Development of a screening assay for surrogate markers of CHK1 inhibitor-induced cell cycle release

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.     

The derivation of clinical-grade human embryonic stem cell lines

The pluripotent nature of human embryonic stem cells (hESC) has attracted great interest in using them as a source of cells or tissue in cell therapy. However, in order to be used in regenerative medicine, the pluripotent hESC lines should be established and propagated according to good manufacturing practice quality requirements. The cultures should be animal substance free in order to exclude the risk of infections and immunogenity. They should also be genetically and epigenetically normal. The detailed molecular mechanisms of their pluripotency are still not defined. Using human feeder cells, a medium containing only human proteins, the mechanical isolation of the inner cell mass and mechanical passaging of hESC, is a safe option until a functional defined medium containing physiological concentrations of regulatory factors is available.     

The generation of GLP-grade human embryonic stem cell banks from four clinical-grade cell lines for preclinical research

The Singapore Stem Cell Bank has generated human embryonic stem cell banks from clinical-grade cell lines ESI-017, ESI-035, ESI-049, and ESI-053. All banks were prepared and characterized according to principles of Good Laboratory Practice for quality assurance. Importantly, each cell line has clearly documented and approved ethical provenance and meets recognized standards for performance and safety. The banks are intended to facilitate the translation of stem cell research to clinical medicine by enabling early phase research and development with high-quality, low-cost cells that are also available as clinical-grade stocks.     

Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine

Live attenuated influenza vaccines (LAIVs) targeting seasonal influenza are produced in embryonated eggs and formulated as a trivalent preparation of three live attenuated vaccine strains, one A(H1N1) strain, one A(H3N2) strain and one type B strain. In this study, we describe an egg-based potency assay for estimating the 50% Egg infectious dose (EID50) of individual strains in the trivalent preparation by selective neutralisation of two strains and then estimating the infective titres of the non-neutralised strain. The test is highly specific, and no cross interference of heterologous antisera is observed in the estimation of individual titres. Individual strains with titres in the range of 6.5-7.0 log EID50 per 0.5 ml show intra-assay and inter-assay coefficients of variance ranging from 1.25% to 2.95%. This assay was developed to establish a simple, reliable and inexpensive egg-based assay for estimating the potency of individual strains in a trivalent preparation.     

Low angiogenic potency induced by the implantation of ex vivo expanded CD117(+) stem cells

Ex vivo expansion of stem cells might be a feasible method of resolving the problem of limited cell supply in cell-based therapy. The implantation of expanded CD34(+) endothelial progenitor cells has the capacity to induce angiogenesis. In this study, we tried to induce angiogenesis by implanting expanded CD117(+) stem cells derived from mouse bone marrow. After 2 wk of culture with the addition of several growth factors, the CD117(+) stem cells expanded approximately 20-fold and had an endothelial phenotype with high expression of CD34 and vascular endothelial-cadherin. However, >70% of these ex vivo expanded cells had a senescent phenotype by beta-galactosidase staining, and their survival and incorporation were poor after implantation into the ischemic limbs of mice. Compared with the PBS injection only, the microvessel density and the percentage of limb blood flow were significantly higher after the implantation of 2 x 10(5) freshly collected CD117(+) cells (P < 0.01) but not after the implantation of 2 x 10(5) expanded CD117(+) cells (P > 0.05). These data indicate that ex vivo expansion of CD117(+) stem cells has low potency for inducing therapeutic angiogenesis, which might be related to the cellular senescence during ex vivo expansion.     

Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA

As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.     

Characterization of a single reporter-gene potency assay for T-cell-dependent bispecific molecules

ABSTRACT

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients’ T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.     

Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum

Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.     

The human tumor stem cell assay revisited

The human tumor stem cell assay (HTSCA) is a bilayer soft agar system for growing fresh human tumor specimens in vitro to determine drug sensitivity and improve our understanding of tumor biology. Recent clinical correlations of 60% accuracy for predicting a positive clinical response and a 90% accuracy for predicting a lack of response to therapeutic agents suggest promising clinical usefulness. However, the clinician should be aware of the assay's inherent pitfalls, such as heterogeneity of the tumor specimen, inability to obtain pure single-cell suspensions, low cloning efficiency, unusual drug dose-dependent survival curves, uncertain validity of in vitro pharmacology, non-standardized criteria for in vitro sensitivity, and the variability of in vitro results. A brief summary of the concepts, potential, and limitations of this assay are discussed.     

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis

Background  

Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis.     

Development of assay system for immunoglobulin production regulatory factors using whole cell cultures of mouse splenocytes

We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, beta-lactoglobulin, alpha-casein, and beta-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.     

Application of methods for viral clearance in stem cell production

Regenerative medicine therapies will allow in the future the transplant of cells of human origin in some diseases that until now have been incurable. The assurance of the safety and quality, especially from a microbiological point of view, is very important for these therapeutic products. Depending on the starting material, there are several sources of pathogen presence, mainly human viruses. Also, the use of feeders of animal origin as layers in which the stem cells can grow may permit the transmission of animal pathogens to these cells. However, cell sources are limited due to the low availability of spare in vitro fecundation human embryos and the low rate of success in the derivation of human stem cell lines. Thus, in several cases, it will be necessary to evaluate the possibility of removing or inactivating these microorganisms. In this paper, we summarize the main methods of viral clearance and we have provided an overview of the main features taking into account in the viral clearance techniques.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

Planarian stem cells specify fate yet retain potency during the cell cycle

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Xeno-free chondrogenesis of bone marrow mesenchymal stromal cells: towards clinical-grade chondrocyte production

Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.     

Quantitative turbidimetric assay for potency evaluation of colchicine-like drugs.

A modified Shelanski method has been applied to quantitatively compare cochicine-like drugs with regard to their inhibition of the rate of rat brain tubulin polymerisation $\text{in vitro}$. The potency of six known microtubule-disrupting drugs was in increasing order: griseofulvin < colchicine < podophyllotoxin < rotenone < vinblastine < vincristine. Two other drugs which have been claimed to interfere with microtubules, melatonin and isopropyl-n-phenylcarbamate were inactive. Methyl benzimidazol-2-yl-carbamate showed only a marginal effect. The usefulness of the method in screening colchicine-like drugs is discussed.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.