技术资料

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes,but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However,the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore,we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo.backslashnbackslashnMETHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte,Laguna Hills,CA,USA) devices into diabetic mice.backslashnbackslashnRESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing textgreater80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices,despite lacking direct contact with the host environment,and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells.backslashnbackslashnCONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device,yielding glucose-responsive insulin-producing cells capable of reversing diabetes. View Publication

Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers and is associated with aggressive disease and poor outcomes. Our recent findings have shown that NR1D1 and the peroxisome proliferator-activated receptor-γ (PPARγ)-binding protein (PBP) act through a common pathway in upregulating several genes in the de novo fatty acid synthesis network,which is highly active in ERBB2-positive breast cancer cells. NR1D1 and PBP are functionally related to PPARγ,a well-established positive regulator of adipogenesis and lipid storage. Here,we report that inhibition of the PPARγ pathway reduces the aldehyde dehydrogenase (ALDH)-positive population in ERBB2-positive breast cancer cells. Results from in vitro tumorsphere formation assays demonstrate that the PPARγ antagonists GW9662 and T0070907 decrease tumorsphere formation in ERBB2-positive cells,but not other breast cells. We show that the mechanism by which GW9662 treatment causes a reduction in ALDH-positive population cells is partially due to ROS,as it can be rescued by treatment with N-acetyl-cysteine. Furthermore,global gene expression analyses show that GW9662 treatment suppresses the expression of several lipogenic genes,including ACLY,MIG12,FASN and NR1D1,and the stem-cell related genes KLF4 and ALDH in BT474 cells. Antagonist treatment also decreases the level of acetylation in histone 3 and histone 4 in BT474 cells,compared with MCF7 cells. In vivo,GW9662 pre-treatment inhibits the tumor-seeding ability of BT474 cells. Together,these results show that the PPARγ pathway is critical for the cancer stem cell properties of ERBB2-positive breast cancer cells. View Publication

Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome,slow,and variable. Alternatively,human fibroblasts can be directly converted into induced neuronal (iN) cells. However,with present techniques conversion is inefficient,synapse formation is limited,and only small amounts of neurons can be generated. Here,we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin,form mature pre- and postsynaptic specializations,and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples,our approach enables large-scale studies of human neurons for questions such as analyses of human diseases,examination of human-specific genes,and drug screening View Publication

Lymphocyte activation is regulated by costimulatory and inhibitory receptors,of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably,it remains unclear how HVEM functions with each of its ligands during immune responses. In this study,we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT,Lymphotoxin-α,or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2,resulting in increased IFN-γ and TNF-α secretion,and tumor cell-expressed HVEM activated CD160 in a human NK cell line,causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast,HVEM activation of BTLA reduced cytolysis of target cells. Together,our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA. View Publication

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However,fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs,and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs,allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs. View Publication

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines,immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere,adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity,consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes,although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly,self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures,suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia,including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like,basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype,or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall,cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells,suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells,has utility as one of a suite of functional assays that provide a read-out of progenitor activity. View Publication

We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium,hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium,possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further,these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful,objective,measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny,particularly changes induced by the cellular environment. View Publication

PURPOSE FK506-binding protein like (FKBPL) and its peptide derivative,AD-01,have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here,we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC). EXPERIMENTAL DESIGN Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75),primary patient samples,and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays,quantitative PCR (qPCR),and immunofluorescence. RESULTS AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers,Nanog,Oct4,and Sox2,were also significantly reduced. Furthermore,we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor,DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally,FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs,highlighting a role for endogenous FKBPL in stem cell signaling. CONCLUSIONS AD-01 has dual antiangiogenic and anti-BCSC activity,which will be advantageous as this agent enters clinical trial. View Publication

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics,and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is,however,traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer,and as a result,possible xenogeneic contamination,contribution of unlabeled amino acids by the feeders,interlaboratory variability of MEF preparation,and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used,chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison,WI) licensed to STEMCELL Technologies (Vancouver,Canada)]. This medium,together with adjustments to the culturing protocol,facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View Publication

Myocardial infarction is a leading cause of mortality and morbidity worldwide,and current treatments fail to address the underlying scarring and cell loss,which is a major cause of heart failure after infarction. The novel strategy,therapeutic angiogenesis and/or vasculogenesis with endothelial progenitor cells transplantation holds great promise to increase blood flow in ischemic areas,thus rebuild the injured heart and reverse the heart failure. Given the potential of self-renewal and differentiation into virtually all cell types,human embryonic stem cells (hESCs) may provide an alternate source of therapeutic cells by allowing the derivation of large numbers of endothelial cells for therapeutic angiogenesis and/or vasculogenesis of ischemic heart diseases. Moreover,to fully understand the fate of implanted hESCs or hESC derivatives,investigators need to monitor the motility of cells in living animals over time. In this chapter,we describe the application of bioluminescence reporter gene imaging to track the transplanted hESC-derived endothelial cells for treatment of myocardial infarction. The technology of inducing endothelial cells from hESCs will also be discussed. View Publication

In this study,a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension,only a few aggregated cells were observed. However,after 3 days,culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry,immunocytochemistry and quantitative RT-PCR,and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium,expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore,the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A,BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes,including HCN4,MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes,including pacemakers. Moreover,when cardiac cell sheets were fabricated using differentiated cardiomyocytes,they beat spontaneously and synchronously,indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. View Publication

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition,including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients,we demonstrate that cell viability can be preserved throughout differentiation. In addition,modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique,as well as gene expression patterns,indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm. View Publication