Introduction: Human induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or\r\nin vitro modelling of neurodegenerative disorders like Parkinsonâ��s disease. Currently, widely used cell sources for the\r\ngeneration of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the\r\npotential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs\r\nduring reprogramming which may influence the functionality of cells differentiated from them. The aim of our\r\nstudy was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from\r\nhuman cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation\r\nin vitro.\r\nMethods: The differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-�Ÿ and bone\r\nmorphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation\r\nwas carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative\r\nreal time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable\r\nprior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell\r\npatch-clamp recordings and calcium imaging.\r\nResults: A Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using\r\na newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic\r\nneurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited\r\nvoltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse\r\nformation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal\r\nchanges in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during\r\nmaturation in vitro.\r\nConclusions: This study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to\r\ngenerate a large number of functional neurons including dopaminergic cells which may serve for the development of\r\nnovel regenerative treatment strategies.
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