技术资料

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening,biomarker discovery,and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here,we describe the (1) retroviral reprogramming strategies in the generation of human iPSC,(2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality,and (3) protocol of cardiac differentiation by modulation of Wnt signaling and $\$-catenin pathway. View Publication

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study,we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly,Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore,GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes,revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement,with initial repression of Nanog and Esrrb,then Sox2,and finally Oct4,alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes,suggesting that Gata6 functions as both a direct repressor and activator. Together,this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. View Publication

Despite major advances in the pathophysiological understanding of peripheral nerve damage,the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end,we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival,migration,and guide neurite extension. View Publication

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T,$\$-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate,the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%,whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular,atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified,functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling,drug discovery,and regenerative medicine. 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 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

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 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

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

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

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

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