Development of a central nervous system axonal myelination assay for high throughput screening.

BACKGROUND: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. RESULTS: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. CONCLUSIONS: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

IL-17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells.

Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 have differing effects on the viability and growth of OPCs, however the effects of IL-17A are largely unknown. Primary murine OPCs were stimulated with IL-17A and their viability, proliferation, and maturation were assessed in culture. IL-17A-stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin-specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL-17A. Downstream, IL-17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL-17A exposure stimulates OPCs to mature and participate in the inflammatory response.

Development of a high throughput drug screening assay to identify compounds that protect oligodendrocyte viability and differentiation under inflammatory conditions.

BACKGROUND: Newly proliferated oligodendrocyte precursor cells (OPCs) migrate and surround lesions of patients with multiple sclerosis (MS) and other demyelinating diseases, but fail to differentiate into oligodendrocytes (OLs) and remyelinate remaining viable axons. The abundance of secreted inflammatory factors within and surrounding these lesions likely plays a major inhibitory role, promoting cell death and preventing OL differentiation and axon remyelination. To identify clinical candidate compounds that may protect existing and differentiating OLs in patients, we have developed a high throughput screening (HTS) assay that utilizes purified rat OPCs. RESULTS: Using a fluorescent indicator of cell viability coupled with image quantification, we developed an assay to allow the identification of compounds that promote OL viability and differentiation in the presence of the synergistic inflammatory cytokines, tumor necrosis factor α and interferon-γ. We have utilized this assay to screen the NIH clinical collection library and identify compounds that protect OLs and promote OL differentiation in the presence of these inflammatory cytokines. CONCLUSION: This primary OL-based cytokine protection assay is adaptable for HTS and may be easily modified for profiling of compounds in the presence of other potentially inhibitory molecules found in MS lesions. This assay should be of use to those interested in identifying drugs for the treatment of MS and other demyelinating diseases.

A high throughput drug screening assay to identify compounds that promote oligodendrocyte differentiation using acutely dissociated and purified oligodendrocyte precursor cells.

BACKGROUND: Multiple sclerosis is caused by an autoimmune response resulting in demyelination and neural degeneration. The adult central nervous system has the capacity to remyelinate axons in part through the generation of new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. RESULTS: Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library. We have identified twenty-seven hit compounds which were validated by dose response analysis and the generation of half maximal effective concentration (EC50) values allowed for the ranking of efficacy. The assay identified novel promoters of OL differentiation which we attribute to (1) the incorporation of an OL toxicity pre-screen to allow lowering the concentrations of toxic compounds and (2) the utilization of freshly purified, non-passaged OPCs. These features set our assay apart from other OL differentiation assays used for drug discovery efforts. CONCLUSIONS: This acute primary OL-based differentiation assay should be of use to those interested in screening large compound libraries for the identification of drugs for the treatment of MS and other demyelinating diseases.

Rapid structure-activity and selectivity analysis of kinase inhibitors by BioMAP analysis in complex human primary cell-based models.

Rapid, quantitative methods for characterizing the biological activities of kinase inhibitors in complex human cell systems could allow the biological consequences of differential target selectivity to be monitored early in development, improving the selection of drug candidates. We have previously shown that Biologically Multiplexed Activity Profiling (BioMAP) permits rapid characterization of drug function based on statistical analysis of protein expression data sets from complex primary human cellbased models of disease biology. Here, using four such model systems containing primary human endothelial cells and peripheral blood mononuclear cells in which multiple signaling pathways relevant to inflammation and immune responses are simultaneously activated, we demonstrate that BioMAP analysis can detect and distinguish a wide range of inhibitors directed against different kinase targets. Using a panel of p38 mitogen-activated protein kinase antagonists as a test set, we show further that related compounds can be distinguished by unique features of the biological responses they induce in complex systems, and can be classified according to their induction of shared (on-target) and secondary activities. Statistical comparisons of quantitative BioMAP profiles and analysis of profile features allow correlation of induced biological effects with chemical structure and mapping of biological responses to chemical series or substituents on a common scaffold. Integration of automated BioMAP analysis for prioritization of hits and for structure-activity relationship studies may improve and accelerate the design and selection of optimal therapeutic candidates.

Long-term culture of human embryonic stem cells in feeder-free conditions.

We have demonstrated previously that human embryonic stem (hES) cells possess a characteristic morphologic, antigenic, and molecular profile that can be used to assess the state of ES cells (Carpenter et al., [2004] Dev Dyn 229:243-258). In this manuscript, we have examined the long-term stability of three hES cell lines in feeder-free culture. We demonstrate that the expression of antigens and transcription factors, telomerase activity, telomere length, and karyotype appear stable for all three hES cell lines after continuous culture for over 1 yr. All three lines retained pluripotent differentiation in vitro and in vivo. Although hES cell lines were remarkably stable over the period of analysis, a detailed quantitative analysis of antigen expression by flow cytometry and gene expression by microarray suggested that cell lines show subtle differences in the expression of small subsets of genes upon long-term culture.

Properties of four human embryonic stem cell lines maintained in a feeder-free culture system.

Several laboratories have begun evaluating human ES (hES) cell lines; however, direct comparisons between different hES cell lines have not been performed. We have characterized the properties of four human cell lines maintained in feeder-free culture conditions. Quantitative assessment of surface markers, microarray analysis of gene expression patterns, expression of SOX-2, UTF-1, Rex-1, OCT3/4, CRIPTO, and telomerase activity demonstrated similar patterns in all hES cell lines examined. Undifferentiated hES cells do not respond to neurotransmitters such as acetylcholine, glutamate, and gamma-aminobutyric acid. In addition, the undifferentiated hES cells possess gap junctions. Although similarities in marker expression were observed, allotyping showed that all four lines have a distinct HLA profile, predicting differences in transplantation responses. These data provide the first detailed comparison of different hES cell lines and demonstrate remarkable similarities among lines maintained in identical culture conditions.