技术资料

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-β[2-(di-n-butylamino)-6-naphthyl]butadienylquinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses,we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision,generating nearly identical values for AP duration (AP durations at 10%,50%,and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure,with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally,di-4-ANBDQBS allowed for moderate-throughput analyses,increasing throughput textgreater10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription,thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs,which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ˜15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together,our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species. View Publication

Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. To better understand the molecular effect of alcohol on the process of neural differentiation,we have performed gene expression microarray analysis on human embryonic stem cells being directed to neural rosettes and neural precursor cells in the presence of ethanol treatment. Here we provide detailed experimental methods,analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE56906. Our data provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development. View Publication

Despite major progress in the management of human liver disease,the only cure for a critically failing organ is liver transplantation. While a highly successful approach,the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore,the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments,there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process,offering new treatment or preventative strategies. With both approaches in mind,we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly,our procedure is highly efficient (∼ 90%) and delivers active,drug-inducible,and predictive human hepatocyte populations. View Publication

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation,we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling,paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types,indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus,iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover,altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

Magnesium (Mg) is a promising conductive metallic biomaterial due to its desirable mechanical properties for load bearing and biodegradability in human body. Controlling the rapid degradation of Mg in physiological environment continues to be the key challenge toward clinical translation. In this study,we investigated the effects of conductive poly(3,4-ethylenedioxythiophene) (PEDOT) coating on the degradation behavior of Mg substrates and their cytocompatibility. Human embryonic stem cells (hESCs) were used as the in vitro model system to study cellular responses to Mg degradation because they are sensitive and can potentially differentiate into many cell types of interest (e.g.,neurons) for regenerative medicine. The PEDOT was deposited on Mg substrates using electrochemical deposition. The greater number of cyclic voltammetry (CV) cycles yielded thicker PEDOT coatings on Mg substrates. Specifically,the coatings produced by 2,5,and 10 CV cycles (denoted as 2×-PEDOT-Mg,5×-PEDOT-Mg,and 10×-PEDOT-Mg) had an average thickness of 31,63,and 78 µm,respectively. Compared with non-coated Mg samples,all PEDOT coated Mg samples showed slower degradation rates,as indicated by Tafel test results and Mg ion concentrations in the post-culture media. The 5×-PEDOT-Mg showed the best coating adhesion and slowest Mg degradation among the tested samples. Moreover,hESCs survived for the longest period when cultured with the 5×-PEDOT-Mg samples compared with the non-coated Mg and 2×-PEDOT-Mg. Overall,the results of this study showed promise in using PEDOT coating on biodegradable Mg-based implants for potential neural recording,stimulation and tissue engineering applications,thus encouraging further research. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however,further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal,we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference,pluripotency,and karyotype. Critically,XF transitioned H9 hESC,Shef4 hESC,and iPS6-9 retained pluripotency (Oct-4 and NANOG),proliferation (MKI67 and PCNA),and normal karyotype. Subsequently,XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors,which could differentiate into astrocytes and neurons,but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly,adherent hNSC derived from H9,Shef4,and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (∼20-30%) with robust proliferation; however,very few GalC+ cells were observed (∼2-4%),indicative of early oligodendrocytic lineage commitment. Overall,these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions,and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events,including differentiation. In this study,we analyzed acetylated forms of histones H2A,H2B,and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-,di-,and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes,mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs,we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-,di-,and tri-acetylation of H4 were reduced,manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation,whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4. View Publication

OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases. View Publication