Directed Differentiation of Dopaminergic Neuronal Subtypes from Human Embryonic Stem Cells

Department of Anatomy, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, Wisconsin 53705, USA.
Stem Cells (Impact Factor: 6.52). 05/2005; 23(6):781-90. DOI: 10.1634/stemcells.2004-0365
Source: PubMed


How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with gamma-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro-generated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.

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    • "Conversely, when a GSK3β inhibitor, which activates canonical Wnt signaling, is applied during neural differentiation from PSCs, neural progenitors of different rostrocaudal identity are induced in a dose-dependent manner – higher levels of inhibitor induce cells with more caudal identity, such as the midbrain (Kirkeby et al., 2012). FGF8, which is secreted from the isthmic organizer located in the midbrainhindbrain boundary in vivo (Allodi and Hedlund, 2014; Stern, 2001), is a potent inducer of midbrain precursors from PSCs (Allodi and Hedlund, 2014; Elkabetz et al., 2008; Kriks et al., 2011; Perrier et al., 2004; Soldner et al., 2009; Yan et al., 2005). These precursors are in turn terminally differentiated into dopaminergic neurons under the effect of other signals (Fig. 1D). "
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    ABSTRACT: The human brain is arguably the most complex structure among living organisms. However, the specific mechanisms leading to this complexity remain incompletely understood, primarily because of the poor experimental accessibility of the human embryonic brain. Over recent years, technologies based on pluripotent stem cells (PSCs) have been developed to generate neural cells of various types. While the translational potential of PSC technologies for disease modeling and/or cell replacement therapies is usually put forward as a rationale for their utility, they are also opening novel windows for direct observation and experimentation of the basic mechanisms of human brain development. PSC-based studies have revealed that a number of cardinal features of neural ontogenesis are remarkably conserved in human models, which can be studied in a reductionist fashion. They have also revealed species-specific features, which constitute attractive lines of investigation to elucidate the mechanisms underlying the development of the human brain, and its link with evolution.
    Development 09/2015; 142(18):3138-3150. DOI:10.1242/dev.120568 · 6.46 Impact Factor
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    • "Brain developmental and functional processes are highly dependent on interactions between different neural cell types and between different regions of the brain (for example, De Marco Garcia et al., 2011; Nishi, 2003). A great deal of attention has, for example, been focused on differentiation of human mDA neurons from hPSCs (Kriks et al., 2011; Perrier et al., 2004; Vazin et al., 2009; Yan et al., 2005; Zeng et al., 2004) because of their potential for use in transplantation therapy for Parkinson's disease. The development and formation of mDA neurons does not, however, occur in isolation , and DA systems and their targets structures are highly interdependent (Halliday et al., 2000; Hemmendinger et al., 1981; Hoffman et al., 1983; Parish et al., 2001; Prasad and Pasterkamp, 2009; Shalaby et al., 1984). "
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    ABSTRACT: We describe a technique for independently differentiating neocortical and mesencephalic dopaminergic (mDA) neurons from a single human pluripotent stem cell (hPSC) line, and subsequently allowing the two cell types to interact and form connections. Dopaminergic and neocortical progenitors were differentiated in separate vessels, then separately seeded into the inner and outer compartments of specialized cell culture vessels designed for in vitro studies of wound healing. Cells were further differentiated using dopamine-specific and neocortex-specific trophic factors, respectively. The barrier was then removed, and differentiation was continued for three weeks in the presence of BDNF. After three weeks of differentiation, neocortical and mDA cell bodies largely remained in the areas into which they had been seeded, and the gap between the mDA and neocortical neuron populations could still be discerned. Abundant tyrosine hydroxylase (TH)-positive projections had extended from the area of the inner chamber to outer chamber neocortical area. We have developed a hPSC-based system for producing connections between neurons from two brain regions, neocortex and midbrain. Future experiments could employ modifications of this method to examine connections between any two brain regions or neuronal subtypes that can be produced from hPSCs in vitro.
    04/2015; 33(3). DOI:10.3233/RNN-140488
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    • "However, we were not able to yield as high percentages of cells (50% to 100%) with inducible APs as studies using hESCs [74] or methods to directly convert fibroblast to DA neurons (71% to 83%; [66,67]). The AP amplitudes of 51 mV on the other hand were similar to previous observations for hiPSC-derived neurons (50 mV [12]) and within the range of studies with hESC-derived nerve cells (35 to 50 mV [74]; 32 mV [44]; 74 to 84 mV [60]) and direct reprogrammed fibroblasts (approximately 45 mV [67]; 78 mV [66]). AP durations (3 ms) were also in the reported range of 1 to 7 ms [44,60,66,67,74]. "
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    ABSTRACT: 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 are mutations that accumulate over lifetime and are transfered 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 form 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-beta and bone morphogenetic protein signalling 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.
    Stem Cell Research & Therapy 03/2014; 5(2):35. DOI:10.1186/scrt423 · 3.37 Impact Factor
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