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Mechanical loading to direct stem cell differentiation

Final Report Summary - CSKFINGERPRINTS (Mechanical loading to direct stem cell differentiation)

The main goal of this project is to characterize at the single-cell level the differentiation of human
mesenchymal stem cells induced by mechanical loads. OBJECTIVES: This proposal has three main
objectives: (1) to identify early fingerprints of cytoskeletal networks that predict lineage commitment
of hMSC; (2) to characterize cytoskeletal reorganization and early lineage commitment of hMSC
induced by mechanical loads; and (3) to verify rearrangement of chromatin in hMSCs as a result of
mechanical load, in particular its temporal correlation with early cytoskeletal reorganization. WORK PERFORMED: We have created a robust and automated image quantification pipeline that allows us to extract >15 multiplex descriptors of cytoskeletal and nuclear organization for single cell data. In addition, we have optimized custom-built machine learning algorithms to define the developmental trajectory of differentiating cells. These two pipelines now work in series to obtain information of the time-evolution of a population of cells, while still maintaining the single-cell information. Given the complexities of stretching the cells in the agarose constructs, we have rather focused on characterizing the differentiation process of cells cultured on agarose gels of different concentrations and challenged with adipogenic or osteogenic media. Of note, the changes in agarose concentration lead to not only significant changes in substrate stiffness, but even larger changes in substrate viscosity. We have taken advantage of this phenomenon to characterize the effect of substrate viscosity on stem cell differentiation towards adipogenic and osteogenic precursors. Finally, we have also characterized how the changes are transmitted to the cell nucleus and how the nucleus changes its mechanical state, together with the condensation of chromatin, as the cell differentiates.
MAIN RESULTS: We have created a robust framework to identify and characterise evolving cell populations based on cytoskeletal changes. This is based on pipelines fully developed in-house. This has brought the realization that cytoskeletal organization can be used as a reliable biomarker of cellular state and cellular processes. Using this approach, we have characterized cellular changes as cells differentiate towards osteogenic and adipogenic lineages when cultured on substrates of different viscoelasticity. We also observe parallel changes in the mechanical state of the nucleus. As part of this project, we have serendipitously observed that live-cell actin probes (Lifeact in particular), may produce artifacts and significantly alter the very structure they are meant to probe.
IMPACT: The impact of the research carried out is two-fold, and it can be divided into technical advances and new cell biology understanding. On as technical perspective, we have identified the minimum number of cells needed to characterize an evolving cellular population based on cytoskeletal biomarkers. Surprisingly, the number is low, and few hundreds of cells are needed to characterize the whole developmental trajectory of the differentiation process. This figure is important, because it allows us to define what high-throughput truly means. In these terms, throughput is not defined by the amount of data an instrument can generate, but rather the amount of data needed to produce meaningful results using said instrument. Accordingly, in our hands, to achieve high-throughput using cytoskeletal imaging and quantification, less than 500 cells may be needed for a whole experiment. In the perspective of new biological understanding, we have confirmed and expanded the understanding that the mechanical properties of the nucleus and the state of chromatin are modulated by the cytoskeleton. Accordingly, they change during stem cell differentiation probably directed by cytoskeletal changes. Furthermore, we have identified that substrate viscosity plays a key role in helping drive stem cell differentiation, in cells challenged with soluble factors to promote differentiation. This understanding should be taken into account when designing new biomatrices or scaffolds for tissue regeneration strategies. CAREER DEVELOPMENT: The fellow has now a fully established lab (currently 5 PhD students and a postdoc); the first PhD student successfully defended his thesis recently. The fellow has given a number of invited talks world-wide, acts regularly as grant reviewer for important national funding bodies and is now a member of the editoral board of Scientific Reports. TRANSFER OF KNOWLEDGE: We have established multiple collaborations in the host institution and also in the UK to utilize the fellow’s technical expertise to solve open questions in fields related to cancer and aging. Most of those collaborations have led to joint grant submissions (3 successful) or published or under review manuscripts. The transfer of knowledge also includes collaborations with UK-based companies (one SME and 2 corporations). OUTREACH: Dissemination and outreach activities have focused on the promotion of STEM fields to younger individuals, specifically female students at undergraduate and secondary education level. In addition, the fellow is now an elected member of the ‘outreach committee’ within the Royal Microscopical Society.