Periodic Report Summary 1 - 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: During the reporting period, we have optimized cell culture, staining, transfection and imaging conditions. Furthermore, we have also developed novel image analysis methods to track in a fully quantifiable way the changes in the cytoskeleton and the nucleus as cells differentiate. The method allows us to obtain 18 cytoskeletal and nuclear morphological parameters for each cell probed within a large cell sample. We then combine these parameters using machine learning techniques, to define the temporal trajectories undergone by the population of cells. In addition, we have also designed implemented and characterize a method to apply controlled strain and compression onto cells cultured in 2.5D conditions. MAIN RESULTS: We have characterized cellular changes as cells differentiate towards osteogenic and adipogenic lineages. Accordingly, we have uncovered that the differentiation process is not monotonic, but it rather contains short-lived states in which cells resemble neither their undifferentiated nor their differentiated counterparts. In brief, differentiating cells have to reach certain milestones while proceeding towards their committed lineage. EXPECTED FINAL RESULTS: We will characterize the differentiation of stem cells when subjected to mechanical loads. Taking advantage of our single-cell, we will focus on assessing whether particular loading regimes lead to faster or more robust differentiation. IMPACT: Considering our findings so far, future bioreactor-based strategies to achieve fast and robust differentiation should provide cells with a dynamically-changing stimulus (either soluble or mechanical), taking into account the differentiation stage displayed by the cell population at a given time. CAREER DEVELOPMENT: This project has, so far, contributed to a published manuscript and given rise to a manuscript currently under review. In addition, based on the preliminary data obtained so far, we have secured other research grants to work on parallel projects using the methods developed here. TRANSFER OF KNOWLEDGE: We have established collaborations in the host institution 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 (2 successful so far). 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.
Greg Dow, (EU Pre-award officer)
Tel.: +44020 7882 2569
Tel.: +44020 7882 2569
Record Number: 187604 / Last updated on: 2016-08-22