Martin Waterfall

Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

Dielectrophoretic Characterisation of Mammalian Cells above 100 MHz

Abstract Dielectrophoresis (DEP) is a label-free technique for the characterization and manipulation of biological particles - such as cells, bacteria and viruses. Many studies have focused on the DEP cross-over frequency fxo1, where cells in a non-uniform electric field undergo a transition from negative to positive DEP. Determination of fxo1 provides a value for the membrane capacitance from the cell diameter, the means to monitor changes in cell morphology and viability, and the information required when devising DEP cell separation protocols. In this paper we describe the first systematic measurements of the second DEP cross-over frequency fxo2 that occurs at much higher frequencies. Theory indicates that fxo2 is sensitive to the internal dielectric properties of a cell, and our experiments on murine myeloma cells reveal that these properties exhibit temporal changes that are sensitive to both the osmolality and temperature of the cell suspending medium.

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Functional analysis of the porcine USP18 and its role during porcine arterivirus replication.

Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV). We have shown that UBP gene is the ortholog of the murine USP18 (Ubp43) gene and the human ubiquitin specific protease 18 (USP18) gene and encodes a biochemically functional de-ubiquitin enzyme which inhibits signalling pathways that lead to IFN-stimulating response element (ISRE) promotor regulation. Furthermore we have demonstrated that overexpression of the porcine USP18 leads to reduced replication and/or growth of PRRSV. Our data contrast with the conclusion of numerous reports demonstrating that USP18-deficient mice are highly resistant to viral and bacterial infections and to oncogenic transformation by BCR-ABL, and highlight USP18 as a potential target gene that promotes reduced replication of PRRSV.

NET23/STING promotes chromatin compaction from the nuclear envelope

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

Effect of neonatal treatment with a GnRH antagonist on development of the cell-mediated immune response in marmosets

PROBLEM: We examined the effect of neonatal treatment with a gonadotropin‐releasing hormone (GnRH) antagonist (antide) on the development of cell‐mediated immunity in male marmosets.

Cellular and humoral immunity in a wild mammal: Variation with age & sex and association with overwinter survival

Abstract Immune defenses are expected to be crucial for survival under the considerable parasite pressures experienced by wild animals. However, our understanding of the association between immunity and fitness in nature remains limited due to both the complexity of the vertebrate immune system and the often‐limited availability of immune reagents in nonmodel organisms. Here, we use methods and reagents developed by veterinary researchers for domestic ungulates on blood samples collected from a wild Soay sheep population, to evaluate an unusually broad panel of immune parameters. Our evaluation included different innate and acquired immune cell types as well as nematode parasite‐specific antibodies of different isotypes. We test how these markers correlate with one another, how they vary with age‐group and sex, and, crucially, whether they predict overwinter survival either within or among demographic groups. We found anticipated patterns of variation in markers with age, associated with immune development, and once these age trends were accounted for, correlations among our 11 immune markers were generally weak. We found that females had higher proportions of naïve T cells and gamma–delta T cells than males, independent of age, while our other markers did not differ between sexes. Only one of our 11 markers predicted overwinter survival: sheep with higher plasma levels of anti‐nematode IgG antibodies were significantly more likely to survive the subsequent high mortality winter, independent of age, sex, or weight. This supports a previous finding from this study system using a different set of samples and shows that circulating antibody levels against ecologically relevant parasites in natural systems represent an important parameter of immune function and may be under strong natural selection. Our data provide rare insights into patterns of variation among age‐ and sex groups in different T‐cell subsets and antibody levels in the wild, and suggest that certain types of immune response—notably those likely to be repeatable within individuals and linked to resistance to ecologically relevant parasites—may be most informative for research into the links between immunity and fitness under natural conditions.

Initial characterisation of adult human ovarian cell populations isolated by DDX4 expression and aldehyde dehydrogenase activity

The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.