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Forthcoming Article in Stem Cells - Human Embryonic Stem Cells Suffer from Centrosomal Amplification

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By Stuart P. Atkinson

Current protocols for the differentiation of embryonic stem cells (ESC) to clinically relevant cell types are woefully inefficient with many millions or tens of millions of ESC used for each differentiation, only to yield small proportions of the desired cell type. This entails large scale culture and amplification of ESC, often over a large period of time. Unfortunately, long term culture of ESCs is also known to lead to karyotypic instability perhaps rendering these cell types at best less functional and at worst potentially tumorigenic. Therefore, a deeper understanding of the processes which lead to karyotypic instability in cultured ESCs is of major interest, and indeed could allow for formulation of strategies to mediate the repression or inhibition of this instability. One potential mechanism fueling chromosome karyotypic instability is the amplification of centrosomes which can perturb chromosome segregation in mitosis. In the upcoming edition of Stem Cells, researchers from the lab of Aleš Hampl at the Masaryk University, Brno, undertake an analysis of centrosome amplification in hESC. In this work they uncover a link between centrosome amplification and karyotypic instability and suggest the implementation of new culture techniques to inhibit such instability (Holubcová et al).

Initial analysis demonstrated that low passage hESCs (12 independent lines, grown under varying conditions in various labs, from P14 to P39) contained around 10-24% multi-centrosomal mitoses and were invariably linked to major abnormalities in chromosomal positioning. However, centrosome structure and function seemed to be unaltered and alongside the further observation of the association of multicentrosomal mitotic states and increased ploidies, suggests that supernumery centrosomes are caused by over-duplication of centrosomes during a single cell cycle and also by mitotic failure. Further analysis uncovered an association between high levels of multicentrosomal mitoses in low passage hESCs, decreasing to approximately 5% (from 10-24%) at later passages, similar to that observed in ESC-derived cells. This suggests that hESC in their most pluripotent state have an excess number of centrosomes and suggests that long term culture of ESC allows the acquisition of unknown mechanisms to remove or inhibit their number. Next, the means by which centrosomal amplification might be reduced were studied and it was shown that certain culture conditions can lead to such a reduction. Mimicking the effect of stimulation by extracellular matrix proteins by treatment of cultured hESCs with an integrin receptor-activating antibody for 24 led to a decrease in multicentrosomal mitoses, compared to growth on other substrates such as gelatin, suggesting that cell culture on substrate material can have an impact on chromosomal instability. Finally inhibition of Aurora A and CDK2 activities was shown to lead to the reduction in the frequency of multicentrosomal mitoses. Aurora A protein levels and CDK2 activity were both elevated in hESC as compared to somatic cells, and Aurora A has been linked to mitotic failure and CDK2 to the over-amplification of centrosomes in one cell cycle.

Studies in cancer cells have linked centrosomal amplification to chromosomal instability and the associated observed deleterious effects. Although the authors do admit that further studies are required to conclude with certainty that supernumery chromosomes lead to chromosomal instability in hESCs, their current work does suggest that these pose a significant risk to karyotypic stability. However, it is demonstrated that with appropriate culture conditions this risk can be inhibited, and implementing these conditions may allow for the required large scale amplification of karotypically stable hESCs for clinical use.

 

References
Human Embryonic Stem Cells Suffer from Centrosomal Amplification.
Holubcová Z, Matula P, Sedláčková M, Vinarský V, Doležalová D, Bárta T, Dvořák P, Hampl A.
Stem Cells. 2010