技术资料

Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However,by using an engineered reporter construct over-expressing L1,another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications,it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here,we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition,we used a novel sequencing strategy. As opposed to conventional sequencing direction,we sequenced from the 3' end of L1Hs to the genomic DNA,thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells,presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore,these insertions could not be detected in iPSCs by PCR,likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects. View Publication

BACKGROUND: Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells,such as embryonic stem cells and induced pluripotent stem cells,appear to have a hypermethylated status compared with differentiated cells. However,the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally,differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.backslashnbackslashnMETHODOLOGY: Here,we determined the DNA methylation profiles of 10 human cell lines,including 2 ESC lines,4 virally derived iPSC lines,2 episomally derived iPSC lines,and the 2 parental cell lines from which the iPSCs were derived using Illumina's Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness,whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.backslashnbackslashnCONCLUSIONS: This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods,the corresponding somatic cells,and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine. View Publication

Predicting the binding mode of flexible polypeptides to proteins is an important task that falls outside the domain of applicability of most small molecule and protein−protein docking tools. Here,we test the small molecule flexible ligand docking program Glide on a set of 19 non-$$-helical peptides and systematically improve pose prediction accuracy by enhancing Glide sampling for flexible polypeptides. In addition,scoring of the poses was improved by post-processing with physics-based implicit solvent MM- GBSA calculations. Using the best RMSD among the top 10 scoring poses as a metric,the success rate (RMSD ≤ 2.0 Å for the interface backbone atoms) increased from 21% with default Glide SP settings to 58% with the enhanced peptide sampling and scoring protocol in the case of redocking to the native protein structure. This approaches the accuracy of the recently developed Rosetta FlexPepDock method (63% success for these 19 peptides) while being over 100 times faster. Cross-docking was performed for a subset of cases where an unbound receptor structure was available,and in that case,40% of peptides were docked successfully. We analyze the results and find that the optimized polypeptide protocol is most accurate for extended peptides of limited size and number of formal charges,defining a domain of applicability for this approach. View Publication

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far,the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here,we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system,and utilized this approach to genotype mice from F0 to F2 generation,which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus,PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN. View Publication

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue,making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1$\$)-hESC lines using chEF1$\$ system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology,karyotype,and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers,OCT-3/4 and SSEA-4,in undifferentiated mHLA-G(EF1$\$)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors,expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1$\$)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives. View Publication

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here,we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform,under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm²,where they attach,migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521,in combination with E-cadherin,allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. View Publication

Cardiovascular progenitor cells (CVPCs) derived from human embryonic stem cells and human induced pluripotent stem cells represent an invaluable potential source for the study of early embryonic cardiovascular development and stem cell-based therapies for congenital and acquired heart diseases. To fully realize their values,it is essential to establish an efficient and stable differentiation system for the induction of these pluripotent stem cells (PSCs) into the CVPCs and robustly expand them in culture,and then further differentiate these CVPCs into multiple cardiovascular cell types. Here we describe the protocols for efficient derivation,expansion,and differentiation of CVPCs from hPSCs in a chemically defined medium under feeder- and serum-free culture conditions. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS),an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study,we identified that YAP/TAZ,two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway,are activated in KSHV-infected cells in vitro,KS-like mouse tumors and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2,promoting the activation of YAP/TAZ. Furthermore,depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacologic inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS,and also suggests a potential of using YAP inhibitors for KS intervention.Oncogene advance online publication,8 September 2014; doi:10.1038/onc.2014.281. View Publication

Human brain development exhibits several unique aspects,such as increased complexity and expansion of neuronal output,that have proven difficult to study in model organisms. As a result,in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue,so-called cerebral organoids,which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex,ventral telencephalon,choroid plexus and retinal identities,among others,within 1-2 months. This straightforward protocol can be applied to developmental studies,as well as to the study of a variety of human brain diseases. Furthermore,as organoids can be maintained for more than 1 year in long-term culture,they also have the potential to model later events such as neuronal maturation and survival. View Publication

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

Aims Hemophilia A (HA) is a severe,congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years,traditional laboratory animals have been used to study HA and its therapies,although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction. Main methods In this study,we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4,SOX2,SV40LT and KLF4,and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells. Key findings HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining,in vitro assays and in vivo assays. Importantly,we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII,but otherwise functioned normally,recapitulating the phenotype of HA disease in vitro. Significance HA-iPSCs,particular those generated from the urine using a non-viral approach,provide an efficient way for modeling HA in vitro. Furthermore,HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine. textcopyright 2014 Elsevier Inc. View Publication