技术资料

Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free,chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers,similar to those on MatrigelTM. Such well-defined,xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs,but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation. ?? 2013 Elsevier Ltd. All rights reserved. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing,full-length mRNA isoforms are not captured. On the other hand,third-generation sequencing,which yields much longer reads,has current limitations of lower raw accuracy and throughput. Here,we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms,including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover,their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification,even in well-characterized human cell lines and tissues,is likely far from complete. View Publication

A human invitro cardiac tissue model would be a significant advancement for understanding,studying,and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an invitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model,we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices,LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity. textcopyright 2013 Elsevier Ltd. View Publication

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day,7-stage protocol. Cultures were ˜90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17,but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine,but not glucose in perifusion studies. Compared to adult human islets,hESC-derived cells expressed ˜10-fold higher levels of glucose transporter 1 (GLUT1) mRNA,but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However,GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells,whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays,hESC-derived cells displayed mild potassium channel activity,but no responsiveness to glucose,metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells,suggesting a lack of functional KATP channels at the cell surface. In addition,KATP channel subunit transcript levels were not at a 1:1 ratio,as would be expected (SUR1 levels were ˜5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells,and along with lack of GLUT1,may explain the absence of glucose-stimulated insulin secretion.?? 2013 Elsevier B.V. View Publication

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason,it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.$$n$$nMETHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry,RT-PCR,and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT),RPE65,cellular retinaldehyde-binding protein (CRALBP),and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.$$n$$nRESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT,CRALBP,and RPE65. After incubation with all-trans retinol,iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly,after incubation with all-trans retinol,iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.$$n$$nCONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally,the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE. View Publication

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However,the current conventional method leads to poor recovery of hPSCs after thawing. Here,we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing,we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival,whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover,the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and induced pluripotent stem cells,are promising for numerous biomedical applications,such as cell replacement therapies,tissue and whole-organ engineering,and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however,the scalable expansion and differentiation of hPSCs,especially for clinical utilization,remains a challenge. We report a simple,defined,efficient,scalable,and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions,free of any human- or animal-derived factors,and entailing only recombinant protein factors. Under an optimized protocol,the 3D system enables long-term,serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage,for a 1072-fold expansion over 280 d),yield (∼2.0 × 107 cells per mL of hydrogel),and purity (∼95% Oct4+),even with single-cell inoculation,all of which offer considerable advantages relative to current approaches. Moreover,the system enabled 3D directed differentiation of hPSCs into multiple lineages,including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales,from basic biological investigation to clinical development. View Publication

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently,differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately,differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether,our results provide a new platform for the study of kidney diseases and lineage commitment,and open new avenues for the future application of regenerative strategies in the clinic. View Publication

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane,because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation,these methods are not standardized,require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA,or EDTA plus mechanical scraping with an electric toothbrush,or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding,and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2,γ1-γ3 chains,α1/α2 and α6 type IV collagen chains,fibronectin,nidogen-2,and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells,immortalized corneal epithelial cells,and induced pluripotent stem cells. This simple,fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients. View Publication

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However,it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9,HS401 and HS360) on short (2 hours),intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore,the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs,were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover,transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further,MYC protein expression in hypoxia was affected by silencing HIF2α,but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. View Publication