We also determined the safety of transplanting incompletely

We also determined the safety of transplanting incompletely differentiated iPS cells. It has been demonstrated that iPS-CMs are safe after transplantation into the heart, but their proliferation is limited (Nelson et al., 2009). Therefore, the selection of adequate time points during differentiation of iPS entacapone is critical. Flk1-positive cells were selected as cardiac progenitors as they possess the capability to differentiate into cardiac lineages (Kattman et al., 2006). In the present study, EBs of iPS cells at days 4 to 8 of differentiation showed a significant increase in Flk1-positive cells. However, unpurified Flk1-positive cells formed teratomas after injection into the hearts of our AMI mouse models; therefore it is unsafe to inject incompletely differentiated iPS cells into the heart. Expression of the Yamanaka factors was silenced in the iPS cells and was not re-activated during the formation of iPSC-CMs (Supplementary Fig. 1), so engrafts derived from more mature cells seemed to be safe (Yan et al., 2011). However, this raised another important issue of whether iPS cell-derived cardiac myocytes would couple with host cells successfully. Engrafts will promote arrhythmia if injected cells cannot couple or synchronize with host cells (Liao et al., 2010).
Conclusion The present study shows that iPS-CMs express cardiac specific markers, express membrane connexin-43, and display cardiac electrophysiological activity. These results indicate that iPS cells might be a promising cell source for the generation of patient-specific cardiomyocytes (Park et al., 2008; Soldner et al., 2009), even for patients with severe heart disease (Carvajal-Vergara et al., 2010; Han et al., 2014), because the cells can be made available in unlimited amounts and are capable of cardiomyocyte generation. However, further studies are warranted to determine the right subgroup of iPS-CMs at a right stage of differentiation to clarify safety issues.
Conflict of interests The following are the supplementary data related to this article.
Acknowledgments This work was supported by a grant NIHP01 GM081627 to AJC.
Introduction The pluripotent NTERA2/D1 (NT2) cell line, derived from human testicular embryonic carcinoma cells (Andrews, 1984), represents a committed neuronal precursor cell that can be induced to differentiate in vitro into postmitotic cells, known as NT2N (or hNT) neurons. Moreover, NT2N cells have the ability to establish functional synapses and to develop neuronal electrophysiological properties, including generation of action potentials and the presence of spontaneous excitatory and inhibitory postsynaptic currents (Hartley et al., 1999a; Podrygajlo et al., 2010; Öz et al., 2013). Consequently, the NT2N cell type is now widely accepted as a suitable model for the study of neuronal networks and differentiation and synaptogenesis processes. In addition, the NT2 clonal line represents a promising cell source for neuronal replacement therapy applicable to the treatment of some neurological conditions, including neurodegenerative disorders such as Parkinson\'s, Huntington\'s, and Alzheimer\'s diseases and acute brain injury such as stroke and head trauma. To this end, grafts or cells in suspension from embryonic ventral mesencephalic tissue, adrenal medullary cells, other neuronal and non-neuronal cells, and even genetically modified cells have produced encouraging results, but none of them are fully satisfactory (Martínez-Morales et al., 2013). As a consequence, researchers have focused their efforts on either trying to increase the efficacy of existing tissue and cell candidates or searching for alternative sources of donor cells. In this sense, NT2N neurons maintain their neuronal phenotype after transplantation into the rodent brain (Trojanowski et al., 1993; Kleppner et al., 1995; Baker and Mendez, 2005) and spinal cord (Hartley et al., 1999b; Lee et al., 2000). Furthermore, NT2N grafts have been shown to improve motor deficits in animal models of stroke (Borlongan et al., 1998a,b; Saporta et al., 1999) and Huntington\'s disease (Hurlbert et al., 1999), making NT2N neurons excellent candidates for cell replacement therapy in neurological disorders (Trojanowski et al., 1997; Lee et al., 2000; Nelson et al., 2002; Borlongan et al., 2006; Hara et al., 2008). Indeed, in a phase I clinical trial on 12 patients with basal ganglia stroke and stable motor deficits, NT2N cells were seen to integrate with the host brain tissue for long periods and with no detectable tumor formation (Kondziolka et al., 2000; Meltzer et al., 2001; Nelson et al., 2002), leading to a Phase II study in 18 patients with basal ganglia infarct (Kondziolka et al., 2005). Although statistical significance could not be reached due to the small population size analyzed, both studies showed an apparent trend towards functional improvement (Banerjee et al., 2010, for review).