技术资料

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes,both in vitro and in vivo. Beyond uses in cell replacement therapy,patient-specific cardiomyocytes may find applications in drug testing,drug discovery,and disease modeling. Recently,approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs),adult heart-derived cardiac progenitor cells (CPCs),and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts,highlighting potential applications and future challenges. View Publication

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure,Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here,we report that a transcriptional factor,RP58,negatively regulates all four Id genes (Id1-Id4) in developing cerebral cortex. Consistently,Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild-type cortical progenitors. Furthermore,Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally,we determined p57 as an effector gene of RP58-Id-mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis. View Publication

BACKGROUND Toll-like receptors (TLR) 7 and 8 are important in single-stranded viral RNA recognition and may play a role in HIV infection and disease progression. We analyzed TLR7/8 expression and signaling in monocytes from HIV-infected and uninfected subjects to investigate a pathway with new potential for the suppression of HIV replication. METHODS Eighty-one HIV-infected and uninfected subjects from Liaoning and Henan provinces in China participated in this study. Monocytes were isolated from subjects' peripheral blood mononuclear cells by magnetic bead selection. TLR7 and TLR8 mRNA was measured using quantitative real-time reverse transcriptase PCR. R-848 (resiquimod) was used as a ligand for TLR7 and TLR8 in order to 1) assess TLR7/8-mediated monocyte responsiveness as indicated by IL-12 p40 and TNF-α secretion and 2) to examine HIV replication in cultured monocytes in the presence of R-848. RESULTS We found that expression of TLR7/8 mRNA in peripheral blood monocytes decreased with disease progression. TLR7 expression was decreased with stimulation with the TLR7/8 agonist,R-848,in vitro,whereas TLR8 expression was unaffected. Following R-848 stimulation,monocytes from HIV-infected subjects produced significantly less TNF-α than those from uninfected subjects,but trended towards greater production of IL-12 than stimulated monocytes from uninfected subjects. R-848 stimulation also suppressed HIV replication in cultured monocytes. CONCLUSIONS Our study provides evidence that the TLR7 and TLR8 triggering can suppress HIV replication in monocytes and lead to postpone HIV disease progression,thereby offering novel targets for immunomodulatory therapy. View Publication

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression,could be maintained in culture for several weeks,expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice,the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease View Publication

Within high-grade gliomas,the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche,ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis,we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity,while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly,Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further,eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival,while knockdown of Olig2 within Id1(low) cells has a significant survival benefit,underscoring the importance of non-self-renewing lineages in disease progression. View Publication

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count,and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation,using both microarray and sequence-based analyses,provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines,the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly,genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes,such as tumor suppressors and genetic disease genes,do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens,such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents. View Publication

Human embryonic stem (hES) cells have the dual ability to self-renew and differentiate into specialized cell types. However,in order to realize the full potential of these cells it is important to understand how the genes responsible for their unique characteristics are regulated. In this study we examine the regulation of the tropomyosin-related kinase (TRK) genes which encode for receptors important in hES cell survival and self-renewal. Although the TRK genes have been studied in many neuronal cell types,the regulation of these genes in hES cells is unclear. Our study demonstrates a novel regulatory relationship between the TRKC gene and the transcription factor SOX2. Our results found that hES cells highly express full-length and truncated forms of the TRKC gene. However,examination of the related TRKB gene showed a lower overall expression of both full-length and truncated forms. Through RNA interference,we knocked down expression levels of SOX2 in hES cells and examined the expression of TRKC,as well as TRKB. Upon loss of SOX2 we found that TRKC mRNA levels were significantly downregulated but TRKB levels remained unchanged,demonstrating an important regulatory dependence on SOX2 by TRKC. We also found that TRKC protein levels were also decreased after SOX2 knock down. Further analysis found the regulatory region of TRKC to be highly conserved among many mammals with potential SOX binding motifs. We confirmed a specific binding motif as a site that SOX2 utilizes to directly interact with the TRKC regulatory region. In addition,we found that SOX2 drives expression of the TRKC gene by activating a luciferase reporter construct containing the TRKC regulatory region and the SOX binding motif. View Publication

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder,ATR-Seckel syndrome. To understand how ATR functions during neurogenesis,we conditionally deleted Atr broadly throughout the murine nervous system,or in a restricted manner in the dorsal telencephalon. Unexpectedly,in both scenarios,Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest,other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs,in the nervous system,p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion,supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress,our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis. View Publication

Cancer stem cells (CSC) are resistant to radiation and chemotherapy and play a significant role in cancer recurrence and metastatic disease. It is therefore important to identify alternative strategies,such as immunotherapies that can be used to control this refractory population. A CD44(+)CD24(-/low) subpopulation of cells within the B6 PyMT-MMTV transgenic mouse-derived AT-3 mammary carcinoma cell line was identified,which had CSC-like characteristics,including pluripotency and a resistance to chemo- and radiotherapy. Therefore,unlike xenograph models that require immunocompromised settings,this novel system may provide a means to study immune-mediated responses against CSC-like cells. The immunobiology of the AT-3 CSC-like cell population was studied by their surface molecule expression profile and their sensitivity to specified cell death pathways. Comparable levels of Rae-1,CD155,CD54 and higher levels of Fas and DR5 were expressed on the AT-3 CSC-like cells compared to non-CSC-like tumor cells. Expression correlated with an in vitro sensitivity to cell death by NK cells or through the ligation of the death receptors (Fas or DR5),by their ligands or anti-Fas and anti-DR5 mAbs. Indeed,compared to the rest of the AT-3 tumor cells,the CD44(+)CD24(-/low) subpopulation of cells were more sensitive to both Fas- and TRAIL-mediated cell death pathways. Therefore,despite the refractory nature of CSC to other conventional therapies,these CSC-like cells were not inherently resistant to specified forms of immune-mediated cell death. These results encourage the continued investigation into immunotherapeutic strategies as a means of controlling breast CSC,particularly through their cell death pathways. View Publication

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent,have the capacity to differentiate into hematopoietic progenitors,and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease. View Publication

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced,but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5),which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9),is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However,LSD1 is not essential for the maintenance of ESC identity. Instead,ESCs lacking LSD1 activity fail to differentiate fully,and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers,LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex,which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation,which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. View Publication

Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet,iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here,we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4,SOX2,KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions,reprogrammed cells underwent dedifferentiation with mitochondrial restructuring,induction of endogenous pluripotency genes - including NANOG,LIN28,and TERT,and down-regulation of cytoskeletal,MHC class I- and apoptosis-related genes. Notably,derived iPS clones acquired a rejuvenated state,characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors,including Indolactam V and GLP-1,redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus,in elderly T2D patients,reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications. View Publication