Comparison of the changes in chromosome centromere distribution, before (myoblasts Mb24 hrs − red spots) and after (myocytes Mc7d − yellow spots) cell differentiation.

<p>When analysing chromosomes 7 and 11, the observed changes did not reach statistical significance (p>0.05). In other cases, the observed changes in centromere localisation were directed towards the nuclear periphery (chromosomes 1, 3, 12, 17, X).</p

Human myoblasts in <i>in vitro</i> culture.

<p>A – flow cytometry analysis of CD56 expression on human myoblasts. Mean percent of cells positive for the CD56 marker: 85.0+/−5.0%. Panel B <i>- in vitro</i> culture of myoblasts and myocytes (Mb24 hrs – proliferating myoblasts; Mc7d- fused myocytes (myotubes). magnification-100x).</p

Comparison of chromosome centromere distribution in co-centric nuclear shells in myoblasts (24 hrs after cell plating) and in differentiated myocytes (after 7 days in a medium containing 2% horse serum).

<p>From the nuclear interior to the periphery, 5 shells were identified.</p

Cytogenetic maps of chosen chromosomes along with genes (loci) of interest marked.

<p>HSA1(MYOG, CD34), HSA3 (CAV3), HSA7 (AQP1); HSA11 (CD56, MYOD, ACTN3); HSA12 (MYF5, MYF6); HSA17 (MYH); HSAX.</p

Characterisation of Nuclear Architectural Alterations during <i>In Vitro</i> Differentiation of Human Stem Cells of Myogenic Origin

<div><p>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 <i>in vitro</i> 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 <i>in vitro</i> 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.</p></div

Immunofluorescence analysis of desmin (DES), myosin heavy chain (MYH) and alpha-actinin (ACTN) in myoblasts (Mb24 hrs) and myocytes after 7 days of differentiation (Mc7d).

<p>(Magnification in the left and middle columns-200x; in the right column 630x).</p

Morphological comparison of myoblast and myotube nuclei.

<p>Nuclei of differentiated myotubes have been characterised by a smaller volume (A) and increased flattening (B) compared to proliferating myoblasts. In both cases, differences were statistically significant (p<0.00001). C – 3D projection of myoblast D – myotube nucleus with a signal detected for chromosome centromeres.</p