Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Super-orbital re-entry in Australia : laboratory measurement, simulation and flight observation

There are large uncertainties in the aerothermodynamic modelling of super-orbital re-entry which impact the design of spacecraft thermal protection systems (TPS). Aspects of the thermal environment of super-orbital re-entry flows can be simulated in the laboratory using arc- and plasma jet facilities and these devices are regularly used for TPS certification work [5]. Another laboratory device which is capable of simulating certain critical features of both the aero and thermal environment of super-orbital re-entry is the expansion tube, and three such facilities have been operating at the University of Queensland in recent years[10]. Despite some success, wind tunnel tests do not achieve full simulation, however, a virtually complete physical simulation of particular re-entry conditions can be obtained from dedicated flight testing, and the Apollo era FIRE II flight experiment [2] is the premier example which still forms an important benchmark for modern simulations. Dedicated super-orbital flight testing is generally considered too expensive today, and there is a reluctance to incorporate substantial instrumentation for aerothermal diagnostics into existing missions since it may compromise primary mission objectives. An alternative approach to on-board flight measurements, with demonstrated success particularly in the ‘Stardust’ sample return mission, is remote observation of spectral emissions from the capsule and shock layer [8]. JAXA’s ‘Hayabusa’ sample return capsule provides a recent super-orbital reentry example through which we illustrate contributions in three areas: (1) physical simulation of super-orbital re-entry conditions in the laboratory; (2) computational simulation of such flows; and (3) remote acquisition of optical emissions from a super-orbital re entry event.

Are the ‘cave’ minerals archerite (K,NH4)H2PO4 and biphosphammite (K,NH4)H2PO4 identical? A molecular structural study

The molecular structure of the mineral archerite ((K,NH4)H2PO4) has been determined and compared with that of biphosphammite ((NH4,K)H2PO4). Raman spectroscopy and infrared spectroscopy has been used to characterise these ‘cave’ minerals. Both minerals originated from the Murra-el-elevyn Cave, Eucla, Western Australia. The mineral is formed by the reaction of the chemicals in bat guano with calcite substrates. Raman and infrared bands are assigned to H2PO4-, OH and NH stretching vibrations. The Raman band at 981 cm-1 is assigned to the HOP stretching vibration. Bands in the 1200 to 1800 cm-1 region are associated with NH4+ bending modes. The molecular structure of the two minerals appear to be very similar, and it is therefore concluded that the two minerals are identical.

Molecular Structure of the mineral woodhouseite CaAl3(PO4,SO4)2(OH)6

The mineral woodhouseite CaAl3(PO4,SO4)2(OH)6 is a hydroxy phosphate-sulphate mineral belonging to the beudantite subgroup of alunites, and has been characterised by Raman spectroscopy, complimented with infrared spectroscopy. Bands at various wavenumbers were assigned to the different vibrational modes of woodhouseite, which were then associated to the molecular structure of the mineral. Bands were primarily assigned to phosphate and sulphate stretching and bending modes. Two symmetric stretching modes for both phosphate and sulphate supported the concept of non-equivalent phosphate and sulphate units in the mineral structure. Bands in the OH stretching region enabled hydrogen bond distances to be calculated.

Large-Eddy Simulation of physiological pulsatile non-newtonian flow in a channel with double constriction

Physiological pulsatile flow in a 3D model of arterial double stenosis, using the modified Power-law blood viscosity model, is investigated by applying Large Eddy Simulation (LES) technique. The computational domain has been chosen is a simple channel with biological type stenoses. The physiological pulsation is generated at the inlet of the model using the first four harmonics of the Fourier series of the physiological pressure pulse. In LES, a top-hat spatial grid-filter is applied to the Navier-Stokes equations of motion to separate the large scale flows from the subgrid scale (SGS). The large scale flows are then resolved fully while the unresolved SGS motions are modelled using the localized dynamic model. The flow Reynolds numbers which are typical of those found in human large artery are chosen in the present work. Transitions to turbulent of the pulsatile non-Newtonian along with Newtonian flow in the post stenosis are examined through the mean velocity, wall shear stress, mean streamlines as well as turbulent kinetic energy and explained physically along with the relevant medical concerns.

Stochastic models for regulatory networks of the genetic toggle switch

Bistability arises within a wide range of biological systems from the λ phage switch in bacteria to cellular signal transduction pathways in mammalian cells. Changes in regulatory mechanisms may result in genetic switching in a bistable system. Recently, more and more experimental evidence in the form of bimodal population distributions indicates that noise plays a very important role in the switching of bistable systems. Although deterministic models have been used for studying the existence of bistability properties under various system conditions, these models cannot realize cell-to-cell fluctuations in genetic switching. However, there is a lag in the development of stochastic models for studying the impact of noise in bistable systems because of the lack of detailed knowledge of biochemical reactions, kinetic rates, and molecular numbers. In this work, we develop a previously undescribed general technique for developing quantitative stochastic models for large-scale genetic regulatory networks by introducing Poisson random variables into deterministic models described by ordinary differential equations. Two stochastic models have been proposed for the genetic toggle switch interfaced with either the SOS signaling pathway or a quorum-sensing signaling pathway, and we have successfully realized experimental results showing bimodal population distributions. Because the introduced stochastic models are based on widely used ordinary differential equation models, the success of this work suggests that this approach is a very promising one for studying noise in large-scale genetic regulatory networks.