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Raman and AFM Integrated Stem Cell Exploration of Differentiation

Periodic Reporting for period 1 - RAISED (Raman and AFM Integrated Stem Cell Exploration of Differentiation)

Reporting period: 2015-05-15 to 2017-05-14

RAISED, Raman and AFM Integrated Stem Cell Exploration of Differentiation, focused on elucidating the mechanisms triggered by external stimulation which drive stem cell fate. The control of stem cell fate via external stimulation is a vital contribution to the advancement of tissue engineering for regenerative medicine. Almost every kind of living cell will respond to a wide range of external stimuli; the direct stimulation of living cells is particularly interesting for the area of tissue engineering as stem cells can be triggered to differentiate from external stimuli such as electrical and mechanical signals. Delivering this signal to stem cells can be done via conductive biomaterials, designed to support the cells and promote the targeted differentiation. In RAISED we focused on understanding the influence of electrical and mechanical stimulation on human mesenchymal stem cells (hMSC) as they undergo osteogenic differentiation on a conductive polymer biomaterial. For the development of new conductive biomaterials for tissue engineering applications, understanding this fundamental relationship between the cells and the material is vital for progression of the field. The main objectives of RAISED were to use non-invasive single cell characterisation techniques, atomic force microscopy (AFM) and Raman micro-spectroscopy. These techniques allow us to measure changes in cellular properties, such as cell stiffness and biochemical changes, which can be correlated with changes in the intracellular structure. Changes in the intracellular structure are an integral part of stem cell restructuring as they differentiate towards different phenotypes for the formation of different tissue types, and hence this approach using non-invasive characterisation allows us to study this restructuring in real time as the stimulus is applied. This approach offers a much more efficient and informative measurements of stem cell response compared to standard biological assays and immunostaining.

The characterisation techniques developed in RAISED revealed that hMSC respond to electrical and mechanical stimulation on a very short time scale with long reaching differentiation consequences. The real-time characterisation measurements on the live hMSC measured near immediate changes in the cell elasticity, a property which is strongly correlated with intracellular cytoskeleton structure. Electrical stimulation disrupted the intracellular structure, resulting in inhibition of osteogenesis; this was correlated with conventional biological assays. These measurements demonstrated the ability to use cell elasticity measurements to predict the long-term stem cell fate in the presence of controlled external stimulation, and can now be applied to a broader approach in optimising electrical stimulation protocols. RAISED also demonstrated that control of human hMSC fate can be modulated via direct electrical stimulation, and the underlying mechanism behind this is the intracellular restructuring.

Throughout RAISED there was significant transfer of knowledge, specifically in training gained in using Raman micro-spectroscopy and biological culture and analysis, and training given in live cell AFM and other highly advanced AFM techniques and conductive polymer electrochemistry. The multidisciplinary environment at the Stevens Group and Imperial College London provided an excellent support for this very multidisciplinary project, and also fantastic opportunity for career development and growth through the interaction with the vast amount of expertise across a broad range of tissue engineering research within the group. Career development was also facilitated through training and courses through the Postdoctoral Development Centre at Imperial College London.
RAISED used AFM force microscopy in combination with a conductive polymer biomaterial platform to observe intracellular restructuring in real time as the cells are exposed to direct electrical stimulation. The measurements involved AFM force spectroscopy to measure the Young’s modulus of individual hMSCs in order to quantify the change in stem cell elasticity. Measurements were performed on both unstimulated and stimulated conductive polymer biomaterials. The passive conductive polymer biomaterials supported hMSC osteogenic differentiation in osteogenic media; this was quantified by qPCR and immunostaining. The hMSC were observed to increase in stiffness as they underwent osteogenic differentiation on unstimulated substrates over a 14 day period. hMSC on stimulated substrates were observed to have disrupted differentiation, as observed through conventional biological assays. This was complementary to the AFM analysis of the stimulated hMSC as their stiffness was observed to immediately decrease upon the application of stimulation.

The inhibitory effect of electrical stimulation on osteogenic differentiation is proposed to be linked to disruption of the actin cytoskeleton; this can be corroborated by the significant decrease in cell elasticity as measured by AFM. For hMSC differentiation, the formation of the actin cytoskeleton into a well organised structure that forms from the nucleus to the focal adhesions of the cell is vital for osteogenic differentiation. Chondrogenic and adipogenic differentiation involves the actin cytoskeleton restructuring around the periphery of the cell. Hence, modulation of the cytoskeleton structure via electrical stimulation may be applied for stem cell fate control if we can provide further stimulus to encourage the hMSC to form the desired cytoskeletal structure. The advantage of electrical stimulation over conventional biochemical techniques to control stem cell fate include a cheaper pathway without expensive growth factors and sera, ability to upscale for higher throughput of cells, and as it is an externally applied physical stimulus it may be more universally applicable to individual patient’s stem cells for tailored stem cell therapy and tissue engineering.
This project delivered a new approach to characterising and understanding intracellular changes during electrical stimulation, an area where there are many publications and innumerable protocols which have not yet elucidated exactly how the electrical stimulation influences stem cell fate. Using the AFM techniques developed in this project we have been able to progress beyond the state of the art in monitoring the live cell response to external stimulation, and shown that the cells will begin responding and restructuring on a very short time frame (minutes). The knowledge gained from this project will go on to help define optimal electrical stimulation protocols, as the technique can produce impactful results much faster than conventional biological techniques which require days and weeks to culture and assess. The ongoing research within the lab now using the information from this project will help further reveal the fundamental mechanisms that are driven by electrical stimulation. This control over stem cell intracellular structure will also allow for further progress in the field of tissue engineering in tailored stem cell therapy for individual patients; a more efficient protocol to produce the desired cell phenotype from a patient’s hMSC (ie osteogenic, chondrogenic) will be very beneficial for providing patient derived cells for regenerative medicine applications.
Schematic of live cell AFM characterisation platform