Mesenchymal stem cells, neural lineage potential, heparan sulfate proteoglycans and the matrix

Along with the tri-lineage of bone, cartilage and fat, human mesenchymal stem cells (hMSCs) retain neural lineage potential. Multiple factors have been described that influence lineage fate of hMSCs including the extracellular microenvironment or niche. The niche includes the extracellular matrix (ECM) providing structural composition, as well as other associated proteins and growth factors, which collectively influence hMSC stemness and lineage specification. As such, lineage specific differentiation of MSCs is mediated through interactions including cell–cell and cell–matrix, as well as through specific signalling pathways triggering downstream events. Proteoglycans (PGs) are ubiquitous within this microenvironment and can be localised to the cell surface or embedded within the ECM. In addition, the heparan sulfate (HS) and chondroitin sulfate (CS) families of PGs interact directly with a number of growth factors, signalling pathways and ECM components including FGFs, Wnts and fibronectin. With evidence supporting a role for HSPGs and CSPGs in the specification of hMSCs down the osteogenic, chondrogenic and adipogenic lineages, along with the localisation of PGs in development and regeneration, it is conceivable that these important proteins may also play a role in the differentiation of hMSCs toward the neuronal lineage. Here we summarise the current literature and highlight the potential for HSPG directed neural lineage fate specification in hMSCs, which may provide a new model for brain damage repair.

Heparan sulfate proteoglycans and human breast cancer epithelial cell tumorigenicity

Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1–4) and glypicans (GPC1–6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression.

Poly(2-oxazoline) hydrogels as next generation three dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell-cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells towards the ultimate aim of high throughput screening of anti-cancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).

Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions.

Are in vitro estimates of cell diffusivity and cell proliferation rate sensitive to assay geometry?

Cells respond to various biochemical and physical cues during wound–healing and tumour progression. In vitro assays used to study these processes are typically conducted in one particular geometry and it is unclear how the assay geometry affects the capacity of cell populations to spread, or whether the relevant mechanisms, such as cell motility and cell proliferation, are somehow sensitive to the geometry of the assay. In this work we use a circular barrier assay to characterise the spreading of cell populations in two different geometries. Assay 1 describes a tumour–like geometry where a cell population spreads outwards into an open space. Assay 2 describes a wound–like geometry where a cell population spreads inwards to close a void. We use a combination of discrete and continuum mathematical models and automated image processing methods to obtain independent estimates of the effective cell diffusivity, D, and the effective cell proliferation rate, λ. Using our parameterised mathematical model we confirm that our estimates of D and λ accurately predict the time–evolution of the location of the leading edge and the cell density profiles for both assay 1 and assay 2. Our work suggests that the effective cell diffusivity is up to 50% lower for assay 2 compared to assay 1, whereas the effective cell proliferation rate is up to 30% lower for assay 2 compared to assay 1.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

A multi-hierarchical motorway ramp signalling strategy

Ramp signalling is an access control for motorways, in which a traffic signal is placed at on-ramps to regulate the rate of vehicles entering the motorway and thus to preserve the motorway capacity. In general, ramp signalling algorithms fall into two categories: local control and coordinated control by their effective scope. Coordinated ramp signalling strategies make use of measurements from the entire motorway network to operate individual ramp signals for the optimal performances at the network level. This study proposes a multi-hierarchical strategy for coordinated ramp signalling. The strategy is structured in two layers. At the higher layer with a longer update interval, coordination group is assembled and disassembled based on the location of high-risk breakdown flow. At the lower layer with a shorter update interval, individual ramps are hired to serve the coordination and are also released based on the prevailing congestion level on the ramp. This strategy is modelled and applied to the northbound Pacific Motorway micro-simulation platform (AIMSUN). The simulation results show an effective congestion mitigation of the proposed strategy.

Simulation of plant cell shrinkage during drying : A SPH–DEM approach

Plant based dried food products are popular commodities in global market where much research is focused to improve the products and processing techniques. In this regard, numerical modelling is highly applicable and in this work, a coupled meshfree particle-based two-dimensional (2-D) model was developed to simulate micro-scale deformations of plant cells during drying. Smoothed Particle Hydrodynamics (SPH) was used to model the viscous cell protoplasm (cell fluid) by approximating it to an incompressible Newtonian fluid. The visco-elastic characteristic of the cell wall was approximated to a Neo-Hookean solid material augmented with a viscous term and modelled with a Discrete Element Method (DEM). Compared to a previous work [H. C. P. Karunasena, W. Senadeera, Y. T. Gu and R. J. Brown, Appl. Math. Model., 2014], this study proposes three model improvements: linearly decreasing positive cell turgor pressure during drying, cell wall contraction forces and cell wall drying. The improvements made the model more comparable with experimental findings on dried cell morphology and geometric properties such as cell area, diameter, perimeter, roundness, elongation and compactness. This single cell model could be used as a building block for advanced tissue models which are highly applicable for product and process optimizations in Food Engineering.

Breast cancer stem cells and epithelial mesenchymal plasticity : implications for chemoresistance

Tumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is being increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can purportedly differentiate to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to de-differentiate to acquire cancer stem attributes, which also impart chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making it a possible generic therapeutic target in cancer biology. Using breast cancer (BrCa) as an example, we summarise here the current understanding of CSCs, the role of EMP in cancer biology - especially in CSCs and different molecular subtypes, and the implications this has for current and future cancer treatment strategies.

Progress in epithelial-mesenchymal transition research

The field of research of epithelial-mesenchymal transitions, EMT, and its reverse, mesenchymal-epithelial transitions, MET, has expanded very rapidly indeed from its beginnings, heralded by Professor Betty Hay in the 1970s and 1980s. This expansion has involved the realisation that the EMT was not just an interesting phenomenon of early developmental morphogenetic cell behaviour, but bore remarkable resemblance to clinically crucial pathological events in cancer invasion. Not surprisingly, this discipline soon became numerically dominant in the EMT publication field. Simultaneously, the EMT concept has been extended to normal physiological wound healing. Exploration revealed that these resemblances were more than skin deep: the same sets of growth factors, receptors, transcription factors, epigenetic marks and signalling pathways turned up repeatedly in EMTs and METs in a variety of contexts, both pathological and normal. This molecular genetic research in turn uncovered similarities of the EMT signature to that of fibrosis, a set of diseases which is of enormous clinical importance, rivalling that of cancer. Most recently, and more surprisingly, the EMT signature has shown considerable similarity to that found in stem cell and cancer stem cell biology.

Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Automatic classification of human epithelial type 2 cell indirect immunofluorescence images using cell pyramid matching

This paper describes a novel system for automatic classification of images obtained from Anti-Nuclear Antibody (ANA) pathology tests on Human Epithelial type 2 (HEp-2) cells using the Indirect Immunofluorescence (IIF) protocol. The IIF protocol on HEp-2 cells has been the hallmark method to identify the presence of ANAs, due to its high sensitivity and the large range of antigens that can be detected. However, it suffers from numerous shortcomings, such as being subjective as well as time and labour intensive. Computer Aided Diagnostic (CAD) systems have been developed to address these problems, which automatically classify a HEp-2 cell image into one of its known patterns (eg. speckled, homogeneous). Most of the existing CAD systems use handpicked features to represent a HEp-2 cell image, which may only work in limited scenarios. We propose a novel automatic cell image classification method termed Cell Pyramid Matching (CPM), which is comprised of regional histograms of visual words coupled with the Multiple Kernel Learning framework. We present a study of several variations of generating histograms and show the efficacy of the system on two publicly available datasets: the ICPR HEp-2 cell classification contest dataset and the SNPHEp-2 dataset.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.