Human iPS Cell-derived Neurons Research Articles

Comprehensive analysis of long noncoding RNA expression in dorsal root ganglion reveals cell-type specificity and dysregulation after nerve injury.

Dorsal root ganglion (DRG) neurons provide connectivity between peripheral tissues and the spinal cord. Transcriptional plasticity within DRG sensory neurons after peripheral nerve injury contributes to nerve repair but also leads to maladaptive plasticity, including the development of neuropathic pain. This study presents tissue and neuron-specific expression profiling of both known and novel long noncoding RNAs (LncRNAs) in the rodent DRG after nerve injury. We have identified a large number of novel LncRNAs expressed within the rodent DRG, a minority of which were syntenically conserved between the mouse, rat, and human, and including, both intergenic and antisense LncRNAs. We have also identified neuron type-specific LncRNAs in the mouse DRG and LncRNAs that are expressed in human IPS cell-derived sensory neurons. We show significant plasticity in LncRNA expression after nerve injury, which in mice is strain and gender dependent. This resource is publicly available and will aid future studies of DRG neuron identity and the transcriptional landscape in both the naive and injured DRG. We present our work regarding novel antisense and intergenic LncRNAs as an online searchable database, accessible from PainNetworks (http://www.painnetworks.org/). We have also integrated all annotated gene expression data in PainNetworks, so they can be examined in the context of their protein interactions.

Artificial intelligence and PCA analysis of the electrical activity in cultured human iPS cell-derived neurons for the prediction of convulsive toxicity and action mechanisms of drugs

Artificial intelligence and PCA analysis of the electrical activity in cultured human iPS cell-derived neurons for the prediction of convulsive toxicity and action mechanisms of drugs

Refinement of a neuronal differentiation protocol predominantly yields human iPS cell-derived dopaminergic neurons for the investigation of neurodegenerative pathomechanisms in vitro

Refinement of a neuronal differentiation protocol predominantly yields human iPS cell-derived dopaminergic neurons for the investigation of neurodegenerative pathomechanisms in vitro

Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

ヒトiPS細胞由来神経細胞を用いた微小電極アレイシステムによる痙攣・てんかん評価法確立の試み－CSAHi 神経チーム

ヒトiPS細胞由来神経細胞を用いた微小電極アレイシステムによる痙攣・てんかん評価法確立の試み－CSAHi 神経チーム

Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome.

Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and behavioral deficits.

ヒトiPS細胞由来神経細胞の特性（神経突起）に関する基礎的検討（ヒトiPS細胞応用安全性評価コンソーシアム）

ヒトiPS細胞由来神経細胞の特性（神経突起）に関する基礎的検討（ヒトiPS細胞応用安全性評価コンソーシアム）

Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd.

Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.

Modeling Neurological Disease with Human iPS Cell-Derived Neurons Containing a KCNT1 Mutation

The sodium-activated potassium channel Slack, encoded by the gene KCNT1, is expressed in neurons throughout many brain regions, including the frontal cortex, and mediates a sodium-sensitive potassium current (IKNa). This outward current regulates neuronal excitability and determines how neurons respond to repeated high frequency stimulations, both of which are aspects of memory and learning. Not surprisingly, mutations in KCNT1 and alterations to the IKNa current have patho-physiological consequences. Recent studies have described the emerging role of Slack channels in cognitive deficits, and several reports have found KCNT1 mutations in patients with severe early onset “childhood” epilepsies.The development of better therapies for neurological disorders has been hindered by limited access to clinically-meaningful cell models for research and drug development. The advent of induced pluripotent stem (iPS) cell technology provides a platform to facilitate increased understanding of disease mechanisms in a physiologically-relevant environment. We have leveraged this technology to generate human neurons that express the KCNT1 P924L mutation in the Slack channel. To introduce this alteration, we genetically engineered a “control” iPS cell line from an apparently healthy donor with no family history of neurological disorders and generated highly purified (>95% TUJ1-positive), terminally differentiated cortical neurons from the two separate but isogenic iPS cell lines.Here, we present data on the characterization of these human neurons, including expression of a standard set of neuronal markers at both the genetic and protein level. We also highlight functional testing of the cells, with a specific focus on electrophysiological readouts such as multi-electrode array (MEA) analysis. The ability to model neurological disorders through editing the genome of an iPS cell and subsequently produce previously inaccessible human neurons has revolutionized the way we approach studying and treating diseases of the central nervous system.

Kv7.2/7.3 Channels are Enhanced During Striatal Development and Promote Neuronal Functional Maturation of iPS Cell-Derived Neurons

M-currents, encoded by Kv7.2/7.3 genes, are low-threshold, voltage-gated potassium channels which contribute to resting membrane potential (Vm) and regulate neuronal excitability. Here, we tested the hypothesis that the development of mouse striatal neurons is associated with enhanced expression and activity of M-currents and that determined whether forced expression of Kv7.2/7.3 accelerates the functional maturation of neurons differentiating from human induced pluripotent stem (iPS) cells. Neurons from the developing mouse striatum were isolated at E13, E15 and E17 and cultured in vitro for up to 14 days; functional profiles of the neurons were explored using whole-cell patch-clamp. In some experiments, subventricular and mantle zones were separated by laser microdissection and channel subunit ontogenies determined by gene chip array. Shortly after isolation (1 day in vitro), there was a clear in vivo development-dependent hyperpolarisation of Vm (E13=-24.8±2.3 mV (n=26), E15=-40.0±2.2 mV (n=12), E17=-47.2±2.4 mV (n=9)). Although Vm continued to hyperpolarise in vitro, by 7 days these differences has disappeared (e.g. E13=-52.1±2.8 mV (n=8), E17 =-54.7±1.9 (n=5)). Associated with these observations, were parallel and progressive increases in M-current activity during in vivo and in vitro development, as evidenced by more pronounced responses to retigabine and XE991. Further, both Kv7.2 and 7.3 genes were progressively enriched in the mantle zone during in vivo development. Importantly, human iPS cell-derived neurons nucleofected with a concatenated Kv7.2/7.3 cDNA plasmid demonstrated a significantly hyperpolarized Vm (from −39.9±2.2 mV (n=12) to −51.3±1.1 mV (n=11)) and a dramatic increase in the proportion of cells generating action potentials spontaneously. Taken together, these data indicate that development of striatal neurons is associated with increased M-current expression and that this might be causative as suggested by the effect of forced Kv7.2/7.3 expression in the iPS cell-derived neurons.

Prevention of β-amyloid induced toxicity in human iPS cell-derived neurons by inhibition of Cyclin-dependent kinases and associated cell cycle events

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline due to the selective neuronal loss in the cortex and hippocampus of the brains. Generation of human induced pluoripotent stem (hiPS) cells holds great promise for disease modeling and drug discovery in AD. In this study, we used neurons with forebrain marker expression from two unrelated hiPS cell lines. As both populations of neurons were vulnerable to β-amyloid 1–42 (Aβ1–42) aggregates, a hallmark of AD pathology, we used them to investigate cellular mediators of Aβ1–42 toxicity. We observed in neurons differentiated from both hiPS cell lines that Aβ induced toxicity correlated with cell cycle re-entry and was inhibited by pharmacological inhibitors or shRNAs against Cyclin-dependent kinase 2 (Cdk2). As one of the hiPS cell lines has been developed commercially to supply large quantities of differentiated neurons (iCell® Neurons), we screened a chemical library containing several hundred compounds and discovered several small molecules as effective blockers against Aβ1–42 toxicity, including a Cdk2 inhibitor. To our knowledge, this is the first demonstration of an Aβ toxicity screen using hiPS cell-derived neurons. This study provided an excellent example of how hiPS cells can be used for disease modeling and high-throughput compound screening for neurodegenerative diseases.

Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease.

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.Methodology/Principal FindingsWe differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.Conclusions/SignificanceThese results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.