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• W4212773994 abstract "What factors and conditions control stem cell differentiation is currently one of the major questions in biomedical research. What factors ultimately influence the cells’ decision is unclear but it has been shown, that stem cells differentiate reversibly through mechanical clues alone, expressing specific tissue markers as early as several days after plating on a substrate with similar stiffness. Stem cells cultured on elastic substrates with different Young’s moduli E exhibit distinct structure formation of their cytoskeletal organization already within the first 24 hours. Whether this is a solely mechanical response or is also accompanied and impacted by early transcriptome changes, is an open question. To assess this, I investigate and quantify both structural changes of the cell’s cytoskeleton and focal adhesions as well as transcriptomic alterations in cells seeded on polyacrylamide (PAA) gels of different stiffness. For a quantitative analysis of the cytoskeleton, I further developed the FilamentSensor software for near real-time stress fiber detection. Besides runtime improvement, I added features for the assessment of the mechanical active area and multi-cell separation. I quantified the experimental effects of the lot-to-lot variation, various culture conditions, actin vs. NMM2a staining, and intrinsic variations of mesenchymal stem cells (hMSCs) as they are primary cells. For a data set of 690 time-lapse movies of live hMSCs on different stiffnesses, I showed that cell area, aspect ratio, filament number per cell, and filament width are reliable parameters to distinguish differentiating hMSC on substrates of different stiffness. To enhance the understanding of the mechanical interplay between cell and matrix, I used Metal-Induced and Förster Resonance Energy Transfer (MIET) to understand stress fiber arrangement in Z and the Focal Adhesion Filament Cross-correlation Kit (FAFCK), a novel functionality of the FilamentSensor, to investigate the correlation of stress fibers and focal adhesions. To investigate the effect of substrate stiffness on the transcriptomic level, I first enucleated cells to rule out de novo transcription and second did full transcriptome RNA-Sequencing of hMSCs after 24 hours on different substrates as well as qRT-PCR of selected targets for multiple time points. Cells seeded on PAA gels show up-regulation in ECM constituent secretion and stress response, but down-regulation in proliferation pathways. The down-regulation of proliferation is more pronounced in higher passage numbers." @default.
• W4212773994 created "2022-02-24" @default.
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• W4212773994 date "2022-02-21" @default.
• W4212773994 modified "2023-09-27" @default.
• W4212773994 title "Multi-parameter assessment of mechano-sensitivity driven differentiation of human mesenchymal stem cells" @default.
• W4212773994 doi "https://doi.org/10.53846/goediss-8377" @default.
• W4212773994 hasPublicationYear "2022" @default.
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