技术资料

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols,immaturity,and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional,and thereby stable,optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons,and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level. View Publication

UNLABELLED Parkinson's disease (PD) is characterized by the accumulation of α-synuclein (α-syn) within Lewy body inclusions in the nervous system. There are currently no disease-modifying therapies capable of reducing α-syn inclusions in PD. Recent data has indicated that loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase) represent an important risk factor for PD,and can lead to α-syn accumulation. Here we use a small-molecule modulator of GCase to determine whether GCase activation within lysosomes can reduce α-syn levels and ameliorate downstream toxicity. Using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from synucleinopathy patients with different PD-linked mutations,we find that a non-inhibitory small molecule modulator of GCase specifically enhanced activity within lysosomal compartments. This resulted in reduction of GCase substrates and clearance of pathological α-syn,regardless of the disease causing mutations. Importantly,the reduction of α-syn was sufficient to reverse downstream cellular pathologies induced by α-syn,including perturbations in hydrolase maturation and lysosomal dysfunction. These results indicate that enhancement of a single lysosomal hydrolase,GCase,can effectively reduce α-syn and provide therapeutic benefit in human midbrain neurons. This suggests that GCase activators may prove beneficial as treatments for PD and related synucleinopathies. SIGNIFICANCE STATEMENT The presence of Lewy body inclusions comprised of fibrillar α-syn within affected regions of PD brain has been firmly documented,however no treatments exist that are capable of clearing Lewy bodies. Here,we used a mechanistic-based approach to examine the effect of GCase activation on α-syn clearance in human midbrain DA models that naturally accumulate α-syn through genetic mutations. Small molecule-mediated activation of GCase was effective at reducing α-syn inclusions in neurons,as well as associated downstream toxicity,demonstrating a therapeutic effect. Our work provides an example of how human iPSC-derived midbrain models could be used for testing potential treatments for neurodegenerative disorders,and identifies GCase as a critical therapeutic convergence point for a wide range of synucleinopathies. View Publication

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease,therapeutic drug screening,and transplant-based regenerative medicine. However,methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here,we present a novel,small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2),we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC),sporadic FTD,and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid,efficient,and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy,terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient,novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases. View Publication

Increasing evidence suggests that cellular stress may underlie mood disorders such as bipolar disorder and major depression,particularly as lithium and its targets can protect against neuronal cell death. Here we describe N-methyl-D-aspartate (NMDA)-induced filamentous actin reorganization (NIFAR) as a useful in-vitro model for studying acute neurocellular stress and investigating the effects of mood stabilizers. Brief incubation of cultured neurons with NMDA (50 µM,5 min) induces marked reorganization of F-actin within the somatodendritic domain of a majority of neurons. During NIFAR,F-actin is rapidly depleted from dendritic spines and aberrantly aggregates within the dendrite shaft. The widely used mood stabilizer lithium chloride prevented NIFAR in a time-dependent and dose-dependent manner,consistent with its known efficacy in treating bipolar disorder. Inhibitors of the lithium target glycogen synthase kinase 3 and its upstream activator phosphoinositide-3-kinase also prevented NIFAR. The antidepressant compounds imipramine and fluoxetine also attenuated NIFAR. These findings have potential relevance to neuropsychiatric diseases characterized by excessive glutamate receptor activity and synaptotoxicity. We propose that protection of the dendritic actin cytoskeleton may be a common mechanism shared by various mood stabilizers. View Publication

A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage,and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However,the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely,during activity-dependent spine growth,LIM kinase stimulates cofilin phosphorylation,which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity. View Publication

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature,post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline,the vast majority of cells are post-mitotic,and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f,we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1,ETV4,SPRY4,and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3,SYT2,and GALR1. Since several of these genes have been implicated in MDD previously,these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. View Publication

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

Neurotoxicity testing still relies on ethically debated,expensive and time consuming in vivo experiments,which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds,such as picrotoxin (PTX) and endosulfan,and the neurotoxicant methylmercury (MeHg). However,the chemical-induced effects on different parameters for neuronal activity,such as mean spike rate (MSR) and mean burst rate (MBR),may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening. View Publication

The essential functions of Polycomb Repressive Complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs),but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs which co-precipitate with PRC1 from chromatin and found candidates that impact Polycomb Group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen,CAT7,regulates expression and PcG binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain which shares high sequence similarity to a non-syntenic zebrafish analog,cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA,enhanced by interference of a PRC1 component,and suppressed by interference of a known PRC1 target gene,demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs,and that CAT7/cat7l represent convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci. View Publication