How Do Cells Make Decisions: Engineering Micro- and Nanoenvironments for Cell Migration

Cell migration contributes to cancer metastasis and involves cell adhesion to the extracellular matrix (ECM), force generation through the cell's cytoskeletal, and finally cell detachment. Both adhesive cues from the ECM and soluble cues from neighbouring cells and tissue trigger intracellular signalling pathways that are essential for cell migration. While the machinery of many signalling pathways is relatively well understood, how hierarchies of different and conflicting signals are established is a new area of cellular cancer research. We examine the recent advances in microfabrication, microfluidics, and nanotechnology that can be utilized to engineer micro- and nanoscaled cellular environments. Controlling both adhesive and soluble cues for migration may allow us to decipher how cells become motile, choose the direction for migration, and how oncogenic transformations influences these decision-making processes

Human wharton’s jelly-derived mesenchymal stem cells minimally improve the growth kinetics and cardiomyocyte differentiation of aged murine cardiac c-kit cells in in vitro without rejuvenating effect

Cardiac c-kit cells show promise in regenerating an injured heart. While heart disease commonly affects elderly patients, it is unclear if autologous cardiac c-kit cells are functionally competent and applicable to these patients. This study characterised cardiac c-kit cells (CCs) from aged mice and studied the effects of human Wharton’s Jelly-derived mesenchymal stem cells (MSCs) on the growth kinetics and cardiac differentiation of aged CCs in vitro. CCs were isolated from 4-week- and 18-month-old C57/BL6N mice and were directly co-cultured with MSCs or separated by transwell insert. Clonogenically expanded aged CCs showed comparable telomere length to young CCs. However, these cells showed lower Gata4, Nkx2.5, and Sox2 gene expressions, with changes of 2.4, 3767.0, and 4.9 folds, respectively. Direct co-culture of both cells increased aged CC migration, which repopulated 54.6 ± 4.4% of the gap area as compared to aged CCs with MSCs in transwell (42.9 ± 2.6%) and CCs without MSCs (44.7 ± 2.5%). Both direct and transwell co-culture improved proliferation in aged CCs by 15.0% and 16.4%, respectively, as traced using carboxyfluorescein succinimidyl ester (CFSE) for three days. These data suggest that MSCs can improve the growth kinetics of aged CCs. CCs retaining intact telomere are present in old hearts and could be obtained based on their self-renewing capability. Although these aged CCs with reduced growth kinetics are improved by MSCs via cell–cell contact, the effect is minimal

Targeting Lung Cancer Stem Cells: Research and Clinical Impacts

Lung cancer is the most common cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012. Non-small cell lung cancer (NSCLC), which is one of two types of lung cancer, accounts for 85–90% of all lung cancers. Despite advances in therapy, lung cancer still remains a leading cause of death. Cancer relapse and dissemination after treatment indicates the existence of a niche of cancer cells that are not fully eradicated by current therapies. These chemoresistant populations of cancer cells are called cancer stem cells (CSCs) because they possess the self-renewal and differentiation capabilities similar to those of normal stem cells. Targeting the niche of CSCs in combination with chemotherapy might provide a promising strategy to eradicate these cells. Thus, understanding the characteristics of CSCs has become a focus of studies of NSCLC therapies

Formation of stable Si–O–C submonolayers on hydrogen-terminated silicon(111) under low-temperature conditions

In this letter, we report results of a hydrosilylation carried out on bifunctional molecules by using two different approaches, namely through thermal treatment and photochemical treatment through UV irradiation. Previously, our group also demonstrated that in a mixed alkyne/alcohol solution, surface coupling is biased towards the formation of Si–O–C linkages instead of Si–C linkages, thus indirectly supporting the kinetic model of hydrogen abstraction from the Si–H surface (Khung, Y. L. et al. Chem. – Eur. J. 2014, 20, 15151–15158). To further examine the probability of this kinetic model we compare the results from reactions with bifunctional alkynes carried out under thermal treatment (<130 °C) and under UV irradiation, respectively. X-ray photoelectron spectroscopy and contact angle measurements showed that under thermal conditions, the Si–H surface predominately reacts to form Si–O–C bonds from ethynylbenzyl alcohol solution while the UV photochemical route ensures that the alcohol-based alkyne may also form Si–C bonds, thus producing a monolayer of mixed linkages. The results suggested the importance of surface radicals as well as the type of terminal group as being essential towards directing the nature of surface linkage

Cell migration in the context of immobilised and soluble cues

Cells respond to adhesive and soluble cues in the extracellular matrix by modulating their behaviour including cell adhesion, migration speed and direction. In vivo, cells are exposed to multiple, even conflicting directional cues, however, how cell migration is influenced by multiple spatial cues is still unclear. The effects of adhesive and soluble cues on cell adhesion and migration have often been studied separately. In this thesis, surface-patterning techniques and microfluidics are combined to independently present adhesive and soluble cues. Microcontact printing was used to pattern adhesive cues onto a layer of polyethylene glycol (PEG) that passivated the underlying surfaces. A microfluidic device was employed to deliver a gradient of soluble cues to migrating cells. Migration of Hela cells derived from human cervical cancer and J774 cells, immortalized mouse macrophages, was recorded with live-cell fluorescence microscopy and analysis by single cell tracking. In the first chapter reporting results (Chapter 3), surface passivation and surface patterning of fibronectin via microcontact printing were established and cell migration was observed with a modified wound assay. It is shown that HeLa cells required fibronectin for adhesion and migration and did not migrate into passivated PEG regions of the surface. Only when the cell density on the fibronectin tracks was high did individual HeLa cells migrate into PEG regions. Conversely, J774 cells migrated faster on PEG regions than on fibronectin tracks. The distinct cell morphology indicated that J774 may adopt different migrating modes on fibronectin and PEG surfaces. In Chapter 4, the modified surfaces from Chapter 3 were integrated with a microfluidic device to create a gradient of fetal bovine serum (FBS) or complement 5a (C5a) as a soluble cue for chemotaxis. Hence this chapter describes the role of patterned surfaces and directional cue of soluble gradients on chemotaxis. HeLa cells were found to migrate towards higher concentrations of FBS, but only when a fibronectin track was accessible in that direction. It was concluded that chemotaxis of HeLa cells is strongly adhesion dependent. In contrast, J774 cells migrated faster and more directed towards the source of C5a on fibronectin tracks than on PEG regions, suggesting that chemotaxis of macrophages is enhanced on adhesive cues.In Chapter 5, two competing directional cues were presented to migrating cells. These were (i) gradients of arginine-glycine-aspartic acid (RGD) peptides immobilised onto streptavidin tracks printed on PEG passivated surfaces and (ii) soluble chemoattractant gradient introduced with the microfluidic device used in Chapter 4. When RGD gradients and FBS gradients opposed each other, a greater fraction of HeLa cells was found to migrate towards the source of FBS rather than towards higher concentrations of RGD peptides. Hence for HeLa cells, in the presence of any adhesive cues, soluble cues were dominant in determining the direction of migration. J774 cells on RGD gradients and PEG regions were found to migrate randomly despite the presence of a chemoattractant gradient. In conclusion, combining microcontact printing and microfluidics described in this thesis revealed novel insights of how HeLa and J774 cells integrate multiple spatial cues environment and modulate their migration behaviour accordingly

Fabrication and characterisation of fluidic based memristor sensor for liquid with hydroxyl group

Two types of memristor sensor were fabricated using two different TiO2 deposition methods of sputtering and sol-gel spin coating. The surface morphology of the sensors and the behaviour of the sensors were analysed by using scanning electron microscopy with energy dispersive x-ray system and I-V characterisation system respectively. The sensors were applied with liquid with hydroxyl group to check the capability of this sensor in sensing different concentration of hydroxyl ion inside the liquid. For that purpose, D-glucose liquid with four concentrations of 10 mM, 20 mM, 30 mM and 40 mM were chosen. The liquids dispensed onto the TiO2 surface to act as sensing material. The TiO2 surface was initially covered with polydimethylsiloxane to control the liquid. The sensing capability of the sensors was determined via the current-voltage measurement and off-on resistance ratio. The sensitivity of the sensors was analysed from the off-on resistance ratio analysis. Type II memristor sensor which was fabricated using sol-gel spin coating technique recorded high sensitivity of 120.65 (mM)−1, while Type I sensor fabricated using the sputtering technique recorded low sensitivity of 0.035 (mM)−1. However, SEM-EDX image illustrated that the sputtering technique produced more uniform TiO2 thin film than sol-gel spin coating technique with larger atomic number of oxygen through the sol-gel spin coating technique. This indicates Type II sensor that has large number of oxygen atom produced more reaction with hydroxyl ion inside the liquid. While, Type I sensor produced less reaction compared with Type II and thus produced smaller off-on resistance ratio

Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells

Objective Myocardial infarction remains the number one killer disease worldwide. Cellular therapy using cardiac c-kit cells (CCs) are capable of regenerating injured heart. Previous studies showed mesenchymal stem cell-derived (MSC) extracellular matrices can provide structural support and are capable of regulating stem cell functions and differentiation. This study aimed to evaluate the effects of human MSC-derived matrices for CC growth and differentiation. Methods Human Wharton's Jelly-derived MSCs were cultured in ascorbic acid supplemented medium for 14 days prior to decellularisation using two methods. 1% SDS/Triton X-100 (ST) or 20 mM ammonia/Triton X-100 (AT). CCs isolated from 4-week-old C57/BL6N mice were cultured on the decellularised MSC matrices, and induced to differentiate into cardiomyocytes in cardiogenic medium for 21 days. Cardiac differentiation was assessed by immunocytochemistry and qPCR. All data were analysed using ANOVA. Results In vitro decellularisation using ST method caused matrix delamination from the wells. In contrast, decellularisation using AT improved the matrix retention up to 30% (p < 0.05). This effect was further enhanced when MSCs were cultured in cardiogenic medium, with a matrix retention rate up to 90%. CCs cultured on cardiogenic MSC matrix (ECMcardio), however, did not significantly improve its proliferation after 3 days (p < 0.05), but the viability of CCs was augmented to 67.2 ± 0.7% after 24-h exposure to H2O2 stress as compared to 42.9 ± 0.5% in control CCs (p < 0.05). Furthermore, CCs cultured on cardiogenic MSC matrices showed 1.7-fold up-regulation in cardiac troponin I (cTnI) gene expression after 21 days (p < 0.05). Conclusion Highest matrix retention can be obtained by decellularization using Ammonia/Triton-100 in 2-D culture. ECMcardio could rescue CCs from exogenous hydrogen peroxide and further upregulated the cardiac gene expressions, offering an alternate in vitro priming strategy to precondition CCs which could potentially enhance its survival and function after in vivo transplantation