技术资料

Huntington's disease (HD) is a fatal neurodegenerative disease,caused by expansion of polyglutamine repeats in the Huntingtin gene,with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however,the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines,which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity,as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed,blocking glutamate signaling,not just through the NMDA but also mGlu and AMPA/Kainate receptors,completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together,these results provide important insight into HD mechanisms at early developmental time points,which may suggest novel approaches to HD therapeutics. View Publication

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD,little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD,we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially,no significant phenotypic differences were observed between iPSCs derived from the different family members. However,upon directed neural differentiation,we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity,including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3,a known regulator of WNT signaling,was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together,these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention. View Publication

The role of intermediate methylation states in DNA is unclear. Here,to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity,we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers,exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation,are predominantly allele-independent and are conserved across individuals and between mouse and human,suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons,highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. 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

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

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs),and their neuronal progeny,will play an important role in disease modeling,drug screening tests,central nervous system development studies,and may even become valuable for regenerative medicine treatments. Nonetheless,it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs,and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here,we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days,and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS),leaving the NP population nearly free of PSCs. View Publication

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells,including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses,such as Japanese encephalitis virus (JEV),provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition,glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast,only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition,functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover,we discover that vimentin intermediate filament,reported as a putative neurovirulent JEV receptor,is highly expressed in NPCs and glial cells,but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally,we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection. View Publication

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor,with dismal survival outcomes. Recently,cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here,we have investigated the expression of TNC in tissue microarrays including 17 GBMs,18 WHO grade III astrocytomas,15 WHO grade II astrocytomas,4 WHO grade I astrocytomas,and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas,whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs,where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population. 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

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton,cornea,peripheral nervous system,and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),further modifications are required to improve the robustness,efficacy,and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions,the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons,glia,melanocytes,and corneal endothelial cells. In addition,cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine. View Publication

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here,neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1,Sox2,Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains,respectively,of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes,epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together,these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays. View Publication

Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2??-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined,these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here,we show that low O2 tension and hypoxiainducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies,we implicate O2 tension as a modifier of a key cell fate decision,namely whether neural progenitors differentiate toward neurons or glia. Furthermore,our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC,a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage.We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types. View Publication