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To reduce damage to stem cells due to replication stress, CNIO scientists use a dual approach:
(1) increasing the production of the Chk1 protein, to repair DNA damage ; and (2) supplement
the medium with nucleoside, both source compounds that build DNA.

Building a safer stem cell

Spanish scientists find a way to generate safer stem cells in the laboratory. Their project represents a major step forward in the possible therapeutic use of stem cells.

Regeneration of damaged tissue to treat cardiovascular diseases, diabetes, and neurodegenerative diseases, is one of the ambitious scenarios for regenerative medicine. The focal point of which is the use of stem cells capable of producing different types of cells and potentially replacement tissues.

2006 marked a turning point in this field, when for the first time, scientist Shinya Yamanaka of Japan managed to generate pluripotent stem cells in his lab. Yamanaka received the Nobel Prize for his discovery that mature cells can be reprogrammed to become pluripotent. Pluripotent stem cells are capable of becoming any type of cell, whether insulin-producing beta cells in the pancreas or cardiomyocytes in the heart, and are known as induced pluripotent stem cells or iPS cells. This cell reprogramming technique eliminated a great ethical dilemma: until then, pluripotent stem cells could only be obtained from 3 to 4 day old human embryos left by donor parents to be destroyed or given to science.

However, there was another problem with iPS cells.

"The drawback of his new technology was that Yamanaka's method damaged the stem cell genome, leading to possible mutations within the cells."

Óscar Fernández-Capetillo PhD, Director, Genomic Instability Group Spanish National Cancer Research Centre (CNIO)

While the fact that his method damaged the DNA of iPS cells was known, the reasons were not. According to María Blasco PhD, Telomeres and Telomerase Group, Spanish National Cancer Research Centre, Madrid, and published in Nature Communications, the damage to the iPS genome is due to the stress cells are subjected to during reprogramming. This replication stress occurs when cells are made to increase their pace of division.

Based on these findings, the team developed strategies to reduce reprogramming stress. Almost a decade since first developed, there is now a more efficient way of obtaining iPS cells with more stability.

Damage to the DNA in iPS cells was due to rearrangement of large fragments of chromosomes which could lead to potentially dangerous mutations if the cells were used clinically.

In a paper published in Nature in 2009, the team led by María Blasco, with the collaboration of Fernández-Capetillo's group, described how damage to the DNA had limited the cell reprogramming process making it less efficient.

Now a team headed by Fernández-Capetillo has not only identified the origin of the damage, replication stress, but has managed to reduce it significantly; potentially improving the safety of induced stem cells for use in biomedicine.

To reduce damage to stem cells and thus achieve more stable genomes, the scientists used two approaches: (1) genetics to increase the production of the Chk1 protein which repairs DNA damage due to replication stress; and (2) supplementing nucleoside into the medium on which iPS cells grow. Both Chk1 protein and nucleoside are source compounds which build DNA.

"Based on previous research performed by the group, we saw that input of additional nucleoside reduces the stress of replication. Probably by facilitating the replication of DNA, nucleoside increases the rate of cell divisions during the reprogramming process."

Sergio Ruiz PhD, first author on the paper

The simplicity of this nucleoside-based strategy means that it can be implemented easily by laboratories around the world working with iPS cells, and thus contribute significantly to the field of regenerative biology, one of the greatest aspirations of biomedicine this century.

Nature Communications Abstract: Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.

The CNIO Telomeres and Telomerase groups, headed by María Blasco, and the Tumoral Suppression Group, headed by Manuel Serrano have also participated in this study, together with groups from the Pasteur Institute in Paris, Toronto University and the Pompeu Fabra University in Barcelona.

The study was jointly funded by the European Union through the European Research Council (ERC), the Ministry of Economy and Competition of the Government of Spain, the Howard Hughes Medical Institute, and the Botín Foundation and Banco Santander, through Santander Universities, among others.

Reference article: Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells. Sergio Ruiz, Andres J. Lopez-Contreras, Mathieu Gabut, Rosa M. Marion, Paula Gutierrez-Martinez, Sabela Bua, Oscar Ramirez, Iñigo Olalde, Sara Rodrigo-Perez, Han Li, Tomas Marques-Bonet, Manuel Serrano, Maria A. Blasco, Nizar N. Batada, Oscar Fernandez-Capetillo. Nature Communications (2015). doi: 10.1038/ncomms9036

Nature Abstract - A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity
The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates1, 2, probably reflecting the existence of ‘reprogramming barriers’ that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells.

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