技术资料

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

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

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies. View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders,and /`a disease of synapses/' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies,little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare,multiply affected,large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and,furthermore,dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders. View Publication

Nature Neuroscience 17,1180 (2014). doi:10.1038/nn.3787 View Publication

AbstractBack to TopbackslashnNeurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However,it remains unclear to what extent these new mechanistic insights will translate to animal models,an important step in the validation of new targets. Previously,we found that glia from mice carrying the SOD1G93A mutation,a model of ALS,were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore,we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span,decreased microglial activation,and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo. View Publication

Adaptive immunity to self-antigens causes autoimmune disorders,such as multiple sclerosis,psoriasis and type 1 diabetes; paradoxically,T- and B-cell responses to amyloid-$\$(A$\$) reduce Alzheimer's disease (AD)-associated pathology and cognitive impairment in mouse models of the disease. The manipulation of adaptive immunity has been a promising therapeutic approach for the treatment of AD,although vaccine and anti-A$\$ approaches have proven difficult in patients,thus far. CD4(+) T cells have a central role in regulating adaptive immune responses to antigens,and A$\$-specific CD4(+) T cells have been shown to reduce AD pathology in mouse models. As these cells may facilitate endogenous mechanisms that counter AD,an evaluation of their abundance before and during AD could provide important insights. A$\$-CD4see is a new assay developed to quantify A$\$-specific CD4(+) T cells in human blood,using dendritic cells derived from human pluripotent stem cells. In tests of textgreater50 human subjects A$\$-CD4see showed an age-dependent decline of A$\$-specific CD4(+) T cells,which occurs earlier in women than men. In aggregate,men showed a 50% decline in these cells by the age of 70 years,but women reached the same level before the age of 60 years. Notably,women who carried the AD risk marker apolipoproteinE-ɛ4 (ApoE4) showed the earliest decline,with a precipitous drop between 45 and 52 years,when menopause typically begins. A$\$-CD4see requires a standard blood draw and provides a minimally invasive approach for assessing changes in A$\$ that may reveal AD-related changes in physiology by a decade. Furthermore,CD4see probes can be modified to target any peptide,providing a powerful new tool to isolate antigen-specific CD4(+) T cells from human subjects. View Publication