| dc.description.abstracteng | Since decades, the differentiation potential of adult human mesenchymal stem cells (hMSCs)
is investigated. They feature the ability for differentiation into various cell types, like
cartilage, fat, nerve, muscle and bone cell lineages. Ten years ago, it has been shown that
physical stimuli in terms of substrate elasticity are sufficient to specifically guide hMSC
differentiation. Key players are contractile stress fibres composed of actin filaments, crosslinkers
and myosin motor-proteins, which generate and transmit forces throughout the
cell. Interestingly, already 24 hours after seeding of hMSCs on polyacrylamide substrates
of defined stiffnesses, distinct stress fibre patterns evolve. These significantly different
cytoskeleton structures serve as early morphological markers in stem cell differentiation.
In this thesis, a massive parallel live-cell imaging set-up was established to record the
dynamics of stress fibre formation under physiological conditions for up to 48 hours. The
cells are kept at 5% CO2 and 37 C. To minimise disturbance of the native acto-myosin
system, we optimized lifeact-TaqRFP transfection of hMSCs and recorded movies on
elastic PAA gels exhibiting Young’s moduli of 1 kPa, 10 kPa and 30 kPa. We minimised
bleaching, by choosing time intervals of ten minutes between two subsequent images. This
provides a good signal-to-noise ratio, while we are not loosing structural information
about stress fibre pattern rearrangement. We found that a resting time after transfection
of 48 instead of 24 hours leads to more reliable results. To robustly detect and track
stress fibres in cells from the long-term live-cell imaging movies, we developed in close
collaboration with mathematicians from the Statistics Department a sophisticated filament
tracking program to gain a deeper understanding of stress fibre formation dynamics in
early stem cell differentiation. We show how the individual patterns develop and whether
the formation processes can be distinguished. We can statistically significantly (99%
confidence) distinguish the development of hMSCs on 1 kPa PAA substrates from hMSCs
on 10 kPa and 30 kPa PAA gels. Cells on 10 kPa and 30 kPa PAA gels are evolving
similarly. However, cells on 30 kPa show a change in migration pattern at 15 hours, which
is reflected by the order parameter and long and short axis development. Starting from
about 15 hours after seeding, cells on 10 kPa PAA gels supersede cells on 30 kPa by order
parameter increase, while cells on 30 kPa catch up stretching. After 24 hours, hMSCs on
10 kPa reach a higher order parameter than cells on 30 kPa PAA gels, but are comparable
in length. In summary, this thesis could show that live-cell imaging with sufficient high
cell numbers yields statistical significant results for primary cells. | de |