TY - BOOK
T1 - Viscoelastic and dynamic properties of embryonic stem cells
AU - Ritter, Christine
PY - 2017
Y1 - 2017
N2 - Stem cells are often referred to as the ‘holy grail’ of regenerative medicine, because they possessthe ability to develop into any cell type. The use of stem cells within medicine is currently limited bythe effectivity of differentiation and cell reprogramming protocols, making it therefore imperative tounderstand stem cells’ differentiation mechanisms better. Studies have shown that mechanical cuescan have an influence on stem cell fate decision. However, in order to understand the reaction of stemcells to mechanical input, one should first investigate and understand the mechanical properties ofthe cells themselves. In this thesis, the viscoelastic properties of mouse embryonic stem cells primedeither toward the epiblast (Epi) or the primitive endoderm (PrE) lineage were investigated.Optical tweezers were used to measure the fluctuations of endogenous lipid granules and therebydraw conclusions on the viscoelasticity of the cells themselves. A stem cell line with a sensitive reporterfor the primitive endoderm, Hhex, was used. The optical tweezers were incorporated into aconfocal microscope, thereby allowing the determination of the state of the cells by their fluorescentsignal. The fluctuations of lipid granules both within the nucleus and in the cytoplasm of Epi- andPrE-primed cells were measured for 3 s and the microrheological scaling exponent a was extractedfrom the power spectrum of the recorded time series. Actin was disrupted with latrunculin B andthe effect of the disruption on the cells’ viscoelasticity was determined. To assess the effect of actindisruption on gene expression, time-lapse microscopy was employed to follow single, LatB treated,PrE-primed cells over a course of 24h, and qPCR was used to determine the effect on the expressionof Nanog, a pluripotency marker, and Gata6, another marker for the primitive endoderm. The underlyingdiffusive process was determined by means of the ensemble and time averaged mean squareddisplacement, the amplitude scatter, higher order moments and the velocity autocorrelation func-tion.It was found that PrE-primed cells were significantly more elastic than Epi-primed cells. In both celltypes, the nucleus was more elastic than the cytoplasm. After actin disruption, both populations becamesignificantly more viscous. The viscoelasticity of the nucleus of Epi-primed cells did not changeupon depolymerizaƟon of acƟn. The periphery of Epi-primed cells, as well as the nucleus and the peripheryof PrE-primed cells became more viscous upon actin disruption. While 24 h time-lapse imagingconfirmed a significant drop in Hhex levels of actin disrupted PrE-primed cells, this result could notbe exactly confirmed with qPCR. Furthermore, the underlying diffusive process was determined to becontinuous time random walk (CTRW).Upon exciting pluripotency, changes occur in the nucleus of stem cells. Chromatin remodeling,the recruitment of lamin A to the nucleoskeleton and stiffening of the cells were reported changescaused by differentiation. These modifications could explain the difference between Epi- and PrEprimedcells. Disruption of actin by LatB treatment indicated that the differences between the twopopulations could be caused by different actin levels. As actin had been associated with chromatin.
AB - Stem cells are often referred to as the ‘holy grail’ of regenerative medicine, because they possessthe ability to develop into any cell type. The use of stem cells within medicine is currently limited bythe effectivity of differentiation and cell reprogramming protocols, making it therefore imperative tounderstand stem cells’ differentiation mechanisms better. Studies have shown that mechanical cuescan have an influence on stem cell fate decision. However, in order to understand the reaction of stemcells to mechanical input, one should first investigate and understand the mechanical properties ofthe cells themselves. In this thesis, the viscoelastic properties of mouse embryonic stem cells primedeither toward the epiblast (Epi) or the primitive endoderm (PrE) lineage were investigated.Optical tweezers were used to measure the fluctuations of endogenous lipid granules and therebydraw conclusions on the viscoelasticity of the cells themselves. A stem cell line with a sensitive reporterfor the primitive endoderm, Hhex, was used. The optical tweezers were incorporated into aconfocal microscope, thereby allowing the determination of the state of the cells by their fluorescentsignal. The fluctuations of lipid granules both within the nucleus and in the cytoplasm of Epi- andPrE-primed cells were measured for 3 s and the microrheological scaling exponent a was extractedfrom the power spectrum of the recorded time series. Actin was disrupted with latrunculin B andthe effect of the disruption on the cells’ viscoelasticity was determined. To assess the effect of actindisruption on gene expression, time-lapse microscopy was employed to follow single, LatB treated,PrE-primed cells over a course of 24h, and qPCR was used to determine the effect on the expressionof Nanog, a pluripotency marker, and Gata6, another marker for the primitive endoderm. The underlyingdiffusive process was determined by means of the ensemble and time averaged mean squareddisplacement, the amplitude scatter, higher order moments and the velocity autocorrelation func-tion.It was found that PrE-primed cells were significantly more elastic than Epi-primed cells. In both celltypes, the nucleus was more elastic than the cytoplasm. After actin disruption, both populations becamesignificantly more viscous. The viscoelasticity of the nucleus of Epi-primed cells did not changeupon depolymerizaƟon of acƟn. The periphery of Epi-primed cells, as well as the nucleus and the peripheryof PrE-primed cells became more viscous upon actin disruption. While 24 h time-lapse imagingconfirmed a significant drop in Hhex levels of actin disrupted PrE-primed cells, this result could notbe exactly confirmed with qPCR. Furthermore, the underlying diffusive process was determined to becontinuous time random walk (CTRW).Upon exciting pluripotency, changes occur in the nucleus of stem cells. Chromatin remodeling,the recruitment of lamin A to the nucleoskeleton and stiffening of the cells were reported changescaused by differentiation. These modifications could explain the difference between Epi- and PrEprimedcells. Disruption of actin by LatB treatment indicated that the differences between the twopopulations could be caused by different actin levels. As actin had been associated with chromatin.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01010311047&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Viscoelastic and dynamic properties of embryonic stem cells
PB - The Niels Bohr Institute, Faculty of Science, University of Copenhagen
ER -