Stanslowsky Nancy, Haase Alexandra, Martin Ulrich, Naujock Maximilian, Leffler Andreas, Dengler Reinhard, Wegner Florian
Stem Cell Res Ther. 2014 Mar 17;5(2):35. doi: 10.1186/scrt423.
Human induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson's disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro.
The differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-β and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging.
A Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro.
This study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.
人类诱导多能干细胞（hiPSC）在再生疗法或帕金森病等神经退行性疾病的体外建模方面具有巨大潜力。目前，用于生成hiPSC的广泛使用的细胞来源是从老年个体获得的体细胞。然而，关于这些iPSC潜在临床应用的一个关键问题是在一生中积累并在重编程过程中转移到iPSC上的突变，这可能会影响从它们分化而来的细胞的功能。我们研究的目的是建立一种分化策略，以有效地从人脐带血来源的iPSC（hCBiPSC）作为年轻细胞来源生成包括多巴胺能细胞在内的神经元，并在体外证明它们的功能成熟。
在进行最终成熟之前，通过使用小分子 dorsomorphin 和 SB 431542 抑制转化生长因子-β 和骨形态发生蛋白信号传导来启动 hCBiPSC 的分化。通过免疫细胞化学和定量实时聚合酶链反应对 hCBiPSC 和分化的神经元进行表征。由于在临床应用之前对 hCBiPSC 衍生神经元的功能研究是必不可少的，我们使用全细胞膜片钳记录和钙成像对基本离子通道特性进行了详细分析。
使用新建立的分化方案从 hCBiPSC 中有效诱导出 Sox1 和 Pax6 阳性神经元祖细胞群体。神经元祖细胞可以进一步成熟为表达酪氨酸羟化酶、多巴胺转运体和 engrailed 1 的多巴胺能神经元。分化的 hCBiPSC 表现出电压门控离子电流，能够产生动作电位并显示出突触活动，表明突触形成。神经递质 GABA、谷氨酸和乙酰胆碱的应用在神经元细胞中诱导了去极化钙信号变化，为这些配体门控离子通道在体外成熟过程中的兴奋作用提供了证据。
本研究首次证明 hCBiPSC 可作为年轻细胞来源，用于生成大量包括多巴胺能细胞在内的功能性神经元，这可能有助于开发新的再生治疗策略。
