Meningeal cells induce dopaminergic neurons from embryonic stem cells

Neural induction of midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells can be achieved by culturing them on a bone marrow-derived stromal cell line, PA6, which possesses stromal cell-derived inducing activity (SDIA). The mechanism of SDIA is unknown, but clinical application of ES cell transplantation requires the use of defined factors for DA neuron induction. Here, we demonstrate that meningeal cells harvested from the developing dura can induce DA neuron differentiation from mouse and human ES cells, as assessed by midbrain DA marker expression and secretion of DA in response to potassium stimuli. Intriguingly, the inductive strength of meningeal cells depends on their developmental stage, with those harvested from embryonic day 18 embryos showing the highest activity. Among six soluble factors known to be involved in DA neuron differentiation, only Wnt-5a and transforming growth factor-β3 were expressed by both meningeal and PA6 cells, and the expression of Wnt-5a correlated with the DA neuron induction activity of these cells. Furthermore, the induction of DA neuron differentiation by PA6 cell-conditioned medium was reversed by addition of a Wnt-5a neutralizing antibody, whereas recombinant Wnt-5a promoted DA neuron induction when cells were cultured on Matrigel. These results indicate that meningeal cells can be used as feeders to induce DA neurons from ES cells, and that Wnt-5a plays an important role in DA neuron induction by SDIA.     

Generation of dopaminergic neurons from embryonic stem cells

Neuronal transplantation is considered to be a promising therapeutic approach to neurodegenerative diseases. In addition to fetal tissues and neural stem cells, embryonic stem cells are good candidates for the creation of neurons. We have recently identified a stromal cell-derived inducing activity that promotes neural differentiation of mouse embryoric stem cells. This activity accumulated on the surface of PA6 stromal cells and induced efficient neuronal differentiation of co-cultured embryonic stem cells under serum-free conditions without the use of either retinoic acid or embryoid bodies. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained. Induction of neurons with stromal cell-derived inducing activity may be a useful new method for basic neuroscience research and therapeutic applications, including cell transplantation therapy for Parkinson's disease.     

Efficient production of neural stem cells and neurons from embryonic stem cells

We have developed a simple method to efficiently produce a large number of neural stem cells and neurons from mouse embryonic stem (ES) cells. When cultured in astrocyte-conditioned medium (ACM) with mitogens (FGF-2 and EGF) under free-floating conditions, colonies of undifferentiated ES cells give rise to neural stem spheres (NSSs), composed of plentiful neural stem cells. Subsequent culture of the NSSs on an adhesive substrate with mitogens results in the migration of neural stem cells onto the substrate. These cells can be expanded, preserved by freezing, and differentiated into functional neurons. Neural stem cells and neurons provided by this NSS method may be valuable as potential donor cells for neuronal transplantation and also as convenient alternatives to tissue-derived neural cells.     

Derivation of homogeneous GABAergic neurons from mouse embryonic stem cells

Embryonic stem cells (ESCs) promise an unlimited source of defined cells for cell transplantation therapy, while protocols for derivation of homogeneous populations of desirable cell types are yet to be developed and/or refined. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, and disturbed GABAergic signaling is associated with a host of neurological conditions. We developed a simple ES cell differentiation protocol which led to the production of uniform GABAergic neurons in  2 weeks. The differentiation protocol involved treatment of embryoid bodies (EBs) with high concentrations (10⁻⁵–10⁻⁴ M) of all-trans-retinoic acid (RA) for 3 days. After plating these EBs on attached dishes in neural supportive medium, 93–96% of the cells became GABA-positive neurons in 7–11 days. These cells also expressed immature neuronal markers with voltage-gated delayed rectifier potassium currents, suggesting that they were immature GABAergic neurons. The technology may have implications for modeling and treatment of GABAergic signaling-related diseases and injuries.     

Rapid and efficient differentiation of dopaminergic neurons from mouse embryonic stem cells

We have developed a fast and effective method for the differentiation of dopaminergic neurons from mouse embryonic stem cells. Neuronal precursors are obtained by formation of embryonic bodies or neural stem spheres via free-floating culture in the presence of the mitogens basic fibroblast growth factor and epidermal growth factor together with L-ascorbic acid. Subsequent culturing of the precursor cells in medium containing epidermal growth factor, FGF8b, SHH and ascorbic acid induces cell proliferation, following withdrawal of the growth factors leads differentiation into predominantly dopaminergic neurons. Mature neurons are obtained within 10 days of replacing the proliferation to differentiation medium. Embryonic stem-derived dopaminergic neurons are purified by cell sorting and may serve as a convenient source for the study of molecular, genetic and cellular properties of dopaminergic neurons.     

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.     

Adipocytes derived from PA6 cells reliably promote the differentiation of dopaminergic neurons from human embryonic stem cells

The PA6 stromal cell line comprises a heterogeneous population of cells that can induce both mouse and human embryonic stem cells to differentiate into dopaminergic neurons. This ability of PA6 cells has been termed stromal cell‐derived inducing activity (SDIA). The level of SDIA has been found to vary considerably between and within batches of PA6 cells. Not only are the molecular mechanisms that underlie SDIA unknown but also the cell type(s) within the heterogeneous PA6 cultures that underlie SDIA remain poorly defined. In this study, we reveal that adipocytes, which are present within the heterogeneous PA6 cell population, robustly release the factors mediating SDIA. Furthermore, we report that the coculture of human embryonic stem cells with PA6‐derived adipocytes reliably induces their differentiation into midbrain dopaminergic neurons. © 2014 Wiley Periodicals, Inc.     

胚胎干细胞定向分化运动神经元的基因调控

本文着重就胚胎干细胞 (ESCs)定向诱导分化为脊髓运动神经元过程中两个重要阶段及相关基因的表达调控进行综述。运动神经元分化的基因调控研究将有利于阐明其发育和分化的模式 ,为其应用于脊髓损伤修复和运动神经元退行性疾病的替代治疗提供理论依据     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

Clonal neural stem cells from human embryonic stem cell colonies

Clonal cell culture is crucial for experimental protocols that require growth or selection of pure populations of cells. High-density derivation of neural progenitors from human embryonic stem cells (hESCs) can lead to incomplete differentiation, and transplantation of resulting heterogeneous cell mixtures can cause proliferation of tumorigenic clusters in vivo. We have identified the neural precursor that resides among normal hESC colonies as a TRA-1-60(-)/SSEA4(-)/SOX1(+) cell and developed a method that allows for the clonal expansion of these FACS-selected progenitors to neural stem cells (NSCs) in serum-free conditions. Single TRA-1-60(-)/SSEA4(-)/SOX1(+) cells grown in serum-free media give rise to multipotent NSCs with an efficiency of 0.7%. The fate of the TRA-1-60(-)/SSEA4(-)/SOX1(+) neural precursor becomes specified in maintenance conditions by inhibition of BMP signaling. This clonal culture method can be scaled up to produce NSCs for differentiation and use in cell therapies.     

Silver nanoparticles exhibit coating and dose-dependent neurotoxicity in glutamatergic neurons derived from human embryonic stem cells

Silver nanoparticles (AgNPs) are used extensively as anti-microbial agents in various products, but little is known about their potential neurotoxic effects. In this study, we used glutamatergic neurons derived from human embryonic stem cells as a cellular model to study 20 nm citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated AgNPs (AgSPs) induced neurotoxicity. AgSCs significantly damaged neurite outgrowths; increased the production of reactive oxygen species and Ca²⁺ influxes; reduced the expression of MAP2, PSD95, vGlut1 and NMDA receptor proteins at concentrations as low as 0.1 μg/ml. In contrast, AgSPs exhibited neurotoxicity only at higher concentration. Furthermore, our results showed that AgSCs induced glutamate excitotoxicity by the activation of calmodulin and the induction of nitric oxide synthase; increased the phosphorylation of glycogen synthase kinase-3 α/β at Tyr²¹⁶ and Tau at Ser³⁹⁶ and reduced the expression of Tau46, which are typically observed in Alzheimer’s disease. This study indicated that stem cells can provide an excellent platform for studying nanoparticle induced neurotoxicity.     

BMP4 induction of sensory neurons from human embryonic stem cells and reinnervation of sensory epithelium

In mammals, hair cells and auditory neurons lack the capacity to regenerate, and damage to either cell type can result in hearing loss. Replacement cells for regeneration could potentially be made by directed differentiation of human embryonic stem (hES) cells. To generate sensory neurons from hES cells, neural progenitors were first made by suspension culture of hES cells in a defined medium. The cells were positive for nestin, a neural progenitor marker, and Pax2, a marker for cranial placodes, and were negative for alpha-fetoprotein, an endoderm marker. The precursor cells could be expanded in vitro in fibroblast growth factor (FGF)-2. Neurons and glial cells were found after differentiation of the neural progenitors by removal of FGF-2, but evaluation of neuronal markers indicated insignificant production of sensory neurons. Addition of bone morphogenetic protein 4 (BMP4) to neural progenitors upon removal of FGF-2, however, induced significant numbers of neurons that were positive for markers associated with cranial placodes and neural crest, the sources of sensory neurons in the embryo. Neuronal processes from hES cell-derived neurons made contacts with hair cells in denervated ex vivo sensory epithelia and expressed synaptic markers, suggesting the formation of synapses. In a gerbil model with a denervated cochlea, the ES cell-derived neurons engrafted in the auditory nerve trunk and sent out neurites that grew toward the auditory sensory epithelium. These data indicate that hES cells can be induced to form sensory neurons that have the potential to treat neural degeneration associated with sensorineural hearing loss.     

Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials,akin to fertilized embryo-derived embryonic stem cells.The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells.Before differentiation,karyotype analysis was performed,with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells.Sex chromosomes were identified as XX.Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene,Oct4,at both the mRNA and protein levels,indicating pluripotent differentiation potential of the two embryonic stem cell subtypes.Embryonic stem cells were induced with retinoic acid to form embryoid bodies,and then dispersed into single cells.Single cells were differentiated in N2 differentiation medium for 9 days.Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin,βIII-tubulin and myelin basic protein.Quantitative real-time PCR found expression of neurogenesis related genes(Sox-1,Nestin,GABA,Pax6,Zic5 and Pitx1) in both types of embryonic stem cells,and Oct4 expression was significantly decreased.Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells.Thus,our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.     

Potential of embryonic stem cell-derived neurons for synapse formation with auditory hair cells

Recent studies have indicated that embryonic stem cells (ESCs) can be a source for the replacement of spiral ganglion neurons (SGNs), auditory primary neurons, and neurite projections from ESC-derived neurons to auditory sensory epithelia. However, the potential of ESC-derived neurons for synapse formation with auditory hair cells (HCs) has not been elucidated. The present study therefore aimed to examine the ability of ESC-derived neurons to form synaptic connections with HCs in vitro. Mouse ESC-derived neural progenitors expressing enhanced green fluorescence protein (EGFP) were cocultured with explants of cochlea sensory epithelia obtained from postnatal day 3 mice. After a 7-day culture, neurites of ESC-derived neurons predominantly elongated toward inner hair cells (IHCs), which play a crucial role in sound transmission to SGNs. Immunohistochemical analyses revealed the expression of synapsin 1 and synaptophysin in the nerve endings of ESC-derived neurons adjacent to IHCs, indicating the formation of synaptic connections. Transmission electron microscopy demonstrated synaptic contacts between nerve endings of ESC-derived neurons and IHCs. The present findings show that ESC-derived neurons can make synaptic connections with IHCs.