技术资料

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected,and the functional consequences,have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51),but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD),a model of global brain ischemia,as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity,the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore,GDNF protected hippocampal neurons when present before,during or after OGD,and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source with pluripotency and capacity to differentiate into all human somatic cell types. Designing simple and safe biomaterials with an innate ability to induce osteoblastic lineage from hPSCs is desirable to realize their clinical adoption in bone regenerative medicine. To address the issue,here we developed a fully defined synthetic peptides-decorated two dimensional (2D) microenvironment assisted via polydopamine (pDA) chemistry and subsequent carboxymethyl chitosan (CMC) grafting to enhance the culture and osteogenic potential of hPSCs in vitro. The hPSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were successfully cultured on the peptides-decorated surface without Matrigel- and ECM protein-coating and underwent promoted osteogenic differentiation in vitro,determined from the alkaline phosphate (ALP) activity,gene expression,and protein production as well as calcium deposit amount. It was found that directed osteogenic differentiation of hPSCs could be achieved through a peptides-decorated niche. This chemical-defined and safe 2D microenvironment which facilitates proliferation and osteo-differentiation of hPSCs,not only helps to accelerate the translational perspectives of hPSCs,but also provides tissue-specific functions such as directing stem cell differentiation commitment,having great potential in bone tissue engineering and presenting new avenues for bone regenerative medicine. View Publication

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

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles,we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform,mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred,thus increasing screening throughput by textgreater30-fold. Compared to conventional methods,micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. 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

BACKGROUND Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver,lungs,thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1,2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry,real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG),definitive endoderm (SOX17,CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures. RESULTS Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG,accompanied by an increase expression of the DE genes SOX17,CXCR4 and GATA4. Importantly,the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment. View Publication

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ),but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method,we have identified small molecules that can enhance CRISPR-mediated HDR efficiency,3-fold for large fragment insertions and 9-fold for point mutations. Interestingly,we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells. View Publication

About half of the human genome consists of highly repetitive elements,most of which are considered dispensable for human life. Here,we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation,thereby generating hundreds of co-expressed,ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements,indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription,many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary,we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos. View Publication

The intestinal mucosa undergoes a continual process of proliferation,differentiation and apoptosis,which is regulated by multiple signaling pathways. Notch signaling is critical for the control of intestinal stem cell maintenance and differentiation. However,the precise mechanisms involved in the regulation of differentiation are not fully understood. Previously,we have shown that tuberous sclerosis 2 (TSC2) positively regulates the expression of the goblet cell differentiation marker,MUC2,in intestinal cells. Using transgenic mice constitutively expressing a dominant negative TSC2 allele,we observed that TSC2 inactivation increased mTORC1 and Notch activities,and altered differentiation throughout the intestinal epithelium,with a marked decrease in the goblet and Paneth cell lineages. Conversely,treatment of mice with either Notch inhibitor dibenzazepine (DBZ) or mTORC1 inhibitor rapamycin significantly attenuated the reduction of goblet and Paneth cells. Accordingly,knockdown of TSC2 activated,whereas knockdown of mTOR or treatment with rapamycin decreased,the activity of Notch signaling in the intestinal cell line LS174T. Importantly,our findings demonstrate that TSC2/mTORC1 signaling contributes to the maintenance of intestinal epithelium homeostasis by regulating Notch activity. View Publication

Airway epithelial cells are of great interest for research on lung development,regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation,and full maturation of the cells in air-liquid interface cultures occurs in textless5 weeks. This protocol can be used for drug discovery,tissue regeneration or disease modeling. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast,humans with NEUROG3 mutations are born with endocrine pancreas function,calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly,we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover,NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast,a 75-90% knockdown of NEUROG3 caused a reduction,but not a loss,of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. View Publication