技术资料

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

Biophysical properties of the scaffolds such as the elastic modulus,have been recently shown to impact stem cell lineage commitment. On the other hand,the contribution of the Poisson's ratio,another important biophysical property,to the stem cell fate decision,has not been studied. Scaffolds with tunable Poisson's ratio (ν) (termed as auxetic scaffolds when Poisson's ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis,in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion,the auxetic scaffolds supported smaller aggregate formation and higher expression of β-tubulin III upon neural differentiation. The influences of pore structure,Poisson's ratio,and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poisson's ratio may confound the effects of elastic modulus,and auxetic scaffolds with proper pore structure and Poisson's ratio enhance neural differentiation. This study demonstrates that tuning the Poisson's ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader,more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation. STATEMENT OF SIGNIFICANCE Biophysical signaling from the substrates and scaffolds plays a critical role in neural lineage commitment of pluripotent stem cells. While the contribution of elastic modulus has been well studied,the influence of Poisson's ratio along with microstructure of the scaffolds remains unknown largely due to the lack of technology to produce materials with tailorable Poisson's ratio. This study fabricated auxetic polyurethane scaffolds with different elastic modulus,Poisson's ratio and microstructure and evaluated neural differentiation of pluripotent stem cells. The findings add a novel angle to understand the impact of biophysical microenvironment on stem cell fate decisions. View Publication

In fragile X syndrome (FXS),CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However,FXS siblings have been identified with more than 200 CGGs,termed unmethylated full mutation (UFM) carriers,without gene silencing and disease symptoms. Here,we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However,a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore,we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs,and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population. View Publication

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice,and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor,Osteocrin (OSTN),that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition,we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that,in response to sensory input,OSTN regulates features of neuronal structure and function that are unique to primates. View Publication

OBJECTIVE The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors,including PAX8 and NKX2-1,which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells,and TAZ interacts directly with both PAX8 and NKX2-1,leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS The use of a small molecule,ethacridine,recently identified as a TAZ activator,in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First,endodermal cells were derived from hES cells using Activin A,followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH,the thyroid-specific genes TG,TPO,TSHR,and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes,thyroid follicle formation and abundant TG protein expression were observed. Furthermore,such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine,a TAZ activator,which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes,without any gene transfection or complex culture conditions,by directly manipulating the transcriptional machinery without interfering with intermediate signaling events. View Publication

Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. View Publication

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is,however,limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel),0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker,MATH5. Furthermore,0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1,CRX,RCVRN,AP2α or VSX2) as determined by qRT-PCR,or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE,but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation,transport and transplantation of neural retina and RPE,and may also enhance formation of other pigmented,neural or epithelial tissue. STATEMENT OF SIGNIFICANCE The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However,derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs,whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented,neural or epithelial tissue. View Publication

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca(2+) level ([Ca(2+)] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus,the presence of active GABA-A receptors,associated with phenotype determination via Ca(2+)-signalling was demonstrated in differentiating human DA neurons. View Publication

In this study,because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes,we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology,genomics,and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all,respectively,CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding,we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. View Publication