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Characterisation of nuclear architectural alterations during in vitro differentiation of human stem cells of myogenic origin.

Rozwadowska N, Kolanowski T, Wiland E, Siatkowski M, Pawlak P, Malcher A, Mietkiewski T, Olszewska M, Kurpisz M - PLoS ONE (2013)

Bottom Line: Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells.The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning.Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

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Related in: MedlinePlus

Scheme of 3D analysis of interphase nuclei.A – stack of pictures collected in the Z-axis; B – model of 3D reconstruction of the nucleus with a schematic view of the co-centric shells; C – evaluation of centromere position in the 3D nucleus environment.
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pone-0073231-g001: Scheme of 3D analysis of interphase nuclei.A – stack of pictures collected in the Z-axis; B – model of 3D reconstruction of the nucleus with a schematic view of the co-centric shells; C – evaluation of centromere position in the 3D nucleus environment.

Mentions: The stacks of pictures for 3D FISH analysis (50+/−8 cell nuclei/cell population) were derived using an Axiovert 200 fluorescent confocal microscope (Zeiss, Jena, Germany), analysed by LSM Image Browser (Zeiss, Jena, Germany) and subsequently by NEMO [12]. For each cell, volume and flattening were measured. The relative distance of the centromere hybridisation signal was calculated from the nuclear centre, based on the division of the nuclear diameter on equal sections (Figure 1). Volume and flattening samples for both, myoblasts and myotubes, did not undergo a normal distribution (Shopiro-Wilk test for normality, p-value<0.001 for all four samples), therefore, we decided to use a non-parametrical analysis. Statistical significance of a morphological analysis of the nuclei volume and the flattening was calculated by Mann-Whitney U test. The nucleus of each cell was divided into five co-centric shells of the same volume. Then, centromeres signals were assigned to appropriate shells, creating a signal distribution in cell nuclei. The centromere signal distribution was then analyzed with the chi-square test.


Characterisation of nuclear architectural alterations during in vitro differentiation of human stem cells of myogenic origin.

Rozwadowska N, Kolanowski T, Wiland E, Siatkowski M, Pawlak P, Malcher A, Mietkiewski T, Olszewska M, Kurpisz M - PLoS ONE (2013)

Scheme of 3D analysis of interphase nuclei.A – stack of pictures collected in the Z-axis; B – model of 3D reconstruction of the nucleus with a schematic view of the co-centric shells; C – evaluation of centromere position in the 3D nucleus environment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760906&req=5

pone-0073231-g001: Scheme of 3D analysis of interphase nuclei.A – stack of pictures collected in the Z-axis; B – model of 3D reconstruction of the nucleus with a schematic view of the co-centric shells; C – evaluation of centromere position in the 3D nucleus environment.
Mentions: The stacks of pictures for 3D FISH analysis (50+/−8 cell nuclei/cell population) were derived using an Axiovert 200 fluorescent confocal microscope (Zeiss, Jena, Germany), analysed by LSM Image Browser (Zeiss, Jena, Germany) and subsequently by NEMO [12]. For each cell, volume and flattening were measured. The relative distance of the centromere hybridisation signal was calculated from the nuclear centre, based on the division of the nuclear diameter on equal sections (Figure 1). Volume and flattening samples for both, myoblasts and myotubes, did not undergo a normal distribution (Shopiro-Wilk test for normality, p-value<0.001 for all four samples), therefore, we decided to use a non-parametrical analysis. Statistical significance of a morphological analysis of the nuclei volume and the flattening was calculated by Mann-Whitney U test. The nucleus of each cell was divided into five co-centric shells of the same volume. Then, centromeres signals were assigned to appropriate shells, creating a signal distribution in cell nuclei. The centromere signal distribution was then analyzed with the chi-square test.

Bottom Line: Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells.The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning.Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

Show MeSH
Related in: MedlinePlus