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    • br Methods br Results br Discussion

    2018-11-08


    Methods
    Results
    Discussion The structural and functional immaturity of the differentiated cardiomyocytes are major obstacles for developing hPSC-VCM-based cell replacement therapies (Kong et al., 2010; van den Heuvel et al., 2014). Although a number of studies showed that the overall maturation of hPSC-CMs does gradually occur by prolonging culture duration, even without any supplementation of exogenous pro-maturation signals (Lundy et al., 2013; Sartiani et al., 2007), the process is too slow for practical consideration, and the overall phenotype of the prolonged cultured hPSC-CMs was still not comparable to the mature adult counterparts. Pro-maturation strategies including manipulation of ion channels (Lieu et al., 2013) and Ca2+ handling proteins expression (Chen et al., 2015; Liu et al., 2009), miRNAs (Fu et al., 2011), electromechanical conditioning (Hirt et al., 2014), 3-dimensional culturing (Otsuji et al., 2010), and supplementation of growth factors (Yang et al., 2014) have been pursued by various investigators with limited successes reported. During early embryonic development, fetal CMs proliferate rapidly (i.e. hyperplasia). The proliferation ceases in the perinatal period at which the last round of mitosis is a nuclear mitosis without cytokinesis (i.e. acytokinetic mitosis), leaving the majority of cardiomyocytes bi-nucleated in the heart of most species (Li et al., 1997). Such bi-nucleation in metabolically-active nitric oxide synthase inhibitors such as CMs has been postulated as an adaptive feature to allow for an increase generation of RNA and subsequent protein synthesis (Ahuja et al., 2007). In contrast to the normal development, the PEG-induced fusion method that we have adopted for generating FS can be considered a reverse-engineering approach. Nevertheless, cell fusion has been extensively employed for generating hybridomas (Lane, 1985) and for studying cellular reprogramming (Pereira et al., 2008; Blau et al., 1983). Viable fused multi-nucleated FS doubled in size compared to the unfused counterparts were generated with hPSC-VCMs for the first time to address whether an acute increase of the physical size and nucleation status would directly impact on the hPSC-VCM functional properties/level of maturation. Spontaneous firing activity (i.e. automaticity) is common in hPSC-CMs owing to a relatively depolarized MDP, regardless of cell lines and differentiation protocols used (van den Heuvel et al., 2014; Liu et al., 2016). Using spontaneous firing rate and AP profile as the primary indices of electrophysiological maturity (Liu et al., 2016), reduced automaticity and moderately prolonged APD were observed in the FS generated, signifying a partially facilitated electrophysiological maturation. However, the lack of improvement in other functional properties such as Ca2+ handling suggested that the increase in cell size and nucleation status alone remained insufficient to drive to an overall maturation. These results highlight the importance of a combinatorial approach deploying multiple pro-maturation strategies (e.g., 3D environmental cues and other non-cell autonomous stimuli) for achieving a more physiologically relevant adult CM-like phenotype.
    Acknowledgements This work was supported by a grant from the Theme-based Research Scheme (T13-706) by the Research Grants Council of the Hong Kong Special Administrative Region, China.
    Introduction A pathological hallmark of Parkinson\'s disease (PD) is degeneration of dopamine (DA) neurons in substantia nigra pars compacta (SNc) of the midbrain (Zigmond and Burke, 2002; Iversen et al., 2010). This causes the motor symptoms of PD such as bradykinesia, resting tremor, postural instability and muscle rigidity (Gelb, 1999). Systemic administration of drugs such as the DA precursor L-3,4-dihydroxyphenylalanine (levodopa or L-DOPA) or DA receptor agonists alleviate motor symptoms by restoring SNc DA neurotransmission towards normal. However, continual use of these drugs produces worsening side-effects such as dyskinesia and impulse control disorders (ICDs), psychoses and anxiety (Obeso et al., 2000; Ahlskog and Muenter, 2001; Fabbrini et al., 2007; Weintraub et al., 2010), probably due to non-physiological delivery of SNc DA and excessive activation of DA receptors in unaffected areas of brain (Weintraub, 2008). Therefore it is generally agreed that more physiological and targeted restoration of SNc DA neurotransmission will more effectively relieve PD motor symptoms without side-effects.