Abstract 1269: Engineering a New Insulin-Like Growth Factor-1 Protein for Embryonic Stem Cell Therapy
Background: Myocardial implantation of embryonic stem cells (ES) differentiated to cardiomyocytes is an exciting potential therapy, but survival after implantation is a problem, as in many types of cell therapy. Here we show that IGF-1 improves survival of ES-derived cardiomyocytes and report the development of a novel heparin binding (HB)-IGF-1 fusion protein engineered to improve survival of injected cells.
Method and Results: To minimize teratoma formation, we studied ES cells committed to the cardiomyocyte lineage. Mouse ES cells, stably transfected with α-cardiac myosin heavy chain promoter-driven enhanced green fluorescent protein (EGFP), were differentiated into cardiomyocytes by the hanging drop method and EGFP positive cells were purified by fluorescent cell sorting. In these ES-derived cardiomyocytes, IGF-1 reduced cell death induced by serum deprivation (13.6 +/− 1.9 % vs 25.9 +/− 2.5 % in control, p<0.05) and decreased apoptosis induced by serum deprivation (TUNEL-positive cells 8.0 +/−1.5 % to 4.3 +/− 0.5 % respectively, p<0.05). Furthermore, IGF-1 decreased Doxorubicin (1μM, 24hr) or chelerythrin (3μM, 1hr)-induced apoptosis (p<0.01). The phosphoinositide-3 kinase inhibitor, LY294002 (10μM), inhibited the protective effect of IGF-1 on Doxorubicin-induced apoptosis (p<0.05). Since IGF-1 diffuses rapidly away from injected sites, we then designed and expressed a novel recombinant IGF-1 fusion protein with an N-terminal HB domain. The protein was purified by Nickel-affinity and then subjected to oxidative refolding to restore biological activity. HB-IGF-1 bound to cell surfaces dramatically better than IGF-1 and HB-IGF-1 activated Akt in neonatal cardiac myocytes and 3T3 fibroblasts as potently as native IGF-1.
Conclusions: Because IGF-1 improves survival of ES-derived cardiomyocytes in vitro, this new Heparin-binding IGF-1 has the potential to improve cell therapy by binding to the surfaces of injected cells. This demonstrates the potential for changing the cellular microenvironment through locally-delivered therapeutic proteins.