技术资料

Pluripotent human embryonic stem cells (hESCs) have the capability of differentiating into different lineages based on specific environmental cues. We had previously shown that hESCs can be primed to differentiate into either neurons or glial cells,depending on the arrangement,geometry and size of their substrate topography. In particular,anisotropically patterned substrates like gratings were found to favour the differentiation of hESCs into neurons rather than glial cells. In this study,our aim is to elucidate the underlying mechanisms of topography-induced differentiation of hESCs towards neuronal lineages. We show that high actomyosin contractility induced by a nano-grating topography is crucial for neuronal maturation. Treatment of cells with the myosin II inhibitor (blebbistatin) and myosin light chain kinase inhibitor (ML-7) greatly reduces the expression level of microtubule-associated protein 2 (MAP2). On the other hand,our qPCR array results showed that PAX5,BRN3A and NEUROD1 were highly expressed in hESCs grown on nano-grating substrates as compared to unpatterned substrates,suggesting the possible involvement of these genes in topography-mediated neuronal differentiation of hESCs. Interestingly,YAP was localized to the cytoplasm of differentiating hESCs. Taken together,our study has provided new insights in understanding the mechanotransduction of topographical cues during neuronal differentiation of hESCs. View Publication

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected,and the functional consequences,have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51),but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD),a model of global brain ischemia,as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity,the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore,GDNF protected hippocampal neurons when present before,during or after OGD,and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF. View Publication

Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides,and are relevant to human diseases such as obesity,narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons,including those producing pro-opiolemelanocortin,agouti-related peptide,hypocretin/orexin,melanin-concentrating hormone,oxytocin,arginine vasopressin,corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types,or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo,and are able to integrate into the mouse brain. These neurons could form the basis of cellular models,chemical screens or cellular therapies to study and treat common human diseases. View Publication

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles,we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform,mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred,thus increasing screening throughput by textgreater30-fold. Compared to conventional methods,micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. View Publication

There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods,enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment,such as schizophrenia,epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders,canvassing proven and putative advantages,current constraints,and future prospects of next-generation culture systems for biomedical research and translation. View Publication

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature,differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF,NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. View Publication

Nature Communications 6,(2015). doi:10.1038/ncomms6999 View Publication

To address existing limitations in live neuron imaging,we have developed NeuO,a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications. View Publication

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

Human sensory neurons are inaccessible for functional examination,and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro,we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals,their most salient characteristic in vivo. Strikingly,mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors,which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity. View Publication

OBJECTIVE Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here,we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS iPSC-derived motor neurons exhibited gene and protein expression,ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient,but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities,including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations,including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies. View Publication

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aβ) peptide. Aβ interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aβ,we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aβ + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization,compared with Aβ or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aβ or NMDA,suggesting that mitochondria control Ca(2+) entry through NMDARs. However,incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aβ. Aβ-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil,an antagonist of GluN2B-containing NMDARs. Exposure to Aβ + NMDA further evoked a higher mitCa(2+) retention,which was ameliorated in GluN2B(-/-) cortical neurons,largely implicating the involvement of this NMDAR subunit. Moreover,pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aβ + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether,data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aβ-mediated GluN2B-composed NMDARs activation. View Publication