Abstract 3489: Axon Guidance of Sympathetic Neurons to Native and Induced Pluripotent Stem (iPS) Cell-Derived Cardiomyocytes Using Glial Cell Line-Derived Neurotrophic Factor (GDNF)
[Aim] Appropriate autonomic innervation is crucial to engineer functional cardiac tissues. The present study is to explore a novel technology for sympathetic axon guidance to iPS cell-derived and native cardiomyocytes using neurotrophic factors.
[Methods] Sympathetic neurons (SNs) and cardiomyocytes (N-CMs) were isolated from superior cervical ganglions and hearts of neonatal rats. iPS cell-derived cardiomyosytes (iPS-CMs) were obtained by microdissection from spontaneously beating clusters of embryoid bodies differentiated from murine iPS cells. iPS-CMs and N-CMs were infected with adenovirus carrying GDNF gene (AdGDNF). Then, clusters of SNs were placed between AdGDNF- and mock-transfected iPS-CMs at a close distance (~1 mm) in the same dish. Effects of GDNF on morphological and functional innervation were further estimated by the application of GDNF (10 microM) into co-culture medium for SNs and either of iPS-CMs or N-CMs. Immunofluorescence, electron microscopy and extracellular potential recording using multi-electrode dishes were conducted.
[Results] When SNs were placed between AdGDNF-infected and mock-transfected iPS-CMs, most of the axons were grown toward AdGDNF-iPS-CMs (n=5). Application of GDNF (10 ng/ml) in the culture medium markedly promoted axon growth toward N-CMs as evaluated by neurifilament-M (NFM)-positive area over the total surface area of alpha-actinin (AA)-positive CMs by 8.7-fold increase compared to CONT (without neurotrophic factors) (p<0.05 vs CONT, n=7). In addition, GDNF significantly enhanced synapse formation by 7.8-fold increase compared to CONT (density of synapsin-1 (SYN)-positive area; p<0.01 vs CONT, n=7). Electron microscopic study revealed that typical synapses were formed with dense vesicles in the presence of GDNF. Stimulation of SNs by the application of nicotine (1 μM) significantly increased the spontaneous beating rate of CMs by 200.1% when cultured with GDNF (n=4, p<0.05 vs. CONT).
[Conclusion] GDNF act as an axon guidance molecule for sympathetic neuron to both stem cell-derived and native cardiomyocytes. The results suggest that sympathetic innervation to regenerated myocardium in vivo may be feasible using novel technology with neurotrophic factors.