The advent of induced pluripotent stems cells (iPSCs) has created unprecedented access to primary cell types such as cardiomyocytes and neurons, which may lead to the next-generation of patient-specific disease models and more effective therapeutics. Unfortunately, the iPS reprogramming and differentiation process produces a heterogeneous mixture of cell types, required cell sorting and purification for testing or therapy. In particular, how the mRNA and protein expression in cells determines the evolution of cell lineage over time is currently unclear at the single cell level. We propose to adapt the ?nanostraw? platform developed initially developed in the Melosh lab for intracellular delivery to non-destructively sample cytosolic proteins and mRNA over an extended period of time (1-3 weeks). This platform can scale from individual cells to thousands of cells in parallel, depending on the desired application. The technique will first be assessed using cell lines as proof of principle, with quantitative statistical evaluation of the performance of the platform. Subsequently we will measure somatic iPSCs during differentiation into cardiomyocytes over a 3-week period. The extracted proteins and mRNA will then be analyzed to provide insights into the longitudinal proteins and mRNA concentrations inside the cell and how these relate to the terminal cell phenotype. The goal of this R21 proposal is to validate and quantitatively assess the performance of this system as applied to the differentiation of iPSC cells into cardiomyocytes. Toward this end, we have constructed a team of both engineering and cardiomyocyte iPSC differentiation expertise. If successful, this work will form the foundational technology for non-destructively monitoring intracellular contents, particularly for understanding dynamic cell function changes such as reprogramming and differentiation.
The advent of human induced pluripotent stem cells has generated significant interest among biologists and clinicians for access to a variety of autologous cell types, however these techniques are still held back by large cell phenotype heterogeneity and low efficiency. Our team will develop a nanotechnology platform that can non-destructively sample and analyze the cytosolic contents of a particular cell or group of cells over an extended period of time. We will then quantitatively assess the performance of the platform, culminating in following the intracellular protein and mRNA concentrations during induced pluripotent stem cell differentiation into a cardiomyocyte cell.
