Control of stem cell potency



About

Heart regeneration in the zebrafish / Mechanisms of induced reprogramming to pluripotency / Disease modeling through induced pluripotent stem (iPS) cells / Induced reprogramming to cardiogenic mesoderm / Bioengineering approach to heart muscle differentiation

During embryo development, the potency of the zygote is deployed through coordinated and stereotypical changes in cell behaviors and processes of tissue patterning, ultimately resulting in the formation of an entire, highly complex organism in a relatively short period of time. Throughout this process, the developmental potency of individual cells, i.e. their ability to give rise to cells of a different type than their own, is progressively lost, so that somatic cells in adult individuals retain very limited potency (such as in rare adult stem cells) or show no potency at all.

Human iPS cells aggregated to form embryoid bodies

Human iPS cells aggregated to form embryoid bodies

Our laboratory is interested in understanding the mechanisms that govern the degree of potency of human somatic cells, and how it can be experimentally increased for conditions where doing so may be of biomedical relevance. Specifically, the context in which we investigate these issues is mainly centred on the paradigm of cardiac regeneration/repair.
Heart-related diseases are the main cause of mortality in the world, with ischemic heart disease being the single most frequent condition accounting for the death toll. This results from the very limited ability of the mammalian heart to regenerate on its own, and underscores the pressing biomedical need to finding ways for potentiating this ability (heart regeneration) and/or providing new cardiac cells to replace the lost or damaged ones (heart repair). We pursue the first approach by studying the process of heart regeneration in the zebrafish, an organism with a remarkable capacity for regeneration. Natural regeneration is a biologically fascinating phenomenon in which somatic cells may regain developmental potency, and we hope that understanding the molecular and cellular mechanisms that control this process will help devising strategies to potentiate the regeneration of adult mammalian hearts.

Heart of a transgenic zebrafish showing mosaic recombination

Heart of a transgenic zebrafish showing mosaic recombination

For achieving heart repair, in turn, our research is aimed at generating functional human heart muscle cells that could be used for transplantation. For this purpose, we investigate ways to manipulate the developmental potency of human somatic cells so they become pluripotent (i.e. regain the potency of cells in the early embryo), and then study how these so-called induced pluripotent stem cells (iPSC) can be instructed to differentiate into functional cardiomyocytes. In addition, since iPSC can be generated from disease-carrying cells, many laboratories including ours are exploring the possibility of using patient-specific iPSC to generate disease-relevant cell types in which to investigate the pathogenic mechanisms of disease initiation and/or progression.

Overall, our research takes advantage of a variety of experimental paradigms (zebrafish heart regeneration, human iPSC generation and differentiation), approached from a multi-disciplinary perspective, ranging from bioengineering approaches to 3D stem cell differentiation to single-cell genetic lineage tracing analyses and genetic manipulation of human cells, aimed at tackling important current issues in biology and biomedicine, such as the mechanisms that control the establishment and maintenance of developmental potency, the initiation and progression of the regenerative process, and the differentiation and functional maturation of human cardiomyocytes, and the development of genuinely human models of human disease.


News/Jobs

IBEC group leader to give public lecture on stem cells
27/10/14

Tomorrow night IBEC group leader and ICREA professor Ángel Raya will give a public lecture at the CCCB in Barcelona entitled “Células madre y medicina regenerativa: realidades y promesas” (“Stem cells and regenerative medicine: realities and promises”).


“Izpisúa no s’ha endut cap projecte”
13/02/14

An interview with IBEC group leader Ángel Raya about his new appointment as director of the Centre de Medicina Regenerativa de Barcelona (CMRB) appears in Catalan newspaper Ara today.


“El investigador Ángel Raya destaca el hallazgo de un nuevo modelo basado en células madre para el estudio del párkinson”
29/06/14

Article in Diario Siglo XXI


IBEC group leader to head CMRB
15/01/14

Ángel Raya, ICREA professor and head of IBEC’s Control of Stem Cell Potency group, is to take up up the role of director of Barcelona’s Center of Regenerative Medicine in Barcelona (CMRB).


“1000 Researchers. Five continents. One Day”
14/11/2013

Control of Stem Cell Potency group leader Ángel Raya has been invited to take part in a virtual stem cell meeting today, alongside other experts from all over the world.


Pfizer award for IBEC/Vall d’Hebron collaboration on hemophilia
07/11/2013

A Spanish collaboration, including researchers from IBEC, has been awarded the prestigious ASPIRE Europe 2013 Hemophilia Research Award from Pfizer, the world’s largest research-based pharmaceutical company.


“El laboratori al ‘Connexió’: Institut de Bioenginyeria de Catalunya”
15/07/2013

Control of stem cell potency group leader Ángel Raya appeared on Thursday night’s edition of current events programme Barcelona Connexió on Barcelona Televisió (BTV), in a special report on cloning.


“Creado un hígado a partir de células madre”
04/07/2013

IBEC’s Control of Stem Cell Potency group leader Ángel Raya appears today in La Vanguardia and El Mundo giving his expert opinion on some research published in Nature about a Japanese group that has managed to create a functional human liver using induced pluripotent stem cells.


“Crean embriones humanos con la técnica empleada para clonar a la oveja ‘Dolly’”
27/05/2013

Ángel Raya, group leader of IBEC’s Control of Stem Cell Potency group, gives his expert opinion in several national and local newspapers on last week’s breakthrough in Oregon, where scientists finally managed to derive human embryonic stem cells from adult tissue.


“Un mecanismo biológico podría optimizar el tratamiento del párkinson”
07/03/2013

This week’s press release about Ángel Raya’s Nature Neuroscience paper gained coverage in La Razón, rTVE and many other science and news websites.


Researchers uncover the ‘double-whammy’ that could reveal a promising strategy for Parkinson’s treatments
04/03/2013

An IBEC researcher and his collaborators have revealed an important biological mechanism which could shed new light on how best to develop treatments for Parkinson’s disease.


“Nacen los primeros ratones sanos de ovulos procedentes de celulas madre”
18/10/2012

IBEC group leader Ángel Raya is quoted in articles in online newspapers Público and Materia, giving his expert opinion about some recent research in Japan. The scientists in Kyoto achieved a litter of healthy, fertile mice ‘born’ from undifferentiated stem cells taken from skin.


“Células madre (y padre)”
13/10/2012

El Periódico published an article about Nobel Prize winners John Gurdon and Shinya Yamanaka, who won the prize in medicine or physiology for their achievements in stem cell research. Towards the end of the piece, the author talks about the leading position held by Catalonia in research into stem cells, naming IBEC’s Ángel Raya as one of the region’s pioneers.


A breakthrough in understanding age-related disease
08/03/2012

Researchers at IBEC have made an important leap towards understanding the second most common neurodegenerative illness, Parkinson’s disease (PD), which affects around 5% of the population by age 85.


Lights, camera, action!
14/04/2011

Spanish national TV channel RTVE are paying a visit to IBEC today to interview Ángel Raya, group leader of the Control of Stem Cell Potency group, on the subject of stem cell research for an episode of the science and technology programme ‘Tres14’.


All the fun of the fair
06/04/2011

For the next three days IBEC scientists from the Control of Stem Cell Potency group will be introducing induced pluripotent stem (IPS) cells to the people of Barcelona at the 9th “Live Research” Fair at La Pedrera.


IBEC expands its research program with a new line of stem cell investigation
20/01/2010

The control of stem cell potency is the line of research most recently incorporated into the IBEC program under the leadership of Ángel Raya, a research professor at the Catalan Institute for Research and Advanced Studies (ICREA). This project, which represents an expansion of the institute’s work in the field, now forms part of IBEC’s Cellular Biotechnology research program.

Projects

National projects
Aproximación de bioingeniería a la regeneración/reparación cardiaca I+D-Investigación fundamental no orientada Ángel Raya

Publications

Tekeli, I., Aujard, I., Trepat, X., Jullien, L., Raya, A., Zalvidea, D., (2016). Long-term in vivo single-cell lineage tracing of deep structures using three-photon activation Light: Science and Applications 5, (6), e16084

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

Keywords: Multi-photon microscopy, Photoactivation, Three-photon microscopy, Zebrafish


Gálvez-Montón, C., Fernandez-Figueras, M. T., Martí, M., Soler-Botija, C., Roura, S., Perea-Gil, I., Prat-Vidal, C., Llucià-Valldeperas, A., Raya, A., Bayes-Genis, A., (2015). Neoinnervation and neovascularization of acellular pericardial-derived scaffolds in myocardial infarcts Stem Cell Research and Therapy 6, (1), 108

Engineered bioimplants for cardiac repair require functional vascularization and innervation for proper integration with the surrounding myocardium. The aim of this work was to study nerve sprouting and neovascularization in an acellular pericardial-derived scaffold used as a myocardial bioimplant. To this end, 17 swine were submitted to a myocardial infarction followed by implantation of a decellularized human pericardial-derived scaffold. After 30 days, animals were sacrificed and hearts were analyzed with hematoxylin/eosin and Masson's and Gallego's modified trichrome staining. Immunohistochemistry was carried out to detect nerve fibers within the cardiac bioimplant by using βIII tubulin and S100 labeling. Isolectin B4, smooth muscle actin, CD31, von Willebrand factor, cardiac troponin I, and elastin antibodies were used to study scaffold vascularization. Transmission electron microscopy was performed to confirm the presence of vascular and nervous ultrastructures. Left ventricular ejection fraction (LVEF), cardiac output (CO), stroke volume, end-diastolic volume, end-systolic volume, end-diastolic wall mass, and infarct size were assessed by using magnetic resonance imaging (MRI). Newly formed nerve fibers composed of several amyelinated axons as the afferent nerve endings of the heart were identified by immunohistochemistry. Additionally, neovessel formation occurred spontaneously as small and large isolectin B4-positive blood vessels within the scaffold. In summary, this study demonstrates for the first time the neoformation of vessels and nerves in cell-free cardiac scaffolds applied over infarcted tissue. Moreover, MRI analysis showed a significant improvement in LVEF (P = 0.03) and CO (P = 0.01) and a 43 % decrease in infarct size (P = 0.007).


Martorell, L., Corrales, I., Ramirez, L., Parra, R., Raya, A., Barquinero, J., Vidal, F., (2015). Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: Assessment of in silico prediction tools accuracy Haemophilia 21, (2), 249-257

Summary: Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

Keywords: Haemophilia A, Leucocytes, RNA splicing, Splice site mutation, Synonymous mutation


Notari, M., Pulecio, J., Raya, A., (2015). Update on the pathogenic implications and clinical potential of microRNAs in cardiac disease BioMed Research International 2015, Article ID 105620

miRNAs, a unique class of endogenous noncoding RNAs, are highly conserved across species, repress gene translation upon binding to mRNA, and thereby influence many biological processes. As such, they have been recently recognized as regulators of virtually all aspects of cardiac biology, from the development and cell lineage specification of different cell populations within the heart to the survival of cardiomyocytes under stress conditions. Various miRNAs have been recently established as powerful mediators of distinctive aspects in many cardiac disorders. For instance, acute myocardial infarction induces cardiac tissue necrosis and apoptosis but also initiates a pathological remodelling response of the left ventricle that includes hypertrophic growth of cardiomyocytes and fibrotic deposition of extracellular matrix components. In this regard, recent findings place various miRNAs as unquestionable contributing factors in the pathogenesis of cardiac disorders, thus begging the question of whether miRNA modulation could become a novel strategy for clinical intervention. In the present review, we aim to expose the latest mechanistic concepts regarding miRNA function within the context of CVD and analyse the reported roles of specific miRNAs in the different stages of left ventricular remodelling as well as their potential use as a new class of disease-modifying clinical options.


Navarro, S., Moleiro, V., Molina-Estevez, F. J., Lozano, M. L., Chinchon, R., Almarza, E., Quintana-Bustamante, O., Mostoslavsky, G., Maetzig, T., Galla, M., Heinz, N., Schiedlmeier, B., Torres, Y., Modlich, U., Samper, E., Río, P., Segovia, J. C., Raya, A., Güenechea, G., Izpisua-Belmonte, J. C., Bueren, J. A., (2014). Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: Implications in cell reprogramming and stem cell therapy Stem Cells 32, (2), 436-446

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2Δ27/Δ27), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2Δ27/Δ27 induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2Δ27/Δ27 iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2 Δ27/Δ27 mouse embryonic fibroblasts. Gene-corrected Brca2Δ27/Δ27 iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2Δ27/Δ27 recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2Δ27/Δ27 iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Keywords: Bone marrow aplasia, Cellular therapy, Fanconi anemia, Gene therapy, Hematopoietic stem cells, Induced pluripotent stem cells


Orenstein, Samantha J., Kuo, Sheng-Hang, Tasset, Inmaculada, Arias, Esperanza, Koga, Hiroshi, Fernandez-Carasa, Irene, Cortes, Etty, Honig, Lawrence S., Dauer, William, Consiglio, Antonella, Raya, Angel, Sulzer, David, Cuervo, Ana Maria, (2013). Interplay of LRRK2 with chaperone-mediated autophagy Nature Neuroscience 16, (4), 394-406

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. We found LRRK2 to be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, was poorly degraded by this pathway. In contrast to the behavior of typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins was enhanced in the presence of other CMA substrates, which interfered with the organization of the CMA translocation complex, resulting in defective CMA. Cells responded to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor, as seen in neuronal cultures and brains of LRRK2 transgenic mice, induced pluripotent stem cell–derived dopaminergic neurons and brains of Parkinson's disease patients with LRRK2 mutations. This newly described LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in Parkinson's disease by compromising the degradation of


Sánchez-Danes, A., Benzoni, P., Memo, M., Dell'Era, P., Raya, A., Consiglio, A., (2013). Induced pluripotent stem cell-based studies of Parkinson's disease: Challenges and promises CNS and Neurological Disorders - Drug Targets 12, (8), 1114-1127

A critical step in the development of effective therapeutics to treat Parkinson's disease (PD) is the identification of molecular pathogenic mechanisms underlying this chronically progressive neurodegenerative disease. However, while animal models have provided valuable information about the molecular basis of PD, the lack of faithful cellular and animal models that recapitulate human pathophysiology is delaying the development of new therapeutics. The reprogramming of somatic cells to induced pluripotent stem cells (iPSC) using delivery of defined combinations of transcription factors is a groundbreaking discovery that opens great opportunities for modeling human diseases, including PD, since iPSC can be generated from patients and differentiated into disease-relevant cell types, which would capture the patients' genetic complexity. Furthermore, human iPSC-derived neuronal models offer unprecedented access to early stages of the disease, allowing the investigation of the events that initiate the pathologic process in PD. Recently, human iPSC-derived neurons from patients with familial and sporadic PD have been generated and importantly they recapitulate some PD-related cell phenotypes, including abnormal α-synuclein accumulation in vitro, and alterations in the autophagy machinery. This review highlights the current PD iPSC-based models and discusses the potential future research directions of this field.

Keywords: Human cellular model, Induced pluripotent stem cells, Neurodegenerative disease, Parkinson's disease


Mora, S., Raya, A., (2013). Dedifferentiation, transdifferentiation, and reprogramming Stem Cells in Reproductive Medicine (ed. Simón, C., Pellicer, A., Pera, R.R.), Cambridge University Press (Cambridge, UK) Basic Science and Therapeutic Potential, 152-163

Stem cell science has the potential to impact human reproductive medicine significantly – cutting edge technologies allow the production and regeneration of viable gametes from human stem cells offering potential to preciously infertile patients. Written by leading experts in the field Stem Cells in Reproductive Medicine brings together chapters on the genetics and epigenetics of both the male and female gametes as well as advice on the production and regeneration of gene cells in men and women, trophoblasts and endometrium from human embryonic and adult stem cells. Although focussing mainly on the practical elements of the use of stem cells in reproductive medicine, the book also contains a section on new developments in stem cell research. The book is essential reading for reproductive medicine clinicians, gynecologists and embryologists who want to keep abreast of practical developments in this rapidly developing field.


Sánchez-Danés, A., Richaud-Patin, Y., Carballo-Carbajal, I., Jiménez-Delgado, S., Caig, C., Mora, S., Di Guglielmo, C., Ezquerra, M., Patel, B., Giralt, A., Canals, J. M., Memo, M., Alberch, J., López-Barneo, J., Vila, M., Cuervo, A. M., Tolosa, E., Consiglio, A., Raya, A., (2012). Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease EMBO Molecular Medicine 4, (5), 380-395

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.

Keywords: Autophagy, Disease modeling, LRRK2 mutation, Neurodegeneration, Pluripotent stem cells


McLenachan, S., Menchon, C., Raya, A., Consiglio, A., Edel, M. J., (2012). Cyclin A(1) is essential for setting the pluripotent state and reducing tumorigenicity of induced pluripotent stem cells Stem Cells and Development 21, (15), 2891-2899

The proper differentiation and threat of cancer rising from the application of induced pluripotent stem (iPS) cells are major bottlenecks in the field and are thought to be inherently linked to the pluripotent nature of iPS cells. To address this question, we have compared iPS cells to embryonic stem cells (ESCs), the gold standard of ground state pluripotency, in search for proteins that may improve pluripotency of iPS cells. We have found that when reprogramming somatic cells toward pluripotency, 1%-5% of proteins of 5 important cell functions are not set to the correct expression levels compared to ESCs, including mainly cell cycle proteins. We have shown that resetting cyclin A1 protein expression of early- passage iPS cells closer to the ground state pluripotent state of mouse ESCs improves the pluripotency and reduces the threat of cancer of iPS cells. This work is a proof of principle that reveals that setting expression of certain proteins correctly during reprogramming is essential for achieving ESC- state pluripotency. This finding would be of immediate help to those researchers in different fields of iPS cell work that specializes in cell cycle, apoptosis, cell adhesion, cell signaling, and cytoskeleton.

Keywords: Self-renewal, IPS cells, Ground-state, C-MYC, Generation, Pathway, Disease, Mice, Link, P53


Sánchez-Danés, A., Consiglio, A., Richaud, Y., Rodríguez-Pizà , I., Dehay, B., Edel, M., Bové, J., Memo, M., Vila, M., Raya, A., Izpisua Belmonte, J. C., (2012). Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells Human Gene Therapy 23, (1), 56-69

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer great hope for in vitro modeling of Parkinson’s disease (PD), as well as for designing cell-replacement therapies. To realize these opportunities, there is an urgent need to develop efficient protocols for the directed differentiation of hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we use lentiviral vectors to drive the expression of LMX1A, which encodes a transcription factor critical for ventral midbrain identity, specifically in neural progenitor cells. We show that clonal lines of hESC engineered to contain one or two copies of this lentiviral vector retain long-term selfrenewing ability and pluripotent differentiation capacity. Greater than 60% of all neurons generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9 subtype, compared with *10% in green fluorescent protein–engineered controls, as judged by specific marker expression and functional analyses. Moreover, DA neuron precursors differentiated from LMX1A-engineered hESC were able to survive and differentiate when grafted into the brain of adult mice. Finally, we provide evidence that LMX1A overexpression similarly increases the yield of DA neuron differentiation from human iPSC. Taken together, our data show that stable genetic engineering of hESC/iPSC with lentiviral vectors driving controlled expression of LMX1A is an efficient way to generate enriched populations of human A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD and may be helpful for designing future cell-replacement strategies.


Ranaldo, Gabriella, Richaud-Patin, Yvonne, Lombardo, Angelo, Grosso, Chantal, Talmon, Maria, Raya, Angel, Naldini, Luigi, Schinco, Piercarla, Follenzi, Antonia, (2012). A novel iPSC-based strategy to correct the bleeding phenotype in Hemophilia A Molecular Therapy 15th Annual Meeting of the American Society of Gene and Cell Therapy (ASGCT) , Nature Publishing group (Philadelphia, USA) 20, S251-S251

Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Currently, there is no defi nitive cure. Therefore, cell and gene therapy may represent powerful solutions for the permanent treatment of HA. Reprogramming of genetically corrected somatic cells can be used to generate high amount of autologous, disease-free induced Pluripotent Stem Cells (iPSC), which can be then differentiated into progenitor cells relevant for gene and cell therapy applications. Towards this goal, we fi rst generated iPSC from human fi broblasts derived from healthy donors by retroviral transduction with four factors (OCT4, KLF4, SOX2 and c-MYC). These cells were phenotypically similar to human embryonic stem cells (hESC): colonies were compact, uniform and with defi ned borders when grown on feeder cells and expressed specifi c stem cell markers such as alkaline phosphatase, Nanog, Oct3/4, Sox2, TRA-1-60, TRA-1-81 and SSEA 3/4. iPSC were competent for differentiation into cell types of the three germ layers. Importantly, iPSC differentiated into endothelial cells (EC), a cell type that, when transplanted in HA mice, allows correcting the hemorrhagic phenotype of this model. iPSC can differentiate into EC acquiring a typical endothelial-like morphology with increased expression of CD31, KDR and FVIII. Moreover, after differentiation these cells were amenable to gene transfer by LV expressing the clotting human B-domain-deleted FVIII (hBDD-FVIII) under control of an endothelial-specifi c VE-cadherin promoter. Using this method we obtained iPSC, but they cannot be used for future therapeutic approach for the risk of reactivation of the reprogramming genes in the iPSC progeny. Thus, we recently reprogrammed human and mouse fi broblasts with a Cre-excisable LV expressing OCT4, KLF4 and SOX2. One month after transduction, colonies displayed a hESC-like morphology and stained positive for embryonic stem cell markers. RT-PCR and WB analyses showed activation of the endogenous reprogramming factors in iPSC. Given these results, we reprogrammed HA mouse fi broblasts into iPSC both before and after correction with a LV expressing hBDD-FVIII under control of the PGK promoter. Importantly, corrected iPSC expressed detectable BDD-FVIII in reprogrammed cells that were differentiated in EC. However, in hemophilic patients, to harvest fi broblasts from skin biopsies is risky; for this reason, we utilized peripheral blood cells as an easy-to-access source of cells and reprogrammed mononuclear cells from donors and hemophilic patients with HA. After genetic correction with LV and Cre-mediated excision of the reprogramming vector, the iPSC will be differentiated into EC and transplanted into NOD-SCID HA mice. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSC to confi rm the suitability of this approach for hemophilia gene-cell-therapy.


Woods, N. B., Parker, A. S., Moraghebi, R., Lutz, M. K., Firth, A. L., Brennand, K. J., Berggren, W. T., Raya, A., Belmonte, J. C. I., Gage, F. H., Verma, I. M., (2011). Brief report: Efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines Stem Cells 29, (7), 1158-1164

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% +/- 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.

Keywords: Differentiation, Hematopoiesis, Hematopoietic progenitors, Pluripotent stem cells


Veiga, A., Raya, A., Izpisúa, J. C., (2010). Stem cell research in Spain Revista Iberoamericana de Fertilidad y Reproduccion Humana XXVIII Congreso Nacional SEF (Sociedad Española de Fertilidad) , SEF (Valencia, Spain) 27, (Supplement 1), 81-83

A partir de cambios legi s l at ivos y de cambios en las estrategias de financiación, la investigación en células madre en España ha experimentado una progresión espectacular, combinándose la investigación básica con la translacional. Los avances en este campo pueden suponer enormes beneficios en pos del desarrollo de la medicina regenerativa.

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Equipment

  • hES/iPS cell culture station
  • Zebrafish transgenesis
  • Molecular biology facilities
  • Stereomicroscope for picking hES colonies
  • Cell culture facilities

Collaborations

  • Lorenzo Monserrat Complejo Hospitalario Universitario A Coruña, Spain
  • Ana Maria Cuervo Albert Einstein College of Medicine, Bronx, USA
  • Ludovic Jullien Ecole Normale Supérieure, Paris, France
  • Anne Weber/Anne Dubart Inserm, Le Kremlin-Bicêtre Cedex (France)
  • Manuel Galiñanes Hospital Universitari Vall d’Hebron, Barcelona
  • Patrizia Dell’Era Università degli Studi di Brescia (Italy)
  • Miquel Vila Institut de Recerca, Hospital Universitari Vall d’Hebron, Barcelona (Spain)
  • Eduard Tolosa Hospital Clínic, Barcelona (Spain)
  • Pedro Muniesa Facultad de Veterinaria, Zaragoza (Spain)
  • José López Barneo IBiS, Sevilla (Spain)
  • Daniel Grinberg/Lluïsa Vilageliu University of Barcelona (Spain)
  • Rafael Garesse Instituto de Investigaciones Biomédicas “Alberto Sols”/UAM (Spain)
  • Dra. Antonia Follenzi Universita’ del Piemonte Orientale, Novara (Italy)
  • Sheng Ding Scripps Research Institute, La Jolla (USA)
  • Jordi Barquinero Institut de Recerca, Hospital Universitari Vall d’Hebron, Barcelona (Spain)
  • Jordi Alberch/Josep M. Canals IDIBAPS, University of Barcelona (Spain)
  • Antoni Bayés-Genís Hospital de la Santa Creu i Sant Pau, Barcelona
  • Jerónimo Blanco & Núria Rubio Centro de Investigación Cardiovascular CSIC-ICCC, Barcelona
  • Francisco J. Blanco Complejo Hospitalario Universitario A Coruña
  • Juan Bueren CIEMAT, Madrid
  • Antonella Consiglio IBUB, Universitat de Barcelona

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