技术资料

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

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker,TBR2,and also glial marker,S100β. In contrast,inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers,PAX6 and EAAT1,respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. View Publication

BACKGROUND Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling,drug screening,and heart repair. Here,we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS We systematically investigated cell composition,matrix,and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological,functional,and transcriptome analyses to benchmark maturation of EHM. RESULTS EHM demonstrated important structural and functional properties of postnatal myocardium,including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction,cardiomyocyte hypertrophy,cardiomyocyte death,and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition,we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined,serum-free conditions. View Publication

Cerebral organoids recapitulate human brain development at a considerable level of detail,even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here,we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions,we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected,suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this,we demonstrate the presence of forebrain organizing centers that express secreted growth factors,which may be involved in dorso-ventral patterning within organoids. Furthermore,we demonstrate the timed generation of neurons with mature morphologies,as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro. View Publication

BACKGROUND CHD8 (chromodomain helicase DNA-binding protein 8),which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors,is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8(+/-)) vs isogenic controls (CHD8(+/-)),including the SZ and bipolar disorder (BD) candidate gene TCF4,which was markedly upregulated in CHD8(+/-) neuronal cells. METHODS In the current study,RNA-seq was carried out on CHD8(+/-) and isogenic control (CHD8(+/+)) cerebral organoids,which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. RESULTS TCF4 expression was,again,significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis,neuronal differentiation,forebrain development,Wnt/β-catenin signaling,and axonal guidance,similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8(+/-) DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably,the top DEG in our respective studies was the non-coding RNA DLX6-AS1,which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD,a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. CONCLUSIONS Overall,the findings show that distinct ASD,SZ,and BD candidate genes converge on common molecular targets-an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. View Publication

Brain-derived microvascular endothelial cells (BMECs),which play a central role in blood brain barrier (BBB),can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported,they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy,drug screening,and pathological study. In the recent study,an induction method of BMECs from PSCs has been established,making it possible to more precisely study the in vitro human BBB function. Here,using induced pluripotent stem (iPS) cell-derived BMECs,we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function,and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly,TNF-α,which is known to be secreted from astrocytes and microglia in the cerebral ischemia,prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus,we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting,but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific,but not primed-specific,proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus,identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting. View Publication

To better understand transcriptional regulation during human oogenesis and preimplantation development,we defined stage-specific transcription,which highlighted the cleavage stage as being highly distinctive. Here,we present multiple lines of evidence that a eutherian-specific multicopy retrogene,DUX4,encodes a transcription factor that activates hundreds of endogenous genes (for example,ZSCAN4,KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably,mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells,measured here by the reactivation of '2C' genes and repeat elements,the loss of POU5F1 (also known as OCT4) protein and chromocenters,and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus,we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state. View Publication

Human blastocyst-derived,pluripotent cell lines are described that have normal karyotypes,express high levels of telomerase activity,and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months,these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers,including gut epithelium (endoderm); cartilage,bone,smooth muscle,and striated muscle (mesoderm); and neural epithelium,embryonic ganglia,and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology,drug discovery,and transplantation medicine. View Publication

Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However,the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells,for example,are not fully understood. Proteins called growth factors are known to have a role in this process,as are transcription factors,but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons,Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly,they found that a compound called selamectin,which is used to treat parasitic flatworm infections,also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms,but this ion channel is not found in vertebrates,which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA,Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea,drugs that increased GABAA activity stimulated the formation of neurons,whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition,Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons,and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells,the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules,which could aid the treatment of neurodevelopmental and neurodegenerative disorders. DOI: [http://dx.doi.org/10.7554/eLife.00508.002][1] [1]: /lookup/doi/10.7554/eLife.00508.002 View Publication

To study molecular events involved in B lymphocyte development and V(D)J rearrangement,we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here,we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first,they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second,they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third,they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover,the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably,EAB cells possess functional V(D)J recombinase activity. In particular,the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation. View Publication