技术资料

Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments,these three PBMC isolation techniques were compared for cell recovery and viability,PBMC population composition,and cell functionality,aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors,applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures,with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability,assessed by trypan blue exclusion,was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures,for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities,these data may support selection of a specific PBMC isolation technique for downstream analysis. View Publication

We have established an in vitro 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular,we reported that the mature model,entirely constituted of primary cells of human origin,develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable Haemophilus influenzae results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary,we propose our in vitro model as a reliable and potentially customizable system to study mid/long term host-pathogen processes. View Publication

BACKGROUND The Tbx3 transcription factor is over-expressed in breast cancer,where it has been implicated in proliferation,migration and regulation of the cancer stem cell population. The mechanisms that regulate Tbx3 expression in cancer have not been fully explored. In this study,we demonstrate that Tbx3 is repressed by the tumour suppressor miR-206 in breast cancer cells. METHODS Bioinformatics prediction programmes and luciferase reporter assays were used to demonstrate that miR-206 negatively regulates Tbx3. We examined the impact of miR-206 on Tbx3 expression in breast cancer cells using miR-206 mimic and inhibitor. Gene/protein expression was examined by quantitative reverse-transcription-PCR and immunoblotting. The effects of miR-206 and Tbx3 on apoptosis,proliferation,invasion and cancer stem cell population was investigated by cell-death detection,colony formation,3D-Matrigel and tumorsphere assays. RESULTS In this study,we examined the regulation of Tbx3 by miR-206. We demonstrate that Tbx3 is directly repressed by miR-206,and that this repression of Tbx3 is necessary for miR-206 to inhibit breast tumour cell proliferation and invasion,and decrease the cancer stem cell population. Moreover,Tbx3 and miR-206 expression are inversely correlated in human breast cancer. Kaplan-Meier analysis indicates that patients exhibiting a combination of high Tbx3 and low miR-206 expression have a lower probability of survival when compared with patients with low Tbx3 and high miR-206 expression. These studies uncover a novel mechanism of Tbx3 regulation and identify a new target of the tumour suppressor miR-206. CONCLUSIONS The present study identified Tbx3 as a novel target of tumour suppressor miR-206 and characterised the miR-206/Tbx3 signalling pathway,which is involved in proliferation,invasion and maintenance of the cancer stem cell population in breast cancer cells. Our results suggest that restoration of miR-206 in Tbx3-positive breast cancer could be exploited for therapeutic benefit. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

Adipose-derived stem cells (ASCs) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix (ECM) composition and stiffness,migration,and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A,ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225,and MCF10A overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly,ASCs promoted the self-renewal of all cell types except SUM225. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs,IFN$$ and HGF displayed unique actions on epithelial cell hierarchy. IFN$$ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres,whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF,whereas adipocytes expressed higher levels of IFN$$. Since luminal progenitor cells are believed to be prone for transformation,IFN$$ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. IMPLICATIONS This study suggests that the ratio of adipose-derived stem cells to adipocytes influences cancer cell hierarchy,which may impact incidence and progression. View Publication

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS),accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions,differentiated these to functional motor and cortical neurons and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons,decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis,reduced levels of the anti-apoptotic protein Bcl-2,increased endoplasmic reticulum (ER) stress and reduced mitochondrial membrane potential. Furthermore,C9orf72 motor neurons,and also cortical neurons,show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats,which describes a novel pathogenic link between C9orf72 mutations,dysregulation of calcium signalling and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia (FTD). This article is protected by copyright. All rights reserved. View Publication

We asked this question: in normal humans,is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d,each intake for 8-9 d,under strictly controlled,normal metabolic conditions,we measured serum concentrations of 1,25(OH)2D daily,and concentrations of phosphorus hourly throughout a 24-h period,before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but,(d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77,P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range,the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90,P less than 0.001). In the aggregate,the results demonstrate that in normal men,dietary phosphorus throughout a normal range and beyond,can finely regulate the renal production and serum concentration of 1,25(OH)2D,and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus. View Publication

AIM To evaluate the tumor localization and efficacy pH-responsive expansile nanoparticles (eNPs) as a drug delivery system for pancreatic peritoneal carcinomatosis (PPC) modeled in nude rats. METHODS & MATERIALS A Panc-1-cancer stem cell xeno1graft model of PPC was validated in vitro and in vivo. Tumor localization was tracked via in situ imaging of fluorescent eNPs. Survival of animals treated with paclitaxel-loaded eNPs (PTX-eNPs) was evaluated in vivo. RESULTS The Panc-1-cancer stem cell xenograft model recapitulates significant features of PPC. Rhodamine-labeled eNPs demonstrate tumor-specific,dose- and time-dependent localization to macro- and microscopic tumors following intraperitoneal injection. PTX-eNPs are as effective as free PTX in treating established PPC; but,PTX-eNPs result in fewer side effects. CONCLUSION eNPs are a promising tool for the detection and treatment of PPC. View Publication

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury,although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation,respectively,attenuating the activity of the C3/C5 convertases and,consequently,avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study,we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly,FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture,resembling immature MoDCs,lower expression of the maturation marker CD83 and the costimulatory molecules CD40,CD80,and CD86,decreased production of key proinflammatory Th1-cytokines (IL-12,TNF-α,IFN-γ,IL-6,and IL-8),and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover,FH-treated MoDCs show low Ag uptake and,when challenged with LPS,display reduced CCR7 expression and chemotactic migration,impaired CD4(+) T cell alloproliferation,inhibition of IFN-γ secretion by the allostimulated T cells,and,conversely,induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus,this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity,transplantation,and autoimmunity. View Publication

Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase,MLL2 (KMT2B),during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here,we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically,by utilizing dimeric CRISPR RNA-guided nucleases,RFNs (commercially known as the NextGEN™ CRISPR),in combination with an excision-only piggyBac™ transposase,we demonstrate how to generate a point mutation of threonine-542,a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection,and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations� View Publication

BACKGROUND Patient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources,including iPSC lines. However,the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study,we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency. METHODS The IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein,whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates. RESULTS Stem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers,as measured by TEER and fluorescein permeability. However,the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity. CONCLUSION This study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication