技术资料

Mitochondria are critical to neurogenesis,but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons,mitochondria in these neurons exhibit pronounced changes during differentiation,including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis,which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases. View Publication

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here,we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes,respectively. Clusters of Wnt targets showed enrichment in specific biological functions,such as gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover� View Publication

The sodium bicarbonate co-transporter (NBC) is the major bicarbonate-dependent acid-base transporter in mammalian astrocytes and has been implicated in ischemic brain injury. A malfunction of astrocytes could have great impact on the outcome of stroke due to their participation in the formation of blood-brain barrier,synaptic transmission,and electrolyte balance in the human brain. Nevertheless,the role of NBC in the ischemic astrocyte death has not been well understood. In this work,we obtained skin biopsies from healthy human subjects and had their fibroblasts grown in culture and reprogrammed into human-induced pluripotent stem cells (hiPSCs). These hiPSCs were further differentiated into neuroprogenitor cells (NPCs) and then into human astrocytes. These astrocytes express GFAP and S100β and readily propagate calcium waves upon mechanical stimulation. Using pH-sensitive dye BCECF [2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] and qPCR technique,we have confirmed that these astrocytes express functional NBC including electrogenic NBC (NBCe). In addition,astrocytes exposed to an ischemic solution (IS) that mimics the ischemic penumbral environment enhanced both mRNA and protein expression level of NBCe1 in astrocytes. Using IS and a generic NBC blocker S0859,we have studied the involvement of NBC in IS-induced human astrocytes death. Our results show that a 30μM S0859 induced a 97.5±1.6% (n=10) cell death in IS-treated astrocytes,which is significantly higher than 43.6±4.5%,(n=10) in the control group treated with IS alone. In summary,a NBC blocker exaggerates IS-induced cell death,suggesting that NBC activity is essential for astrocyte survival when exposed to ischemic penumbral environment. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. View Publication

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells,methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here,we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped,submillimeter colonies. After 42 h of BMP4 stimulation,cells form self-organized differentiation patterns in concentric radial domains,which express specific markers associated with the embryonic germ layers,reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO),human laminin-521 (LN-521) as extracellular matrix coating,and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. View Publication

Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens,ranging from viruses to bacteria. However,the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study,we describe the recognition of an emerging fungal pathogen,Candida glabrata,by the human NK cytotoxic receptor NKp46 and its mouse ortholog,NCR1. Using NCR1 knockout mice,we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally,we delineated the fungal ligands to be the C. glabrata adhesins Epa1,Epa6,and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors,we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor. View Publication

CD46 is a glycoprotein with important functions in innate and adaptive immune responses. Functionally different isoforms are generated by alternative splicing at exons 7-9 (BC and C isoforms) and exon 13 (CYT-1 and CYT-2 isoforms) giving rise to BC1,BC2,C1 and C2. We developed a novel real-time PCR assay that allows quantitative comparisons between these isoforms. Their relative frequency in CD4(+) T cells from 100 donors revealed a distribution with high interpersonally variability. Importantly,the distribution between the isoforms was not random and although splicing favoured inclusion of exon 8 (BC isoforms),exclusion of exon 8 (C isoforms) was significantly linked to exclusion of exon 13 (CYT-2 isoforms). Despite inter-individual differences,CD4(+) and CD8(+) T cells,B cells,NK cells and monocytes expressed similar isoform profiles intra-individually. However,memory/effector CD4(+) T cells had a significantly higher frequency of CYT-2 when compared with naïve CD4(+) T cells. Likewise,in vitro activation of naïve and total CD4(+) T cells increased the expression of CYT-2. This indicates that although splicing factors determine a certain expression profile in an individual,the profile can be modulated by external stimuli. This suggests a mechanism by which alterations in CD46 isoforms may temporarily regulate the immune response. View Publication

Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases,in particular those affecting the nervous system,which has been difficult to study for its lack of accessibility. In this emerging and promising field,recent iPSCs studies are mostly used as proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development� View Publication

The mechanisms underlying human germ cell development are largely unknown,partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here,we studied NANOS3 and DAZL,which have critical roles in germ cell development in several species,via their over expression in human embryonic stem cells using global transcriptional analysis,in vitro germ cell differentiation,and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition,we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL,our results suggest a post-transcriptional regulation mechanism in hES cells. In addition,we found that DAZL suppressed the translation of OCT4,and affected the transcription of several genes associated with germ cells,cell cycle arrest,and cell migration. Furthermore,DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. View Publication

The blood disorder,β-thalassaemia,is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype,with sustained elevation of blood haemoglobin levels into the normal range,reduced reticulocytosis,reversal of splenomegaly and up to 7% β-globin gene correction in HSCs,with extremely low off-target effects. The combination of nanoparticle delivery,next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration. View Publication

A new method of spatially controlled gene regulation in 3D-cultured human embryonic stem cells is developed using hollow gold nanoshells (HGNs) and near-infrared (NIR) light. Targeted cell(s) are discriminated from neighboring cell(s) by focusing NIR light emitted from a two-photon microscope. Irradiation of cells that have internalized HGNs releases surface attached siRNAs and leads to concomitant gene downregulation. View Publication