Mcleod, Claire Marie
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PublicationSingle Cell Molecular Heterogeneity In Musculoskeletal Differentiation(2017-01-01) Mcleod, Claire MarieMesenchymal stem cells (MSCs) display substantial cell-to-cell variation that manifests across many aspects of cell phenotype and complicates the use of MSCs in regenerative applications. However, most conventional assays measure MSC properties in bulk and, as a consequence, mask this cell-to-cell variation. To better understand MSC heterogeneity and its underlying mechanisms, we quantitatively assessed MSC phenotype within the context of chondrogenesis, amongst clonal populations and single cells. Clonal MSCs differed in their contractility, ability to transmit extracellular strain the nucleus, capacity to form cartilage-like matrix, and transcriptomic signature. RNA FISH measurements of single cell gene expression found that both primary chondrocytes and chondrogenically-induced MSCs showed substantial mRNA expression heterogeneity. Surprisingly, variation in differentiation marker transcript levels only weakly associated with cartilage-like matrix production at the single cell level. This finding suggested that, although canonical markers have very clear functional roles in differentiation and matrix formation, their instantaneous mRNA abundance is only tenuously linked to the chondrogenic phenotype and matrix accumulation at the single cell level. One possible explanation for the apparent disconnect between gene and protein expression is that mRNA and protein exhibit different temporal dynamics. Using stochastic models of single cell behavior, we explored the impact of transcriptional stochasticity and temporal matrix dynamics on the perceived relationship between single cell mRNA and protein abundance. Simulations suggested that considering recent temporal fractions of protein (vs. total protein) increased the correlation between mRNA and protein abundance, and illustrated that mRNA stability was a crucial determinant of the timescale over which any such correlation persisted. Experimentally, non-canonical amino acid tagging was used to visualize and quantify temporal fractions of nascent extracellular matrix with high fidelity. The organization and temporal dynamics of the proteinaceous matrix depended on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Both chondrocytes and MSCs demonstrated marked cell-to-cell heterogeneity in nascent matrix production, consistent with model predictions. Ongoing work aims to combine these experimental measurements of nascent protein expression with instantaneous measures of mRNA abundance to better understand the mRNA-protein relationship, and to harness this understanding to improve regenerative therapies.