The 2nd CEXS-UPF Symposium on Evolutionary Biology that took place in November at the Barcelona Biomedical Research Park (PRBB) opens this edition of El·lipse, the park’s monthly newspaper.
Also on the topic of evolution, Salvador Carranza (IBE) tells us about his research on reptile phylogeny. Other news include new findings on senescence and embryo development, lung cancer diagnosis, ‘mini-kidneys’ created from human stem cells, the benefits of long-term breastfeeding, new molecules involved in metastasis or computational models to decipher biological problems. On a more personal note, Baldomero Oliva (UPF) tells us about his scientific career and the secret to become a good scientist: patience and stubbornness. The current-affairs debate deals with a very topical question, raised by a recent article in The Economist: is there a reliability problem in science? Find out the different opinions of four researchers at the park!
Carles Miquel Colell, coordinator of the Research and Innovation programme for the Generalitat’s Department of Health, is Chairman of the CMRB Ethics Committee (CEIC). Doctor of internal medicine by profession, he started off in the world of healthcare, proceeded to healthcare management, teaching, and currently, research coordination. Dr. Miquel explains the whys and wherefores of the CEIC of the CMRB.
How long has the committee existed?
In Spain in 2006, a unique approval mechanism was created to establish certain safeguards in the use of pre-embryos left over from in vitro fertilization for stem cell research. In particular, it is necessary to ensure that permission be received from the progenitors, that there exists no other research model that would yield the same results and that the research team is fully prepared and has sufficient resources to carry out the project, amongst other things. For this reason the “Commission of safeguards for the donation and use of human tissues and cells” was established in Madrid. And in Catalonia the CMRB CEIC (Clinical Research Ethical Committee) was created to be the sole organisation accredited to authorise these projects before going to the Commission in Madrid.
Who is it made up of?
There are currently 13 people, from different disciplines: a biologist, three pharmacists, a clinical pharmacologist, two nurses, a customer services representative, a gynaecologist, two lawyers, an expert in bioethics and a technician, which is me, and I am the link between the Committee and the Department of Health. We try to make sure there are no CMRB researchers in the committee in order to avoid potential conflicts of interest.
What kind of projects do you evaluate?
Any study that uses stem cells in Catalonia, both embryonic and the newer induced pluripotent cells (iPS). These are obtained from the dedifferentiation of adult cells. But in many countries these types of stem cells are not covered by CEIC specifics, as they are derived from adult, not embryonic or foetal tissue.
What procedure must be followed for a stem cell project? How long does it take?
The researchers have to prepare a series of documents that must first go through the centre’s own ethics committee, if there is one, and later, in Catalonia, through the CMRB CEIC. We assess and improve the project as far as possible before sending it to the Safeguards Committee in Madrid – to which I also belong – who prepare a mandatory report. This can take between 3 and 5 months, depending on whether clarification or further information is requested.
Why is stem cell research important?
The original idea was that of regenerative medicine: to be able to create organs and tissues to substitute damaged or old ones, and researchers are working on that. But even if this promise wasn’t totally fulfilled, these types of cells are still extremely important to the furthering of our scientific knowledge. And in the case of iPSs, they are fantastic models. We can take pluripotent cells from a Parkinson’s sufferer, for example, and use them to study the disease and test potential therapies.
Anna Bigas and Lluís Espinosa, of the Stem Cell and Cancer group of the IMIM are two principal investigators who have joined forces to investigate different aspects of cancer development. Together with their jointed group of 14 researchers, Bigas focuses on hematopoietic stem cells, while Espinosa concentrates on solid cancer and intestinal stem cells.
Bigas aims to understand how a pluripotent stem cell becomes a hematopoietic stem cell during embryogenesis. `It is a great challenge in the regenerative medicine field to understand where these stem cells come from and how they conserve this self- renewing capacity which enables them to maintain a tissue´, she explains. She focuses on a major signalling pathway controlling decisions in both normal and leukemic cells and which is also important for tissue maintenance: the Notch pathway.
Searching for Notch target genes
In order to specify the molecular mechanisms driving an undifferentiated cell towards the hematopoietic lineage or to the leukemic phenotype, the group’s current objective is to find Notch target genes and to describe their mechanism of function.
The researchers use techniques like such as chromatin precipitation and promoter arrays. This results in lists of possible candidate genes from which the real targets have to be isolated and validated in a series of experiments, including FACS to isolate the cells of interest and to determine whether the candidate gene is expressed. Further molecular and biochemical analysis, as well as experiments using mutant mice, help define possible interactions of the target molecules and their effect in the organism.
One target gene is GATA2, an important hematopoietic transcription factor that is not expressed in Notch mutant mice and is altered in human leukaemia. The Bigas group have characterized the GATA2 promoter and found that Notch exerts both positive and negative signals that restrict the intensity and the duration of GATA2 expression in hematopoietic cells.
In parallel, Espinosa is studying whether Notch cooperates with other signalling pathways in different contexts. He found that specific elements of the NF-κB pathway, which is involved in cancer development, directly regulate the transcription of genes which are known to be Notch-dependent.
In a common work Bigas and Espinosa identified a new role of the Notch signalling pathway in the maintenance of leukemic stem cells. Previous studies had shown that both the NF-κB and the Notch pathway are involved in T-cell acute lymphoblastic leukaemia, and future therapeutical strategies may employ both Notch and NF-kB inhibitors to fight this leukaemia.
However only the recent findings of Bigas and Espinosa describe the exact mechanism by which Notch activates the NF-kB transcription factor. These new insights published in Cancer Cell, could be translated to clinical trials and result in better pharmaceutical treatments with less side effects.
Despite all the advances in the field, it is not yet known whether Notch is also important for the leukaemia initiating cells, a question that Bigas would like to answer in the close near future. These cells are of major interest, since they are resistant to standard leukaemia treatments, remain in the organism and ultimately are the source of a recurrent outbreak.
Bigas and Espinosa maintain collaborations both inside and outside of the PRBB. `It is a great advantage to have so many scientists within a few square metreers´ Bigas states, `and we just have a great collaborative work in progress, including involving scientists from the CRG, the UPF, Hospital del Mar and others´.
This article was published in the El·lipse publication of the PRBB.
The 5th Open Day at the Barcelona Biomedical Research Park (PRBB) opens this edition of El·lipse, the park’s monthly newspaper.
Other news include the celebration of the CRG 10th anniversary, new proteins important for cell division or for tumour growth, how stem cell dysfunction links cancer and ageing or a new drug against skin cancer. You will also learn about the “Jennifer Aniston” neurone from Rodrigo Quian, from the University of Leicester (UK), or about the effects of radiations from mobile phones on our health, a subject that Elisabeth Cardis (CREAL) and her group are studying.
Research on human aging is a hot topic nowadays, due to a growing aging population and the consequent prevalence of aging-associated diseases such as Alzheimer’s, arthritis or cardiovascular diseases. Researchers at the CMRB review the use of human induced pluripotent stem cells (hiPSC) to study the fundamental mechanisms underlying aging in this article published in Current Opinion in Cell Biology.
Indeed, hiPSC-based models of aging and aging-related diseases are facilitating the study of the molecular and cellular mechanisms underlying aging. For example, the use of iPSCs from patients with accelerated aging (like those with Hutchinson–Gilford progeria syndrome) could recapitulate the aging process in vitro much faster than the several decades needed for normal human tissue to age. Also, cell and organ derivatives from patient-specific iPSCs can be transplanted into animal models and the integrated human living materials could provide an opportunity to study human tissue and organ aging or disorders in an in vivo context.
Liu GH, Ding Z, Izpisua Belmonte JC. iPSC technology to study human aging and aging-related disorders. Curr Opin Cell Biol. 2012 Sep 18;
Cristina Eguizabal came from Cambridge, UK, to the CMRB two and a half years ago to try to get male haploid cells (spermatozoids) from human pluripotent stem cells. She uses both human embryonic cells (hES) and induced pluripotent stem cells (iPS). The first are obtained through fertility clinics from unused fertilised embryos. The hES are isolated from the embryos and derived into cell lines at the Stem Cell Bank of the CMRB, on the 4th floor of the PRBB, where Eguizabal is working under the coordination of Anna Veiga. iPS cells, on the other hand, can be derived in vitro from skin cells or cord blood, as was shown recently, although the protocol is not optimal and these cells are not yet safe for clinical use.
Once she has the pluripotent stem cells in culture, Eguizabal tries to find the best conditions to differentiate them into primordial germ cells (PGC), which can then give rise to the spermatozoids or oocytes. What would be the practical applications of this, if it works?
Giving hope to parents
The simplest would be to allow people who are sterile to have children with their genetic characteristics. Currently, a sterile man depends upon anonymous semen donations to have a child. With this technique, a simple skin biopsy could lead to the creation of his own iPS cells, which would be differentiated in vitro into PGCs and which would give rise to sperm with his DNA which could be used in “in vitro fertilisation” (IVF) to have his child.
Another related application would be to allow people to have children free of a genetic disease which they themselves suffer. In this case, the procedure would be the same, but once the iPS cells were obtained, their genetic error would be corrected. This would be possible for well-studied monogenic diseases, caused by a single gene, such as cystic fibrosis or Duchene muscular dystrophy.
Many steps to go
“This won’t be happening for 10 or 15 years”, predicts Eguizabal. “Apart from the step I am working on, the differentiation of the iPS cells into PGCs, of which very little is known, all the other steps in the process need more research. For example, deriving the iPS cells from skin cells needs to become safer, as at the moment one of the genes included in this transformation is an oncogene, which could lead to cancer. The genetic correction of the iPS cells is also something people are working hard on”, continues the researcher. Both of these issues are also studied at the CMRB.
The differentiation of stem cells into sexual gametes, spermatozoids and oocytes, is the most difficult, much more so than getting stem cells to become neurones or cardiovascular cells, according to the Basque biologist. “First of all, the sexual gametes are the only cells that go through meiosis, a type of cell division that implies losing half of your genome. And secondly, their aim is to give rise to a living being, so their genetic and epigenetic information must be 100% correct”, she says. During normal physiology, germ cells suffer a huge epigenetic re-programming: their whole genome is methylated and de-methylated again. Methylation is a reversible modification of the DNA which affects gene expression. And in order to get functional gametes, it is important to reproduce this methylation in vitro. But it is not easy. Eguizabal plans to use molecular techniques such as bisulphite sequencing to check the methylation status of the DNA once she gets the cells she is interested in.
This article was published in the El·lipse publication of the PRBB.
Chris Jopling joined the CMRB as a postdoctoral research scientist in June 2007. Since then, he has been investigating heart regeneration in zebrafish. The technicians M. Carme Fabregat and Guillermo Suñé, as well as Veronika Sander, another postdoctoral researcher, collaborate with the English biochemist in this line of research.
They are all trying to find out which genes are involved in heart regeneration in the zebrafish, Danio rerio. “You can cut off up to 20% of a ventricle of an adult fish, and in one month it is completely regenerated”, explains Jopling. Mammals are able to regenerate some tissues, such as blood or liver, but not heart. At least that is what scientists used to think. Earlier this year, it was found that newborn mice were able to regenerate their hearts, even though this ability was lost after just one week. This means that mammals actually do have the potential for heart regeneration.
Not everything is stem cells
For years, researchers have thought that stem cells were responsible for regeneration in the heart, and many groups around the world look for these stem cells. But Jopling and his colleagues at the CMRB showed, in 2010, that heart regeneration in zebrafish did not involve stem cells at all (you can see here the full paper). Rather, it was the cardiomyocytes (heart muscle cells) that dedifferentiated and started proliferating upon heart amputation. “Interestingly, the neonate mice regenerated their heart in the same way that the zebrafish do, that is, through the differentiated heart cells, and not through stem cells”, says Sander.
So far, only five genes have been demonstrated to be directly involved in heart regeneration in zebrafish. The aquatic animals group has identified three more genes that, if mutated or over-activated, block regeneration. In order to find these genes, the researchers followed a specific protocol. “We make a small cut on the ‘chest’ of the fish (just above its heart). If you then squeeze gently, the heart ventricle comes out. You then just grab it with very small forceps and make a cut with the scissors”, explains Jopling. The fish are sent back to their tank, where they continue swimming peacefully. Fourteen and thirty days after the amputation, the researchers check the status of heart regeneration. At the first time point, normal fish are in the peak of proliferation and, after a month, regeneration is complete. When the fish are transgenic or exposed to chemical additives that inhibit genes involved in regeneration, the process is halted.
A long way to go to mend a broken heart
The final aim of the research is to regenerate a human heart after a stroke. But there is still a long way to go. The CMRB group has already planned their next steps. First, they will check how the newly identified genes affect the expression of other genes by using microarrays and comparing the gene expression of a normal regenerating heart with that of a heart in which these genes have been modified. That should help to better understand how these genes regulate regeneration and which pathways they are involved in.
The scientists will then move on to mice. Since most genes in the zebrafish have homologues in mammals, they will check whether the identified genes are able to induce proliferation of cultured mouse cardiomyocytes. Mouse heart muscle cells usually don’t proliferate, so a cell culture can only last about a week. If the researchers are able to get the cells in the culture to replicate and proliferate by using these new genes, they will then generate transgenic mice. And bring us all a step closer to mending a broken heart.
This article was published in the El·lipse publication of the PRBB.
Divide and conquer
In this image by Cristina Morera from the CMRB, taken with a SP5 Leica confocal microscope, a mouse stem cell can be seen dividing. The DNA is highlighted in blue, the pluripotency marker Oct4 in red, and α-tubulin, one of the main components of cell cytoskeleton microtubules, in green. The α-tubulin enables the observation of the cell in metaphase, the stage of cell division where the pairs of chromosomes (blue) are aligned in the centre of the cell. Later, the chromosomes will be separated and divided between the two daughter cells by the mitotic spindle formed of microtubules (green).