The demonstration of induced pluripotency and direct lineage conversion of cells has led to remarkable insights regarding the roles of epigenetic modifications in mediating cell state transitions. However, approaches to elucidate the molecular mechanisms behind induced reprogramming are hindered by the inefficiency of induction and the interplay of reprogramming mechanisms with other cellular processes such as the cell cycle, which leads to variability in induced cell populations and limits the conclusiveness of bulk analyses. Here I propose to develop mass cytometry-based methods to monitor mRNA levels and gene locus-specific epigenetic modifications in single-cells. Thereby, the multi-dimensionality of mass cytometry will enable simultaneous measurements of up to 40 additional single-cell parameters, such as surface markers and intracellular phosphorylation sites. This platform will be used to profile cell populations at specific time-points during reprogramming of human somatic cells into induced pluripotent stem cells. The obtained data will be organized into clusters of similar cell phenotypes resulting in continuous single-cell maps that chart epigenetic changes and accompanying marker expressions during induced reprogramming. These maps will then serve as references to monitor the effects of chromatin-modifying small molecules that affect the efficiency of reprogramming. In combination, these analyses will lead to a more detailed understanding of the hierarchical and dynamic organization of reprogramming and will ideally define gene locus-specific epigenetic events that are rate-limiting for the process. This fine-grained view of reprogramming will then be used to define minimal combinations of early cellular markers that are likely to predict cell fate. In the final stage of the project, long-term single-cell imaging will be used to monitor cells with these previously defined properties to confirm their specific clonal future with absolute certainty.
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