技术资料

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research,diagnostic,industrial vaccine,and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success,a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins,carbohydrates,DNA,and haptens) (see Note 1). Furthermore,in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein. View Publication

In a prior study on electronic cigarette (EC) refill fluids,Cinnamon Ceylon was the most cytotoxic of 36 products tested. The purpose of the current study was to determine if high cytotoxicity is a general feature of cinnamon-flavored EC refill fluids and to identify the toxicant(s) in Cinnamon Ceylon. Eight cinnamon-flavored refill fluids,which were screened using the MTT assay,varied in their cytotoxicity with most being cytotoxic. Human embryonic stem cells were generally more sensitive than human adult pulmonary fibroblasts. Most products were highly volatile and produced vapors that impaired survival of cells in adjacent wells. Cinnamaldehyde (CAD),2-methoxycinnamaldehyde (2MOCA),dipropylene glycol,and vanillin were identified in the cinnamon-flavored refill fluids using gas chromatography-mass spectrometry and high-pressure liquid chromatography (HPLC). When authentic standards of each chemical were tested using the MTT assay,only CAD and 2MOCA were highly cytotoxic. The amount of each chemical in the refill fluids was quantified using HPLC,and cytotoxicity correlated with the amount of CAD/product. Duplicate bottles of the same product were similar,but varied in their concentrations of 2MOCA. These data show that the cinnamon flavorings in refill fluids are linked to cytotoxicity,which could adversely affect EC users. ?? 2013 Elsevier Ltd. View Publication

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases,showing feasibility and safety (and some efficacy) of this approach. However,protocols for isolation and expansion of donor MSCs vary widely between these trials,which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production,which should be evidence-based,regulatory authority-compliant,of good medical practice grade,cost-effective,and clinically practical,so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy,which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods,including materials and protocols for isolation and expansion,are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy. View Publication

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation,to develop cell culture models of liver disease,and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally,hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery,determination of pharmaceutical-induced hepatotoxicity,and evaluation of idiosyncratic drug-drug interactions. Here,we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. View Publication

BACKGROUND & AIMS Reduced generation of all-trans retinoic acid (RA) by CD103(+) intestinal dendritic cells (DCs) is linked to intestinal inflammation in mice. However,the role of RA in intestinal inflammation in humans is unclear. We investigated which antigen-presenting cells (APCs) produce RA in the human intestine and whether generation of RA is reduced in patients with Crohn's disease (CD). METHODS Ileal and colonic tissues were collected from patients with CD during endoscopy or surgery,and healthy tissues were collected from subjects who were undergoing follow-up because of rectal bleeding,altered bowel habits,or cancer (controls). Cells were isolated from the tissue samples,and APCs were isolated by flow cytometry. Retinaldehyde dehydrogenase (RALDH) activity was assessed by Aldefluor assay,and ALDH1A expression was measured by quantitative real-time polymerase chain reaction. Macrophages were derived by incubation of human blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS CD103(+) and CD103(-) DCs and CD14(+) macrophages from healthy human intestine had RALDH activity. Although ALDH1A1 was not expressed by DCs,it was the predominant RALDH enzyme isoform expressed by intestinal CD14(+) macrophages and their putative precursors,CD14(+) monocytes. RALDH activity was up-regulated in all 3 populations of APCs from patients with CD; in CD14(+) macrophages,it was associated with local induction of ALDH1A1 expression. Blocking of RA receptor signaling during GM-CSF-mediated differentiation of monocytes into macrophages down-regulated CD14 and HLA-DR expression and reduced the development of tumor necrosis factor $$-producing inflammatory macrophages. CONCLUSIONS RA receptor signaling promotes differentiation of human tumor necrosis factor $$-producing inflammatory macrophages in vitro. In vivo,more CD14(+) macrophages from the intestinal mucosa of patients with CD than from controls are capable of generating RA,which might increase the inflammatory phenotype of these cells. Strategies to reduce the generation of RA by CD14(+) macrophages could provide new therapeutic options for patients with CD. View Publication

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy,drug screening,physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods,techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results,we adopted a polycistronic cassette encoding Thomson's cocktail OCT4,NANOG,SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones,based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes,ONSL and OCT4,SOX2,KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly,in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM,we indeed observed a remarkable synergy,yielding a reprogramming efficiency of textgreater2%. We present here a drastic improvement of the reprogramming efficiency,which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore,non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency. View Publication

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive,result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs,we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate,formulated as a hypertonic solution,gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium,retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together,this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology,high levels (textgreater80%) of pluripotency markers OCT4,SSEA-4,TRA-1-60 andTRA-1-81,a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers,both in vivo and in vitro. View Publication

Over 60 bromodomains belonging to proteins with very different functions have been identified in humans. Several of them interact with acetylated lysine residues,leading to the recruitment and stabilization of protein complexes. The bromodomain and extra-terminal domain (BET) proteins contain tandem bromodomains which bind to acetylated histones and are thereby implicated in a number of DNA-centered processes,including the regulation of gene expression. The recent identification of inhibitors of BET and non-BET bromodomains is one of the few examples in which effective blockade of a protein-protein interaction can be achieved with a small molecule. This has led to major strides in the understanding of the function of bromodomain-containing proteins and their involvement in diseases such as cancer and inflammation. Indeed,BET bromodomain inhibitors are now being clinically evaluated for the treatment of hematological tumors and have also been tested in clinical trials for the relatively rare BRD-NUT midline carcinoma. This review gives an overview of the newest developments in the field,with a focus on the biology of selected bromodomain proteins on the one hand,and on reported pharmacological inhibitors on the other,including recent examples from the patent literature. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells,especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system,high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1??106cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers,both in static culture and stirred bioreactors,induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea,and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment. ?? 2014. View Publication

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein,we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine,a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas,and their intracardiac injection led to prominent colonization of various organs (bone,lung,liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition),accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity,confirming the tumor suppressor function of this gene in this particular model. Interestingly,we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases,with significant reduction in protein levels as compared to normal tissues. Overall,our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis. View Publication

A priori,a common receptor induced in tumor microvessels,cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis,invasiveness,and CSC anoikis resistance and survival,could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor,DEspR,(formerly named Dear,Chr.4q31.2) supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness,CD133+CSCs,and polar angiogenic features. Unlike normal tissue,DEspR is detected in PDAC and GBM microvessels,tumor cells,and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis,invasiveness,CSC-survival and anoikis resistance in vitro,and decreased Panc1-CSC and U87-CSC xenograft tumor growth,vasculo-angiogenesis and invasiveness in nude(nu/nu) rats,suggesting that DEspR activation would coordinate these tumor progression events. As an accessible,cell-surface 'common receptor coordinator',DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma. View Publication