Temporal Analysis of Human Mesenchymal Stem Cells under External Stimulation with Q-AFM

Human mesenchymal stem cells (hMSCs) are gaining significant interest due to their capabilities of self-renewal and differentiation into various cell types such as osteoblast, adipocyte, chondrocyte and neuron.1–6 These characteristics make hMSCs a promising cell source for tissue engineering aimed at repairing or replacing damaged tissues. Various approaches were developed to regulate stem cell fate, including mechanical forces and chemical treatments. However, the mechanisms underlying cell fate regulation are still unclear, resulting in reduced effectiveness in controlling cell fate and limiting clinical applications. Among external stimulations on cell fate regulation, electrical stimulation (E-STIM) stands out due to its potential to yield different cell types, possibly using the same device with varying inputs. Research has primarily focused on final outcomes of stem cell differentiation, often neglecting the temporal response of cells under stimulation. Limited studies have reported cytoskeleton reorganisation and activation of mechanotransduction in response to E-STIM.7,8 Understanding these temporal dynamics is critical for uncovering the pathways leading to cell fate commitment and designing effective stimulation treatments. The cytoskeleton is crucial in supporting cell structure, signal transmission, and influencing stem cell behaviour, including shape, and fate decisions.9,10 When stem cells are cultured on substrates with varying stiffness, cytoskeletal remodelling occurs. Cell stiffness, largely determined by cytoskeleton, eventually aligns with the substrate stiffness, guiding cells to differentiate into specific types, such as osteoblasts on substrates mimicking bone collagen stiffness (25-40 kPa).11 Fluorescence imaging, commonly used to observe cytoskeletal structures, requires cell fixation, preventing real-time observation of same cells. Other technique like particle tracking maintains cell viability but involves invasive procedures that could alter cell behaviour. Quantitative atomic force microscopy (Q-AFM) offers a non-invasive alternative for characterising live cell mechanics. Q-AFM allows simultaneous cell mechanics and topography characterisation, correlating mechanical properties with intracellular structures. It operates faster and provides higher resolution images than traditional AFM, making it ideal for real-time monitoring cell dynamics. Additionally, FluidFM enables single-cell manipulation such as extraction using hollow tips connected to microfluidic system. By applying negative pressure, fractions of cytosol can be extracted for molecule analysis while maintaining cell viability. This capability allows for monitoring gene expression changes in real-time, providing insights into cell response mechanisms. This thesis aimed to investigate hMSCs behaviour under external stimulation. By integrating stimulation and Q-AFM measurement, we monitored changes in cytoskeletal structure and cell mechanics during and post-stimulation. Developing protocols for cell extraction with FluidFM could enable comprehensive analysis of single-cell behaviour in response to external stimulation, advancing our understanding of the mechanisms governing stem cell fate. After developing protocols for Q-AFM measurements on hMSCs and a customised data processing program for fitting Young's modulus, in collaboration with Dr. Joseph Berry, we correlated Q-AFM measurements with fluorescent imaging. Major features observed in Q-AFM topographical images corresponded to cytoskeletal structures, specifically actin filaments. Young's modulus mappings highlighted features corresponding to actin structures observed in the topographical images, suggesting that cell stiffness was primarily contributed by actin stiffness. To further demonstrate Q-AFM’s capabilities for real-time intracellular measurements, we applied various cytoskeleton disruptors and monitored cytoskeletal structure and cell mechanics. 1 µg/mL Cytochalasin D (CytD) significantly disrupted cytoskeletal network and reduced cell stiffness by 22%. Upon CytD removal, cells began to rebuild their cytoskeletal network, with average stiffness returning to pre-treatment values. This was the first study to monitor changes in intracellular structures and mechanics in single cells with chemical treatment. Next, we conducted two collaborative projects to demonstrate that Q-AFM could be coupled with existing external stimulation devices to monitor the effects of physical stimulation on stem cells. Firstly, in collaboration with Prof. Leslie Yeo, 10 MHz surface acoustic wave (SAW) was applied to hMSCs for 8 minutes. The cytoskeleton alignment towards the cell's original elongation direction was observed immediately post stimulation. Stress fibre remodelling resulted in around 1.5-fold cell stiffening at 65 minutes post-stimulation. Another device used was confined microchannels designed by Asst. Prof. Andrew Holle’s from National University of Singapore. The microchannels (3 and 10 µm width) were modified to fit into 35 mm cell dishes for Q-AFM scanning. Cells became softer after emerging from the channels, as the narrow space broke down the cytoskeleton structure. The narrower the channel, the more significant the breakdown effect. This was the first study quantifying cell mechanics changes with confinement stimulation. Next, we designed our own E-STIM device that fit Q-AFM measurements to monitor cell behaviour with E-STIM. The charge was directly injected into the cells via an electrode made of conductive polymer Polypyrrole. Electrical signals of 0.005V, 0.3V, 0.5V at 1Hz were applied to hMSCs for 1 hour. With the low charge 0.005V, fully adhered hMSCs showed an increase or no significant change in Young's modulus, along with cytoskeleton realignment and stress fibre formation. At 0.3V, thicker stress fibres formed with less aligned cytoskeleton, resulting in overall unchanged cell stiffness. When applying a 0.5V signal, the Young's modulus of all the scanned cells decreased, along with a decrease in the amount of stress fibres. Finally, we developed protocols for intracellular manipulation via FluidFM. A cell-impermeable DNA stain propidium iodide, was successfully injected into nuclear regions of hMSCs using pressure +200 mbar for 20 seconds. The injected cells exhibited red fluorescence. Due to temporary system malfunction, the extraction process was not explored. The primary goal was to extract fractions of cytosol for downstream molecule analysis while maintaining cell viability. This would enable real-time monitoring of gene expression changes with E-STIM, helping comprehensively understand the intracellular response and provide guidance for fine-tuning E-STIM parameters to achieve precise cell fate control. In summary, we established a platform that allows monitoring cell responses under external stimulations. High-resolution topographical images and mechanics mappings can be obtained within 30 minutes using Q-AFM. With the integrated FluidFM system, gene expression changes in single cells can be monitored, providing deeper understandings of the mechanisms behind the external stimulation effects on stem cells. This platform has facilitated wide ranges of collaborations, from cell mechanics measurement to intracellular manipulation, aiding in understanding stem cell behaviour and regulating stem cell commitment for tissue engineering applications.<p></p>