技术资料

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT,BMP,retinoic acid (RA),and hedgehog (Hh),but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology,mouse genetics,and human ES cell cultures,we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally,RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis. View Publication

Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however,little is known about it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally-induced stress. We tested the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH),which was characterized by changes in mitochondrial morphology indicative of fusion,increased mitochondrial membrane potential (MMP),increased ATP levels,increased superoxide production,and increased oxidation of mitochondrial proteins. SIMH was accompanied by a decrease in Fis1 expression,a gene responsible for mitochondrial fission,and a decrease in apoptosis-related genes,including Aifm2,Bbc3 and Bid There was also down regulation of Ucp2,Ucp4 and Ucp5,genes that decrease MMP thereby reducing oxidative phosphorylation,while promoting glycolysis. These effects,which collectively accompany SIMH,are a pro-survival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure to THS caused a reduction in MMP and decreased cell proliferation,which likely leads to apoptosis. View Publication

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages,we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs) to incorporate a reporter cassette at the transcription start site of one of its canonical targets,cytochrome P450 1A1 (CYP1A1). This cell line faithfully reports on CYP1A1 expression,with luciferase levels as its functional readout,when treated with an endogenous AHR ligand (FICZ) at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands,and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed,this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR. View Publication

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD),including tuberous sclerosis,caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here,we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism,suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis. View Publication

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder,which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD,existing pharmaceutical can only relieve its symptoms. Results: Here,induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene,and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro,as evidenced by mutant huntingtin protein aggregation,increased number of lysosomes/autophagosomes,nuclear indentations,and enhanced neuronal death during cell aging. Moreover,store-operated channel (SOC) currents were detected in the differentiated neurons,and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally,the quinazoline derivative,EVP4593,reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Conclusions: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks,providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug. [ABSTRACT FROM AUTHOR] View Publication

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However,how it mediates Ca2+ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here,we aimed to determine the role of P2Rs in mediating Ca2+ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations,respectively. Confocal imaging revealed that Ca2+ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently,the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca2+ transients in hESCs but only partially inhibited those in CVPCs. Moreover,the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca2+ signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca2+ transients only in hESCs but not in CVPCs. Furthermore,IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast,both IP3R2 and IP3R3 contributed to UTP-induced Ca2+ responses while ATP-induced Ca2+ responses were more dependent on IP3R2 in the CVPCs. In conclusion,a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca2+ mobilization between these cells. View Publication

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells. View Publication

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining,leading to reading frame disruption. The approach is applicable to dominant negative disorders,which can be treated simply by knocking out the mutant allele,while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB),which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation,c.80688084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed,respectively,into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting,90% of the iPSCs were edited,and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition,we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders. View Publication

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA,PARK2,LRRK2,and GBA mutations,and one age-matched control. After validation of pluripotency,long-term genome stability,and integration-free reprogramming,eight of these lines (one of each SNCA,LRRK2 and GBA,four PARK2 lines,and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line,nor amongst the patient lines,although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast,iPSC,NSC,and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation,no mutation-specific clustering or difference was observed,though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line,and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease,and highlights the need to use more focused strategies for large-scale data analysis. View Publication

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers,deficiency of enzyme activity,and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes,neurons). In MPS II iPSC clones,the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data,in accordance with the literature,suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time,we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease. View Publication

Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach,sequencing of psoralen crosslinked,ligated,and selected hybrids (SPLASH),that maps pairwise RNA interactions in vivo with high sensitivity and specificity,genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes,including the modular organization of mRNAs,its impact on translation and decay,and the enrichment of long-range interactions in noncoding RNAs. Additionally,intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites,expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology. View Publication