Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

The University of Auckland

Automated image-based and biochemical assays have greatly increased throughput for quantifying cell numbers in in vitro studies. However, it has been more difficult to automate the counting of specific cell types with complex morphologies in mixed cell cultures. We have developed a fully automated, fast, accurate and objective method for the quantification of primary human GFAP-positive astrocytes and CD45-positive microglia from images of mixed cell populations. This method, called the complex cell count (CCC) assay, utilizes a combination of image processing and analysis operations from MetaMorph (Version 6.2.6, Molecular Devices). The CCC assay consists of four main aspects: image processing with a unique combination of morphology filters; digital thresholding; integrated morphometry analysis; and a configuration of object standards. The time needed to analyze each image is 1.82s. Significant correlations have been consistently achieved between the data obtained from CCC analysis and manual cell counts. This assay can quickly and accurately quantify the number of human astrocytes and microglia in mixed cell culture and can be applied to quantifying a range of other cells/objects with complex morphology in neuroscience research.

High throughput quantification of cells with complex morphology in mixed cultures.

Glial scar formation occurs after virtually any injury to the brain. The migration of astrocytes into regions of brain injury underlies the formation of the glial scar. The exact role of the glial scar has yet to be elucidated, although it is likely to impair brain recovery. Understanding astrocyte migration is fundamental to understanding the formation of the glial scar. We have used human astrocytes (NT2A cells), derived from human NT2/D1 precursor cells to study astrocyte migration using an in vitro scratch wound model. Time-lapse microscopy and bromodeoxyuridine labeling revealed that the astrocytes migrated rather than proliferated across the scratch. Time course immunocytochemical studies showed that scratching human astrocytes induced the activation (phosphorylation) of ERK 1/2 at 10 min after scratch. The MEK 1/2 inhibitor U0126 inhibited both the ERK 1/2 phosphorylation and the migration of the astrocytes across the wound after scratch. Thus, the migration of human astrocytes after injury is partly initiated by activation of the MEK-ERK signalling pathway.

Extracellular signal-regulated kinase involvement in human astrocyte migration.

Microglia and astrocytes play vital roles in normal human brain function and in neurological disorders. To study their physiological and pathological roles it is desirable to establish in vitro systems that are derived from the adult human brain. Although several groups have successfully cultured cells from the human brain, the composition of these cultures remains controversial. Using morphological criteria, immunocytochemical analysis and a BrdU incorporation assay we demonstrate the presence of poorly proliferative microglia and astrocytes in cultures derived from epilepsy biopsy tissue. In addition, we characterized a third cell type as fibronectin and prolyl 4-hydroxylase immunopositive fibroblast-like cells, which are highly proliferative and become the predominant cell type after successive sub-culturing. Therefore, although cultures from adult human brain tissue provide an excellent resource for studying human glial cells, careful consideration must be given to their cellular composition when performing studies using these methods.

Cellular composition of human glial cultures from adult biopsy brain tissue.

Mutant protein aggregates are an important biomarker in Huntington's and other neurodegenerative diseases however their quantification has typically relied on manual imaging and counting, or cell-free assays, which do not allow for concurrent analysis of cell viability. Here we describe four automated high throughput image analysis methods, developed using Metamorph software, to quantify mutant huntingtin aggregates in a cellular context. Imaging of aggregate-forming cells was also automated, using a Discovery-1 automated fluorescence microscope. All four analysis methods measured aggregate formation accurately in relation to manual counting, but with differing throughput. Our in-house PolyQ assay gave the highest throughput, processing images at 0.31 s per image. The Cell Scoring assay gave lower throughput, at 19.5s per image, but offered accurate quantification of the proportion of cells which formed aggregates, without bias from cell death. These image analysis tools provide rapid and objective alternatives to manual counting in studies of aggregate formation, to facilitate the discovery of drugs to treat Huntington's and related neurodegenerative diseases.

High throughput quantification of mutant huntingtin aggregates.

Epigenetics is a rapidly growing field and holds great promise for a range of human diseases, including brain disorders such as Rett syndrome, anxiety and depressive disorders, schizophrenia, Alzheimer disease and Huntington disease. This review is concerned with the pharmacology of epigenetics to treat disorders of the epigenome whether induced developmentally or manifested/acquired later in life. In particular, we will focus on brain disorders and their treatment by drugs that modify the epigenome. While the use of DNA methyl transferase inhibitors and histone deacetylase inhibitors in in vitro and in vivo models have demonstrated improvements in disease-related deficits, clinical trials in humans have been less promising. We will address recent advances in our understanding of the complexity of the epigenome with its many molecular players, and discuss evidence for a compromised epigenome in the context of an ageing or diseased brain. We will also draw on examples of species differences that may exist between humans and model systems, emphasizing the need for more robust pre-clinical testing. Finally, we will discuss fundamental issues to be considered in study design when targeting the epigenome.

Pharmacology of epigenetics in brain disorders.

There is increasing demand for automated image analysis of cell nuclei to be fast, objective and informative. Here, we have developed a high content analysis method for quantifying histone acetylation within any given population of cells. To demonstrate the utility of this method we quantified the effect of valproic acid (VPA) on histone H3 acetylation levels in SK-N-SH cells, a human neuroblastomal cell line. VPA, commonly used for treatment of bipolar disorder and epilepsy, has also been shown to act as a histone deacetylase inhibitor (HDACi), and to maintain the N-terminals of susceptible histones in an acetylated and transcriptionally active state. The Discovery-1™ (Molecular Devices) platform was used for automated image acquisition of immunolabelled cells. Multiple parameters of labelled nuclei were analysed in 1.82 s per image using the built-in count nuclei assay from MetaMorph™ (Molecular Devices) image analysis software. Data were presented in two forms: summary graphs or heterogeneity profiles using frequency distributions within GraphPad Prism (SmartDrawNet). Results showed that VPA increased histone H3 acetylation in a concentration- and time-dependent manner in SK-N-SH cells. The same analysis was shown to accurately quantify histone acetylation changes in human tissue sections also. Trichostatin A, a known HDACi was used to validate VPA action. Western blotting was used to validate the specificity of the antibodies. Overall these data demonstrate that this novel method for quantifying average treatment effects and the heterogeneity within any given population of cells, is fast, reproducible and can be applied to many different cellular contexts (immunocyto- and immunohisto-chemistry).

High content analysis of histone acetylation in human cells and tissues.

Tissue microarrays are commonly used to evaluate disease pathology however methods to automate and quantify pathological changes are limited.

Pritika Narayan

School of Biological Sciences,
Biomedical Imaging Research Unit,
Faculty of Medical and Health Sciences,
Biomedical Imaging Research Unit, Faculty of Medical and Health Sciences

The University of Auckland

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Pritika Narayan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Biological Sciences,Biomedical Imaging Research Unit,Faculty of Medical and Health Sciences,Biomedical Imaging Research Unit, Faculty of Medical and Health Sciences

The University of Auckland

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

School of Biological Sciences,
Biomedical Imaging Research Unit,
Faculty of Medical and Health Sciences,
Biomedical Imaging Research Unit, Faculty of Medical and Health Sciences