Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CAiPSC (Determining centromere assembly mechanisms and improving mitotic fidelity during somatic cell reprogramming)

Teaser

Human embryonic stem cells (hESCs) are obtained from a small group of cells found very early in the development of the embryo. Cells taken from one embryo can be made to multiply in the laboratory to create a ‘cell line’, like the cell line we used in our project, which...

Summary

Human embryonic stem cells (hESCs) are obtained from a small group of cells found very early in the development of the embryo. Cells taken from one embryo can be made to multiply in the laboratory to create a ‘cell line’, like the cell line we used in our project, which was derived from an embryo in 1998. hESCs can be used in research to improve our understanding of how the egg can develop into a complex organism. Moreover, in the laboratory hESCs can be differentiated to specialized cells like heart or nerve cells, which can then be studied to understand more about how and why diseases develop. These specialised cells can also be used to study how cells react to new drugs and if these new drugs can be used to treat a specific disease. This is of great importance, as it allows studying cells that are not easily obtained from patients, such as brain cells.

More than a decade ago, there was a major breakthrough in stem cell research - researchers discovered that it is possible to “reprogram” specialised adult cells into cells that behave like ESCs. These cells were termed induced pluripotent stem cells (iPSCs). Human induced pluripotent stem cells (hiPSCs) share common features with hESCs, such as being pluripotent. hiPSCs can be easily obtained by reprogramming, for example, skin cells. The technology is quite new and researchers do not yet know precisely how the process of reprogramming works. Although pluripotent stem cells (PSCs, which include both hESCs and hiPSCs) may become great tools, some concerns have been raised over their safety.

All cells are produced from other cells by the process of cell division. Cell division occurs when one cell divides to produce two new cells (daughter cells). The new cells produced by cell division are genetically identical to the parent cell because they each receive a copy of all the chromosomes it has in its nucleus. PSCs, however, seem to be prone to errors in passing the correct number of chromosomes to daughter cells. Therefore, it is important to understand the molecular mechanisms that ensure that each daughter cell receives the correct number of chromosomes needed to function normally. Although these mechanisms have been extensively studied in differentiated cells, nearly nothing is known in pluripotent cells.

All chromosomes in a cell have a specialised region - termed the centromere – that is essential for the correct segregation of chromosomes to the daughter cells. The centromere is defined by the presence of CENP-A, a chromatin-bound histone protein, and it is the region of the chromosome where the kinetochore can assemble. The kinetochore is a multi-protein complex that serves as the site of attachment for microtubules, which pull sister chromatids apart during cell division, ensuring each daughter cell has the same number of chromosomes. If the centromere is somehow defective, cells can no longer pass the correct number of chromosomes to their daughter cells.

This project aimed to determine centromere assembly, maintenance and kinetochore function in PSCs, to understand if the genome-wide remodelling of chromatin during reprogramming affects centromere assembly and function and to modify centromere composition to understand if it is possible to obtain safer and more stable iPSCs.

Work performed

During the tenure of my Marie Sklodoska-Curie we have revealed that although the total pool of CENP-A is higher in PSCs, when compared to differentiated cells, CENP-A is depleted at the centromere of pluripotent stem cells. PSCs divide much faster by shortening the phase of the cell cycle when the centromere is assembled. In differentiated cells CENP-A assembles in the G1 phase of the cell cycle, we have shown that CENP-A is also assembled in this phase of the pluripotent stem cell cycle. These results indicate that the regulation of CENP-A levels is altered in PSCs, possibly due to differences in the cell cycle.

By further characterising key centromeric and kinetochore proteins we found that other important proteins for the function of these structures are also present at lower levels in PSCs. Our results suggest that PSCs maintain a reduced centromere and kinetochore size, when compared to differentiated cells. Moreover, studying how these structures change during the reprogramming of differentiated cells to iPSCs, we determined that loss of CENP-A at the centromere is an early event when inducing pluripotency, concomitant with genome-wide remodelling of chromatin marks. We are currently attempting to strengthen both centromere and kinetochore function, by modulating the levels or activity of key proteins in PSCs. This will also allow us to assess if the weakened centromere/kinetochore underlies the increased mis-segregation observed in these cells.

We will present our findings in the following scientific meetings:
EMBO Workshop - Chromosome segregation and aneuploidy. Cascais. May, 2019
ISSCR Annual Meeting 2019. Los Angeles. June, 2019

I have also presented my work in seminars within the host institute and I have created and organised the Lisbon area stem cell club, which brings together researchers from six different Institutes in the Lisbon area every two months to share and discuss their science. This project was fundamental for my training in scientific and transferable skills, for enhancing contact networks for me and for the host institution through academic conferences and public engagement events. Moreover, during the tenure of the Marie Sklodoska-Curie fellowship, I was able to obtain highly insightful and promising results that have already allowed me to secure funding for my first project as principal investigator.

Final results

All the results obtained with this Marie Sklodoska-Curie project contributed to increasing the knowledge of pluripotent stem cell biology, as virtually nothing was known about centromere and kinetochore structure and mechanisms of assembly and maintenance in pluripotent stem cells. Understanding the regulation of cell division and the correct allocation of chromosomes is of high importance if these cells are to be used to their full potential, as incorrect number of chromosomes can increase the chances of the cell becoming a cancer cell.

Website & more info

More info: http://www.jansenlab.org/.