + AAMSC transplanted into wild-type female infarcted rat hearts showed significantly higher numbers of Ki67-expressing cells (p < 0.05 vs. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in AAMSC. miR array supported by real-time PCR showed induction of miR-143 in AAMSC (4.73-fold vs. Flow cytometry showed > 10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in AAMSC using EmpMSC as controls. AAMSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt ( AktMSC) and Ang-1 ( Ang-1MSC) transgenes for their individual or simultaneous overexpression ( AAMSC > 5-fold gene level and > 4-fold Akt and Ang-1 protein expression in AAMSC vs. We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor.