技术资料

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers,strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13,ID1,BCL2L1,KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1,nor the downstream effects expected from over-expression of this gene. On the other hand,hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were ∼2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells,leading to apoptosis-resistant cells,and BCL2L1-knock-down in the mutant hESC,resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells,explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells. 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

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

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

Production of human embryonic stem cell (hESC)-derived lung progenitors has broad applicability for drug screening and cell therapy; however,this is complicated by limitations in demarcating phenotypic changes with functional validation of airway cell types. In this paper,we reveal the potential of hESCs to produce multipotent lung progenitors using a combined growth factor and physical culture approach,guided by the use of novel markers LIFRα and NRP1. Lung specification of hESCs was achieved by priming differentiation via matrix-specific support,followed by air-liquid interface to allow generation of lung progenitors capable of in vitro maturation into airway epithelial cell types,resulting in functional characteristics such as secretion of pulmonary surfactant,ciliation,polarization,and acquisition of innate immune activity. This approach provided a robust expansion of lung progenitors,allowing in vivo assessment,which demonstrated that only fully differentiated hESC-derived airway cells were retained in the distal airway,where they aided in physiological recovery in immunocompromised mice receiving airway injury. Our study provides a basis for translational applications of hESCs for lung diseases. View Publication

Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However,existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs,we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs,using size-controlled embryoid bodies (EBs),and addition of triiodothyronine (T3) and taurine to the differentiation medium,with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation,which will help understand retinal diseases and facilitate large-scale preclinical trials. View Publication

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately,with low efficiencies,from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model,elucidate,and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor,basic fibroblast growth factor,and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media,these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells,respectively. Furthermore,we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations. View Publication

High telomerase activity is a characteristic of human embryonic stem cells (hESCs),however,the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion,but stabilized at later passages. We report that the core unit of telomerase,hTERT,was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore,this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation,demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation,suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines,we were able to establish that telomere length remained relatively stable at 12.02±1.01 kb during later passages (15-95). In contrast,telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary,we propose that correct,stable telomere length may serve as a potential biomarker for genetically stable hESCs. View Publication

BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases,including neurodegenerative diseases,as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts.backslashnbackslashnMETHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.backslashnbackslashnRESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents,including Lipofectamine® 2000,FuGENE® HD,and 25 kDa branched polyethylenimine,for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa,and enabled coexpression of exogenously delivered genes,as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation,but not by poly(beta-amino ester) reprogramming,could be differentiated toward the neuronal lineage,specifically pseudostratified optic cups.backslashnbackslashnCONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. View Publication

Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs,the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here,we describe a method based on the use of a small-molecule GSK3?? inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3?? inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover,we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free,efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies,in vitro screenings,and disease modeling. ?? 2013 The Authors. View Publication