技术资料

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children,with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common,with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer,resulting in embryos containing<99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However,some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions,it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition,some haplotypes confer proliferative and growth advantages to cells. Hence,we propose a matching paradigm for selecting compatible donor mtDNA for MRT. View Publication

We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore,the results of the transcriptomic profile,coupled with immunostaining,and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells,hepatocytes like cells,and endothelial like cells. However,the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless,the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented. View Publication

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

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

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

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study,we investigated the effect of the LRRK2 (G2019S) mutation in monocytes,using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant,compared to non-mutant isogenic controls,leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines,demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells,compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision,endorsing the involvement of the immune system in the development of PD. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

In the past decade,tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly,enormous amount of data can be obtained from the cell line macroarray (CLMA) technology,which developed from the TMA using formalin-fixed,paraffin-embedded cell pellets. Here,we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones,which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here,we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer,TissueQuest,as a reliable tool to quickly select the best clones,based upon the level of expression of multiple pluripotent biomarkers. View Publication

BACKGROUND & AIMS One major obstacle of hepatitis B virus (HBV) research is the lack of efficient cell culture system permissive for viral infection and replication. The aim of our study was to establish a robust HBV infection model by using hepatocyte-like cells (HLCs) derived from human pluripotent stem cells. METHODS HLCs were differentiated from human embryonic stem cells and induced pluripotent stem cells. Maturation of hepatocyte functions was determined. After HBV infection,total viral DNA,cccDNA,total viral RNA,pgRNA,HBeAg and HBsAg were measured. RESULTS More than 90% of the HLCs expressed strong signals of human hepatocyte markers,like albumin,as well as known host factors required for HBV infection,suggesting that these cells possessed key features of mature hepatocytes. Notably,HLCs expressed the viral receptor sodium-taurocholate cotransporting polypeptide more stably than primary human hepatocytes (PHHs). HLCs supported robust infection and some spreading of HBV. Finally,by using this model,we identified two host-targeting agents,genistin and PA452,as novel antivirals. CONCLUSIONS Stem cell-derived HLCs fully support HBV infection. This novel HLC HBV infection model offers a unique opportunity to advance our understanding of the molecular details of the HBV life cycle; to further characterize virus-host interactions and to define new targets for HBV curative treatment. LAY SUMMARY Our study used human pluripotent stem cells to develop hepatocyte-like cells (HLCs) capable of expressing hepatocyte markers and host factors important for HBV infection. These cells fully support HBV infection and virus-host interactions,allowing for the identification of two novel antiviral agents. Thus,stem cell-derived HLCs provide a highly physiologically relevant system to advance our understanding of viral life cycle and provide a new tool for antiviral drug screening and development. View Publication

Behçet's disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD,its diagnosis is very difficult and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology,which provides inaccessible disease-relevant cell types,opens a new era for disease treatment. In this study,we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells,we identified eight novel BD-specific genes,AGTR2,CA9,CD44,CXCL1,HTN3,IL-2,PTGER4,and TSLP,which were differentially expressed in BD patients compared with healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore,we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers,as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers,phenotype- or target-driven drug discovery,and patient-tailored therapies. View Publication

A progressive increase in MECP2 protein levels is a crucial and precisely regulated event during neurodevelopment,but the underlying mechanism is unclear. We report that MECP2 is regulated post-transcriptionally during in vitro differentiation of human embryonic stem cells (hESCs) into cortical neurons. Using reporters to identify functional RNA sequences in the MECP2 3' UTR and genetic manipulations to explore the role of interacting factors on endogenous MECP2,we discover combinatorial mechanisms that regulate RNA stability and translation. The RNA-binding protein PUM1 and pluripotent-specific microRNAs destabilize the long MECP2 3' UTR in hESCs. Hence,the 3' UTR appears to lengthen during differentiation as the long isoform becomes stable in neurons. Meanwhile,translation of MECP2 is repressed by TIA1 in hESCs until HuC predominates in neurons,resulting in a switch to translational enhancement. Ultimately,3' UTR-directed translational fine-tuning differentially modulates MECP2 protein in the two cell types to levels appropriate for normal neurodevelopment. View Publication

Alternative models for more rapid compound safety testing are of increasing demand. With emerging techniques using human pluripotent stem cells,the possibility of generating human in vitro models has gained interest,as factors related to species differences could be potentially eliminated. When studying potential neurotoxic effects of a compound it is of crucial importance to have both neurons and glial cells. We have successfully developed a protocol for generating in vitro 3D human neural tissues,using neural progenitor cells derived from human embryonic stem cells. These 3D neural tissues can be maintained for two months and undergo progressive differentiation. We showed a gradual decreased expression of early neural lineage markers,paralleled by an increase in markers specific for mature neurons,astrocytes and oligodendrocytes. At the end of the two-month culture period the neural tissues not only displayed synapses and immature myelin sheaths around axons,but electrophysiological measurements also showed spontaneous activity. Neurotoxicity testing - comparing non-neurotoxic to known neurotoxic model compounds - showed an expected increase in the marker of astroglial reactivity after exposure to known neurotoxicants methylmercury and trimethyltin. Although further characterization and refinement of the model is required,these results indicate its potential usefulness for in vitro neurotoxicity testing. View Publication