技术资料

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

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Spinal and bulbar muscular atrophy (SBMA,Kennedy's disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle,the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells,but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable,with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls,with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9,Isl1,ChAT,and SMI-32,and those with the largest repeat expansions were found to have increased acetylated ??-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated ??-tubulin and HDAC6. Perinuclear lysosomal enrichment,an HDAC6 dependent process,was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease,and the observations of reduced androgen receptor levels,repeat instability,and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy. ?? 2014. View Publication

MED13L is a component subunit of the Mediator complex,an important regulator of transcription that is highly conserved across eukaryotes. Here we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome,moderate intellectual disability (ID),craniofacial anomalies,and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish,med13b,showed early defective migration of cranial neural crest cells (NCCs) that contributed into cartilage structure deformities in the later stage,recapitulating craniofacial anomalies seen in human patients. Notably,we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos,which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system,we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors,and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signalling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency,and emphasizes a clinically recognizable syndromic phenotype in these patients. This article is protected by copyright. All rights reserved. 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

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

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

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases,for example,in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency,which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions,deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template,such as an introduced single-stranded oligo DNA nucleotide (ssODN),allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ,editing by HDR remains inefficient and can be corrupted by additional indels,preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore,targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations,and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB,we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation,whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach,we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons,which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9,facilitating study of human disease. View Publication

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation,but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358,normally added late in NPC biogenesis. SUN1,a torsinA-related molecule implicated in interphase NPC biogenesis,also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice,NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology. View Publication

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders,including Parkinson's disease and Alzheimer's disease,elevated levels of RTP801 have been observed,which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD),an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently,the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here,we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells,mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate,in addition to promoting RTP801 gene expression. Interestingly,silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However,RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice,two HD models that display motor deficits but not neuronal death. Importantly,RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together,our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease. View Publication