Novel IRES-based lentivirus co-expressing IGF1 and BMP2 enhances both cardiomyogenesis and cytoprotection of bone marrow-derived mesenchymal stem cells

Purpose: The in vivo effects of bone marrow mesenchymal stem cells (BM-MSC) in repairing ischemic hearts are limited by poor engraftment, low differentiation rate and, in elderly patients, by reduced paracrine properties. To overcome these limitations, we genetically engineered BM-MSC with a novel bicistronic lentivirus co-expressing IGF1 and BMP2 (IB), two factors involved in both cardiac differentiation and cytoprotection. Methods: We cloned an IRES-based expression cassette into a novel lentiviral vector. Rat BM-MSC were transduced with a control (GFP-MSC) or IB (IB-MSC) virus. Cardiac differentiation was evaluated by RT-qPCR, western blot, and immunocytochemistry for cardiac specific markers such as GATA4, Nkx2.5, Tbx5, Mef2c, Cx43, cTnT, a-MHC, a-SA and a-SR1. Resistance to hypoxia-induced apoptosis was measured after 24hrs of hypoxia. To study the cytoprotective paracrine properties, H9c2 cells were deprived of oxygen and glucose (6hrs), and then reoxygenated (18hrs) in the presence of control medium (CTRL-M) or medium conditioned by either GFP-MSC (GFP-CM) or IB-MSC (IB-CM). Viability was measured by MTS assay, apoptosis by caspase-3 activation, cytotoxicity by LDH release. Transcriptional levels of known pro- and anti-apoptotic genes were measured by RT-PCR. Activation of I and B associated pathways (Akt, ERK1/2, and SMAD1/5/8) was assessed by western blot. Results: The transduction efficiency with our novel vector was extremely high (>90% with one cycle of infection). Compared with GFP-MSC, IB-MSC triggered expression of cardiac markers both at RNA (Tbx5 +3.8, p<0.01; Mef2c +1.7, p<0.05; GATA4 +3.1, p<0.05; Cx43 +7.5, p<0.001; cTnT +6.4, p<0.01; a-SA +4.8, p<0.001) and protein level and favoured the nuclear translocation of cardiac-specific transcription factors. Furthermore, IB-MSC showed increased viability (+26.8%, p<0.01), and marked reduction of apoptosis (-62.9%, p<0.001) and cytotoxicity (-22.9%, p<0.05) vs GFP-MSC after hypoxia. IB-CM also increased H9c2 viability (+44.2% vs CTRL-M, p<0.001; +21.4% vs GFP-CM, p<0.01) and reduced caspase-3 activation (-68% vs CTRL-M, p<0.001; -60.4% vs GFP-MSC, p<0.001) and LDH release (-34% vs CTRL-M, p<0.001; -20% vs GFP-MSC, p<0.05). These cytoprotective effects were matched by a strong activation of Akt, ERK1/2 and SMAD1/5/8 anti-apoptotic pathways, by enhanced expression of Bcl-2, Stat3 and Hsp20 pro-survival genes and by inhibition of FasL and TNFa pro-apoptotic genes. Conclusions: IB overexpression commits BM-MSC toward the cardiac lineage, increases their resistance to hypoxia and also improves their cytoprotective paracrine effects.