Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells

The growth and differentiation of mesenchymal stem cells is controlled by various growth factors, the activities of which can be modulated by heparan sulfates. We have previously underscored the necessity of sulfated glycosaminoglycans for the FGF-2-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of heparan sulfate to cultures of primary rat MSCs stimulates their proliferation leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation and osteogenic differentiation of rMSCs when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. Furthermore, we show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth resulting in the cells being unable to reach the critical density necessary to induce differentiation. Interestingly, blocking FGFR1 signaling in post-confluent osteogenic cultures significantly increased calcium deposition. Taken together our data clearly suggests that FGFR1 signaling plays an important role during osteogenic differentiation, firstly by stimulating cell growth that is closely followed by an inhibitory affect once the cells have reached confluence. It also underlines the importance of HS as a co-receptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Addressing issues in sparseness, ecological bias and formulation of the adjacency matrix in Bayesian spatio-temporal analysis of disease counts

The main objective of this PhD was to further develop Bayesian spatio-temporal models (specifically the Conditional Autoregressive (CAR) class of models), for the analysis of sparse disease outcomes such as birth defects. The motivation for the thesis arose from problems encountered when analyzing a large birth defect registry in New South Wales. The specific components and related research objectives of the thesis were developed from gaps in the literature on current formulations of the CAR model, and health service planning requirements. Data from a large probabilistically-linked database from 1990 to 2004, consisting of fields from two separate registries: the Birth Defect Registry (BDR) and Midwives Data Collection (MDC) were used in the analyses in this thesis. The main objective was split into smaller goals. The first goal was to determine how the specification of the neighbourhood weight matrix will affect the smoothing properties of the CAR model, and this is the focus of chapter 6. Secondly, I hoped to evaluate the usefulness of incorporating a zero-inflated Poisson (ZIP) component as well as a shared-component model in terms of modeling a sparse outcome, and this is carried out in chapter 7. The third goal was to identify optimal sampling and sample size schemes designed to select individual level data for a hybrid ecological spatial model, and this is done in chapter 8. Finally, I wanted to put together the earlier improvements to the CAR model, and along with demographic projections, provide forecasts for birth defects at the SLA level. Chapter 9 describes how this is done. For the first objective, I examined a series of neighbourhood weight matrices, and showed how smoothing the relative risk estimates according to similarity by an important covariate (i.e. maternal age) helped improve the model’s ability to recover the underlying risk, as compared to the traditional adjacency (specifically the Queen) method of applying weights. Next, to address the sparseness and excess zeros commonly encountered in the analysis of rare outcomes such as birth defects, I compared a few models, including an extension of the usual Poisson model to encompass excess zeros in the data. This was achieved via a mixture model, which also encompassed the shared component model to improve on the estimation of sparse counts through borrowing strength across a shared component (e.g. latent risk factor/s) with the referent outcome (caesarean section was used in this example). Using the Deviance Information Criteria (DIC), I showed how the proposed model performed better than the usual models, but only when both outcomes shared a strong spatial correlation. The next objective involved identifying the optimal sampling and sample size strategy for incorporating individual-level data with areal covariates in a hybrid study design. I performed extensive simulation studies, evaluating thirteen different sampling schemes along with variations in sample size. This was done in the context of an ecological regression model that incorporated spatial correlation in the outcomes, as well as accommodating both individual and areal measures of covariates. Using the Average Mean Squared Error (AMSE), I showed how a simple random sample of 20% of the SLAs, followed by selecting all cases in the SLAs chosen, along with an equal number of controls, provided the lowest AMSE. The final objective involved combining the improved spatio-temporal CAR model with population (i.e. women) forecasts, to provide 30-year annual estimates of birth defects at the Statistical Local Area (SLA) level in New South Wales, Australia. The projections were illustrated using sixteen different SLAs, representing the various areal measures of socio-economic status and remoteness. A sensitivity analysis of the assumptions used in the projection was also undertaken. By the end of the thesis, I will show how challenges in the spatial analysis of rare diseases such as birth defects can be addressed, by specifically formulating the neighbourhood weight matrix to smooth according to a key covariate (i.e. maternal age), incorporating a ZIP component to model excess zeros in outcomes and borrowing strength from a referent outcome (i.e. caesarean counts). An efficient strategy to sample individual-level data and sample size considerations for rare disease will also be presented. Finally, projections in birth defect categories at the SLA level will be made.

Comparison of temporal processing and motion perception in emmetropes and myopes

While spatial determinants of emmetropization have been examined extensively in animal models and spatial processing of human myopes has also been studied, there have been few studies investigating temporal aspects of emmetropization and temporal processing in human myopia. The influence of temporal light modulation on eye growth and refractive compensation has been observed in animal models and there is evidence of temporal visual processing deficits in individuals with high myopia or other pathologies. Given this, the aims of this work were to examine the relationships between myopia (i.e. degree of myopia and progression status) and temporal visual performance and to consider any temporal processing deficits in terms of the parallel retinocortical pathways. Three psychophysical studies investigating temporal processing performance were conducted in young adult myopes and non-myopes: (1) backward visual masking, (2) dot motion perception and (3) phantom contour. For each experiment there were approximately 30 young emmetropes, 30 low myopes (myopia less than 5 D) and 30 high myopes (5 to 12 D). In the backward visual masking experiment, myopes were also classified according to their progression status (30 stable myopes and 30 progressing myopes). The first study was based on the observation that the visibility of a target is reduced by a second target, termed the mask, presented quickly after the first target. Myopes were more affected by the mask when the task was biased towards the magnocellular pathway; myopes had a 25% mean reduction in performance compared with emmetropes. However, there was no difference in the effect of the mask when the task was biased towards the parvocellular system. For all test conditions, there was no significant correlation between backward visual masking task performance and either the degree of myopia or myopia progression status. The dot motion perception study measured detection thresholds for the minimum displacement of moving dots, the maximum displacement of moving dots and degree of motion coherence required to correctly determine the direction of motion. The visual processing of these tasks is dominated by the magnocellular pathway. Compared with emmetropes, high myopes had reduced ability to detect the minimum displacement of moving dots for stimuli presented at the fovea (20% higher mean threshold) and possibly at the inferior nasal retina. The minimum displacement threshold was significantly and positively correlated to myopia magnitude and axial length, and significantly and negatively correlated with retinal thickness for the inferior nasal retina. The performance of emmetropes and myopes for all the other dot motion perception tasks were similar. In the phantom contour study, the highest temporal frequency of the flickering phantom pattern at which the contour was visible was determined. Myopes had significantly lower flicker detection limits (21.8 ± 7.1 Hz) than emmetropes (25.6 ± 8.8 Hz) for tasks biased towards the magnocellular pathway for both high (99%) and low (5%) contrast stimuli. There was no difference in flicker limits for a phantom contour task biased towards the parvocellular pathway. For all phantom contour tasks, there was no significant correlation between flicker detection thresholds and magnitude of myopia. Of the psychophysical temporal tasks studied here those primarily involving processing by the magnocellular pathway revealed differences in performance of the refractive error groups. While there are a number of interpretations for this data, this suggests that there may be a temporal processing deficit in some myopes that is selective for the magnocellular system. The minimum displacement dot motion perception task appears the most sensitive test, of those studied, for investigating changes in visual temporal processing in myopia. Data from the visual masking and phantom contour tasks suggest that the alterations to temporal processing occur at an early stage of myopia development. In addition, the link between increased minimum displacement threshold and decreasing retinal thickness suggests that there is a retinal component to the observed modifications in temporal processing.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

Temporal-based support vector machines for facial expression recognition

When classifying a signal, ideally we want our classifier to trigger a large response when it encounters a positive example and have little to no response for all other examples. Unfortunately in practice this does not occur with responses fluctuating, often causing false alarms. There exists a myriad of reasons why this is the case, most notably not incorporating the dynamics of the signal into the classification. In facial expression recognition, this has been highlighted as one major research question. In this paper we present a novel technique which incorporates the dynamics of the signal which can produce a strong response when the peak expression is found and essentially suppresses all other responses as much as possible. We conducted preliminary experiments on the extended Cohn-Kanade (CK+) database which shows its benefits. The ability to automatically and accurately recognize facial expressions of drivers is highly relevant to the automobile. For example, the early recognition of “surprise” could indicate that an accident is about to occur; and various safeguards could immediately be deployed to avoid or minimize injury and damage. In this paper, we conducted initial experiments on the extended Cohn-Kanade (CK+) database which shows its benefits.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population

Emerging evidence supports that prostate cancer originates from a rare sub-population of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (-T3) is one of the vitamin-E constituents which have been shown to have anticancer effects against a wide-range of human cancers. Recently, we have reported that -T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that -T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that -T3 can down-regulate the expression of prostate CSC markers (CD133/CD44) in androgen independent (AI) prostate cancer cell lines (PC-3 & DU145), as evident from western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by -T3 treatment. In addition, pre-treatment of PC-3 cells with -T3 was found to suppress tumor initiation ability of the cells. More importantly, while CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to -T3 treatment as the CD133-depleted population. Our data suggest that -T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Dealing with temporal inconsistency in automated computer forensic profiling

Computer profiling is the automated forensic examination of a computer system in order to provide a human investigator with a characterisation of the activities that have taken place on that system. As part of this process, the logical components of the computer system – components such as users, files and applications - are enumerated and the relationships between them discovered and reported. This information is enriched with traces of historical activity drawn from system logs and from evidence of events found in the computer file system. A potential problem with the use of such information is that some of it may be inconsistent and contradictory thus compromising its value. This work examines the impact of temporal inconsistency in such information and discusses two types of temporal inconsistency that may arise – inconsistency arising out of the normal errant behaviour of a computer system, and inconsistency arising out of deliberate tampering by a suspect – and techniques for dealing with inconsistencies of the latter kind. We examine the impact of deliberate tampering through experiments conducted with prototype computer profiling software. Based on the results of these experiments, we discuss techniques which can be employed in computer profiling to deal with such temporal inconsistencies.

Description and evaluation of a Newton-based electronic appetite rating system for temporal tracking of appetite in human subjects

This study assessed the reliability and validity of a palm-top-based electronic appetite rating system (EARS) in relation to the traditional paper and pen method. Twenty healthy subjects [10 male (M) and 10 female (F)] — mean age M=31 years (S.D.=8), F=27 years (S.D.=5); mean BMI M=24 (S.D.=2), F=21 (S.D.=5) — participated in a 4-day protocol. Measurements were made on days 1 and 4. Subjects were given paper and an EARS to log hourly subjective motivation to eat during waking hours. Food intake and meal times were fixed. Subjects were given a maintenance diet (comprising 40% fat, 47% carbohydrate and 13% protein by energy) calculated at 1.6×Resting Metabolic Rate (RMR), as three isoenergetic meals. Bland and Altman's test for bias between two measurement techniques found significant differences between EARS and paper and pen for two of eight responses (hunger and fullness). Regression analysis confirmed that there were no day, sex or order effects between ratings obtained using either technique. For 15 subjects, there was no significant difference between results, with a linear relationship between the two methods that explained most of the variance (r2 ranged from 62.6 to 98.6). The slope for all subjects was less than 1, which was partly explained by a tendency for bias at the extreme end of results on the EARS technique. These data suggest that the EARS is a useful and reliable technique for real-time data collection in appetite research but that it should not be used interchangeably with paper and pen techniques.

The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized stsytems for temporal tracking of appetite ratings.

This present paper reviews the reliability and validity of visual analogue scales (VAS) in terms of (1) their ability to predict feeding behaviour, (2) their sensitivity to experimental manipulations, and (3) their reproducibility. VAS correlate with, but do not reliably predict, energy intake to the extent that they could be used as a proxy of energy intake. They do predict meal initiation in subjects eating their normal diets in their normal environment. Under laboratory conditions, subjectively rated motivation to eat using VAS is sensitive to experimental manipulations and has been found to be reproducible in relation to those experimental regimens. Other work has found them not to be reproducible in relation to repeated protocols. On balance, it would appear, in as much as it is possible to quantify, that VAS exhibit a good degree of within-subject reliability and validity in that they predict with reasonable certainty, meal initiation and amount eaten, and are sensitive to experimental manipulations. This reliability and validity appears more pronounced under the controlled (but more arti®cial) conditions of the laboratory where the signal : noise ratio in experiments appears to be elevated relative to real life. It appears that VAS are best used in within-subject, repeated-measures designs where the effect of different treatments can be compared under similar circumstances. They are best used in conjunction with other measures (e.g. feeding behaviour, changes in plasma metabolites) rather than as proxies for these variables. New hand-held electronic appetite rating systems (EARS) have been developed to increase reliability of data capture and decrease investigator workload. Recent studies have compared these with traditional pen and paper (P&P) VAS. The EARS have been found to be sensitive to experimental manipulations and reproducible relative to P&P. However, subjects appear to exhibit a signi®cantly more constrained use of the scale when using the EARS relative to the P&P. For this reason it is recommended that the two techniques are not used interchangeably