技术资料

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are,in part,controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis,we have compared gene expression profiles of human erythroblasts,megakaryocytes,B cells,cytotoxic and helper T cells,natural killer cells,granulocytes,and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors,immunoglobulin superfamily members,and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude,ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition,we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg,GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data,which are freely accessible,will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. View Publication

Aberrant neural progenitor cell (NPC) proliferation and self-renewal have been linked to age-related neurodegeneration and neurodegenerative disorders including Alzheimer's disease (AD). Rhizoma Acori tatarinowii is a traditional Chinese herbal medicine against cognitive decline. In this study,we found that the extract of Rhizoma Acori tatarinowii (AT) and its active constituents,asarones,promote NPC proliferation. Oral administration of AT enhanced NPC proliferation and neurogenesis in the hippocampi of adult and aged mice as well as that of transgenic AD model mice. AT and its fractions also enhanced the proliferation of NPCs cultured in vitro. Further analysis identified α-asarone and β-asarone as the two active constituents of AT in promoting neurogenesis. Our mechanistic study revealed that AT and asarones activated extracellular signal-regulated kinase (ERK) but not Akt,two critical kinase cascades for neurogenesis. Consistently,the inhibition of ERK activities effectively blocked the enhancement of NPC proliferation by AT or asarones. Our findings suggest that AT and asarones,which can be orally administrated,could serve as preventive and regenerative therapeutic agents to promote neurogenesis against age-related neurodegeneration and neurodegenerative disorders. View Publication

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain,HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies,independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro,HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation,cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies,HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens,in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation,cytokine secretion,and cytotoxic activity in vitro compared to either compound alone. In conclusion,HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner,without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade. View Publication

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines. View Publication

Human embryonic stem (hES) cells show an atypical cell-cycle regulation characterized by a high proliferation rate and a short G1 phase. In fact,a shortened G1 phase might protect ES cells from external signals inducing differentiation,as shown for certain stem cells. It has been suggested that self-renewal and pluripotency are intimately linked to cell-cycle regulation in ES cells,although little is known about the overall importance of the cell-cycle machinery in maintaining ES cell identity. An appealing model to address whether the acquisition of stem cell properties is linked to cell-cycle regulation emerged with the ability to generate induced pluripotent stem (iPS) cells by expression of defined transcription factors. Here,we show that the characteristic cell-cycle signature of hES cells is acquired as an early event in cell reprogramming. We demonstrate that induction of cell proliferation increases reprogramming efficiency,whereas cell-cycle arrest inhibits successful reprogramming. Furthermore,we show that cell-cycle arrest is sufficient to drive hES cells toward irreversible differentiation. Our results establish a link that intertwines the mechanisms of cell-cycle control with the mechanisms underlying the acquisition and maintenance of ES cell identity. View Publication

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4,SOX2,and either cMYC and KLF4 or NANOG and LIN28. Little is known,however,about the mechanisms by which reprogramming occurs,which is in part limited by the low efficiency of conversion. To this end,we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs),demonstrated by gene expression profiles,DNA methylation status,and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts,we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system,we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells,we obtained secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming." View Publication

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies,particularly in view of the diverse methods for derivation and maintenance of these cell lines. However,characterization methods are generally not standardized and many currently used assays are subjective,making dependable and direct comparison of cell lines difficult. In order to address this problem,we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines,derived in two different laboratories using different derivation techniques,as pathogen free,genetically stable,and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control,a crucial element of successful hESC research and clinical translation. View Publication

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system,including a model driven by PDGF-B,H3.3K27M,and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study,an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R,IR,MET,TRKA,TRKB,AURKA,AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM,significant inhibition of proliferation at a concentration of 1.5 μM,as well as inhibition of AKT activation. Interestingly,IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo,systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50,suggesting that inadequate drug delivery may limit in vivo efficacy. In summary,an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG,but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice. View Publication

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals,thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide,the use of human cell-teratogenicity assays has just started to be explored. Herein,a human pluripotent stem cell test (hPST) for identifying teratogens is described,benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically,71 drug-like compounds with known in vivo effects,including thalidomide,were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore,15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally,to assess the suitability of the hPST for high-throughput screens,a small library of 300 kinase inhibitors was tested,demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus,the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles,controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore,aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals,their variability in aggregate size contributes significantly to poor SERS signal reproducibility,which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally,both functionalities should be obtained from a single molecule. Here,we report the use of 3,5-dimercaptobenzoic acid,a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles,and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe,producing very robust,and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters,these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs). View Publication

IntroductionAngelman Syndrome (AS) is characterized in large part by the loss of functional UBE3A protein in mature neurons. A majority of AS etiologies is linked to deletion of the maternal copy of the UBE3A gene and epigenetic silencing of the paternal copy. A common therapeutic strategy is to unsilence the intact paternal copy thereby restoring UBE3A levels. Identifying novel therapies has been aided by a UBE3A-YFP reporter mouse model. This study presents an analogous fluorescent UBE3A reporter system in human cells.MethodsPreviously derived induced Pluripotent Stem Cells (iPSCs) with a Class II large deletion at the UBE3A locus are used in this study. mGL and eGFP are integrated downstream of the endogenous UBE3A using CRISPR/Cas9. These reporter iPSCs are differentiated into 2D and 3D neural cultures to monitor long-term neuronal maturation. Green fluorescence dynamics are analyzed by immunostaining and flow cytometry.ResultsThe reporter is successfully integrated into the genome and reports paternal UBE3A expression. Fluorescence expression gradually reduces with UBE3A silencing in neurons as they mature. Expression patterns also reflect expected responses to molecules known to reactivate paternal UBE3A.DiscussionThis human-cell-based model can be used to screen novel therapeutic candidates,facilitate tracking of UBE3A expression in time and space,and study human-specific responses. However,its ability to restore UBE3A function cannot be studied using this model. Further research in human cells is needed to engineer systems with functional UBE3A to fully capture the therapeutic capabilities of novel candidates. View Publication