技术资料

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed,iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro,phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation,validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data,typically by relating the obtained levels of gene expression to the levels of expression of a house keeping gene"� View Publication

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine,including individualized,patient-specific stem cell-based treatments. There are,however,few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues,ideally comprising direct-write printing of cells for encapsulation,proliferation,and differentiation. Here,such a method,employing a clinically amenable polysaccharide-based bioink,is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically,There are extrusion printed the bioink including iPSCs,alginate (Al; 5% weight/volume [w/v]),carboxymethyl-chitosan (5% w/v),and agarose (Ag; 1.5% w/v),crosslinked the bioink in calcium chloride for a stable and porous construct,proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm,ectoderm,and mesoderm,or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined,scalable,and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. View Publication

Microglia,the immune cells of the brain,are crucial to proper development and maintenance of the CNS,and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology,we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes,expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca(2+) transients,whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines. View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair,underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here,we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines,including a WT1-2A-eGFP knock-in reporter line,under chemically-defined,xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells,resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo,as determined by morphological and functional assays,including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule 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

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

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