技术资料

Human embryonic stem cells (hESCs) are used as platforms for disease study,drug screening and cell-based therapy. To facilitate these applications,it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However,the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However,certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site,probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein,LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. View Publication

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways,including photoreceptor development,phototransduction,the retinoid cycle,cilia,and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants,two missense variants,and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1,which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383CtextgreaterT [p.Pro128Leu] and c.404TtextgreaterC [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore,CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP,including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore,our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy. View Publication

The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However,generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)-derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons,which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology,we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure,function,and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. View Publication

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival,proliferation,differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2,previously shown to promote Cx32 expression in mature hepatocytes,up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation,resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast,negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast,the p38 MAPK activator,anisomycin,blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation. View Publication

Historically,the routine use of laboratory automation solutions has been prohibitively expensive for many laboratories. As legacy hardware has begun to emerge on the secondary market,automation is becoming an increasingly affordable option to augment workflow in virtually any laboratory. To assess the utility of legacy liquid handling in stem cell differentiation,a used liquid handling robot was purchased at auction to automate a stem cell differentiation protocol that gives rise to CD14 + CD45+ mononuclear cells. To maintain sterility,the automated liquid handling robot was housed in a custom constructed HEPA filtered enclosure. A custom cell scraper and a disposable filter box were designed and 3D printed to permit the robot intricate cell culture actions required by the protocol. All files for the 3D printed labware are uploaded and are freely available. •A used liquid handling robot was used to automate an hESC to monocyte differentiation protocol.•The robot-performed protocol induced monocytes as effectively as human technicians.•Custom 3D printed labware was made to permit certain cell culture actions and are uploaded for free access. View Publication

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions,including motility and epithelial permeability. Perturbations in ENS development or function are common,yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme,differentiated into neurons and glial cells and showed neuronal activity,as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus,had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally,we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is,to the best of our knowledge,the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract. View Publication

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study,whole-exome sequencing (WES) was carried out on patients from three different families that presented with life-threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln),which encodes the trans-2,3-enoyl-CoA reductase-like protein. Both patients had cardiac arrest,stress-induced atrial and ventricular tachycardia,and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1GtextgreaterA) in TECRL Analysis of intracellular calcium ([Ca(2+)]i) dynamics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from this individual (TECRLHom-hiPSCs),his heterozygous but clinically asymptomatic father (TECRLHet-hiPSCs),and a healthy individual (CTRL-hiPSCs) from the same Sudanese family,revealed smaller [Ca(2+)]i transient amplitudes as well as elevated diastolic [Ca(2+)]i in TECRLHom-hiPSC-CMs compared with CTRL-hiPSC-CMs. The [Ca(2+)]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores,evidenced by the decreased magnitude of caffeine-induced [Ca(2+)]i transients. In addition,the decay phase of the [Ca(2+)]i transient was slower in TECRLHom-hiPSC-CMs due to decreased SERCA and NCX activities. Furthermore,TECRLHom-hiPSC-CMs showed prolonged action potentials (APs) compared with CTRL-hiPSC-CMs. TECRL knockdown in control human embryonic stem cell-derived CMs (hESC-CMs) also resulted in significantly longer APs. Moreover,stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom-hiPSC-CMs and treatment with flecainide,a class Ic antiarrhythmic drug,significantly reduced the triggered activity in these cells. In summary,we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT Patient-specific hiPSC-CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias. View Publication

Targeted delivery of siRNA controlled by near-infrared light using hollow gold nanoshells has been demonstrated in cancer and stem cells models. Here,a universal surface module and several functionalization rules for the maximized delivery of short nucleic acids (here,siRNA) applicable for diverse gold nanocarriers are described. Streptavidin is devised as a handle to assemble biotinylated cell penetrating peptides (e.g.,transactivating transcriptional activator (TAT)),as well as an insulator between the positive charge of TAT and the dense negative charge of RNA. However,direct linking of streptavidin to functional siRNA inhibits its silencing activity. The approach then involves the orthogonal assembly of two types of RNA strands: one with biotin modification for cell targeting and penetration (scaffold RNA); the other without biotin as functional RNA (i.e.,siRNA). Initially,flexible single-stranded RNA is used for dense surface-packing,followed by hybridization with the complementary RNA strand to maximize the assembly of the targeting peptide for cellular uptake and siRNA delivery. This orthogonal approach for the delivery of short oligonucleotides,together with novel surface functionalization rules discovered here,should enable the use of these materials for nanomedicinal research and applications. View Publication

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular,this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells),hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here,we described a new platform which enables,rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular,we devised a protocol that,combining the expression of the CRISPR components with neurogenic factors,generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy,fast and that does not require the generation of stable isogenic clones,practice that is time consuming and for some genes not feasible. View Publication

Nucleosome position,density,and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis,we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly,extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters,suggesting developmental stage-specific organization of histone methylation states. View Publication

BACKGROUND Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ),schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ,e.g.,PRODH and DGCR8. However,the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development,we carried out RNA-seq analysis to investigate gene expression in early differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. METHODS Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subjected to RNA sequencing. Using a systems level analysis,differentially expressed genes/gene-modules and pathway of interests were identified. Lastly,we related our findings from in vitro neuronal cultures to brain development by mapping differentially expressed genes to BrainSpan transcriptomes. RESULTS We observed ˜2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value textless 0.05,36 of which reached a false discovery rate textless 0.05). Outside of the deleted region,745 genes showed significant differences in expression between SZ and control neurons (p textless 0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways,such as apoptosis,cell cycle and survival,and MAPK signaling in the SZ and SAD samples. By leveraging transcriptome profiles of normal human brain tissues across human development into adulthood,we showed that the differentially expressed genes converge on a sub-network mediated by CDC45 and the cell cycle,which would be disrupted by the 22q11.2 deletion during embryonic brain development,and another sub-network modulated by PRODH,which could contribute to disruption of brain function during adolescence. CONCLUSIONS This study has provided evidence for disruption of potential molecular events in SZ patient with 22q11.2 deletion and related our findings from in vitro neuronal cultures to functional perturbations that can occur during brain development in SZ. View Publication

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently,human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However,the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages,including enabling production of larger scale lots,decreasing lead time to generate purified iPSC-BMEC cultures,and facilitating use of iPSC-BMECs in large-scale screening. In this study,we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability,express standard endothelial and BBB markers,and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ωtextperiodcenteredcm(2),equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs,as well as stabilized TEER above 800 Ωtextperiodcenteredcm(2) out to 7 days post-thaw. Overall,cryopreservation will ease handling and storage of high-quality iPSC-BMECs,reducing a key barrier to greater implementation of these cells in modeling the human BBB. View Publication