技术资料

To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications,large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However,the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics,we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition,we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components,subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications. View Publication

We report the identification and synthesis of a series of aminopyrimidin-4-one IRAK4 inhibitors. Through high throughput screening,an aminopyrimidine hit was identified and modified via structure enabled design to generate a new,potent,and kinase selective pyrimidin-4-one chemotype. This chemotype is exemplified by compound 16,which has potent IRAK4 inhibition activity (IC50 = 27 nM) and excellent kinase selectivity (textgreater100-fold against 99% of 111 tested kinases),and compound 31,which displays potent IRAK4 activity (IC50 = 93 nM) and good rat bioavailability (F = 42%). View Publication

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1,ANXA11,CST4,PRDX5,PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1,ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. View Publication

Pluripotent stem cells,including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),have the potential to treat type 1 diabetes through cell replacement therapy. However,the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification,and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation,and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified,suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR,and the relationship between one of these miRNAs,miR-151a-5p,and its predicted target gene,SOX17,was investigated by luciferase assay,and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion,these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. View Publication

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins,the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins,and as extension of previous work (Palomo et al.,2014),a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. View Publication

GTF2IRD1 is one of the three members of the GTF2I gene family,clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities,mental retardation,visuospatial deficits and hypersociability of WBS. However,the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here,for the first time,we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner,yeast two-hybrid libraries were screened,isolating 38 novel interaction partners,which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function,as the isolated partners are mostly involved in chromatin modification and transcriptional regulation,whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain,behaviour and human disease. View Publication

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies,drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro-generated CMs. This protocol describes a suspension culture-based strategy for the generation of hPSC-CMs as cell-only aggregates,which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale,providing a well-controlled upscaling platform for CM production. In bioreactors,the generation of 40-50 million CMs per differentiation batch at textgreater80% purity without further lineage enrichment can been achieved within 24 d. View Publication

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However,clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis,a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC,maintained in an optimised cytokine cocktail,to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4,Sox2,Klf4,L-Myc,and Lin28) in a feeder-free system,without any requirement for small molecules,we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells,we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function. View Publication

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here,we report on the comparative cytotoxicity of 80 compounds (neurotoxicants,developmental neurotoxicants,and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC),neurons,and astrocytes. All compounds were tested over a 24-h period at 10 and 100$\$,in duplicate,with cytotoxicity measured using the MTT assay. Of the 80 compounds tested,50 induced significant cytotoxicity in at least one cell type; per cell type,32,38,46,and 41 induced significant cytotoxicity in iPSC,NSC,neurons,and astrocytes,respectively. Four compounds (valinomycin,3,3',5,5'-tetrabromobisphenol,deltamethrin,and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1,10,and 100$\$ using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone,we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally,the cytotoxicity assay was simplified by measuring luciferase activity using lineage-specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain. View Publication

Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however,the introduction of ectopic genes limits the therapeutic applications of such induced neurons (iNs). Here,we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules,bypassing a neural progenitor stage. These human chemical-induced neuronal cells (hciNs) resembled hiPSC-derived neurons and human iNs (hiNs) with respect to morphology,gene expression profiles,and electrophysiological properties. This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together,our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine. View Publication

Recently,direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine,but applications could be limited by the genetic manipulation involved. Here,we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules,with a yield of up to textgreater90% being TUJ1-positive after 16 days of induction. After a further maturation stage,these chemically induced neurons (CiNs) possessed neuron-specific expression patterns,generated action potentials,and formed functional synapses. Mechanistically,we found that a BET family bromodomain inhibitor,I-BET151,disrupted the fibroblast-specific program,while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall,our findings provide a proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation� View Publication

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential,many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. View Publication