La Paz Hospital Research Institute

Madrid, Spain

La Paz Hospital Research Institute

Madrid, Spain
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Alio del Barrio J.L.,Ramon y Cajal Hospital | Chiesa M.,La Paz Hospital Research Institute | Garagorri N.,Tecnalia | Garcia-Urquia N.,Tecnalia | And 8 more authors.
Experimental Eye Research | Year: 2015

Purpose: To evaluate the invivo biocompatibility of grafts composed of sheets of decellularized human corneal stroma with or without the recellularization of human adipose derived adult stem cells (h-ADASC) into the rabbit cornea. Methods: Sheets of human corneal stroma of 90μm thickness were decellularized, and their lack of cytotoxicity was assayed. The recellularization was achieved by the injection of 2×105 labeled h-ADASC in the graft followed by five days of cell culture. The grafts were implanted invivo into a stromal pocket at 50% depth. After a triple-masked three-month follow-up, the animals were euthanized and the biointegration of the graft, the viability of the stem cells and the expression of keratocan (human keratocyte-specific protein) were assessed. Results: The decellularized stromal sheets showed an intact extracellular matrix with a decellularization rate of 92.8% and an excellent recellularization capacity invitro with h-ADASC. A complete and stable graft transparency was observed during the full follow-up, with absence of any clinical sign of rejection. The postmortem analysis demonstrated the survival of the transplanted human stem cells inside the graft and their differentiation into functional keratocytes, as assessed by the expression of human keratocan. Conclusions: We report a new model of lamellar keratoplasty that requires only a simple and safe procedure of liposuction and a donor allogeneic cornea to provide an optically transparent autologous stromal graft with excellent biocompatibility and integration into the host tissue in a rabbit model. © 2015 Elsevier Ltd.


Arnalich-Montiel F.,Ramon y Cajal Hospital | Arnalich-Montiel F.,Vissum Madrid Eye Hospital | Arnalich-Montiel F.,Hospital Ramon Y Cajal Of Madrid | Hernandez-Verdejo J.L.,Vissum Madrid Eye Hospital | And 3 more authors.
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2013

Background: Descemet membrane endothelial keratoplasty (DMEK) is being proposed as the procedure of choice in corneal endothelial disease as it achieves better visual and refractive outcomes than Descemet stripping automated endothelial keratoplasty (DSAEK). Nevertheless, primary graft failure is frequent, especially during the learning curve, and secondary back-up procedure consists on DSAEK. We aim to compare corneal haze and visual acuity of patients undergoing primary DSAEK vs. patients undergoing DSAEK as a back-up procedure after primary DMEK failure. Methods: This study is a comparative case series that included 19 eyes from 16 patients with early stages of corneal failure and limitation of daily activities after primary DSAEK or secondary DSAEK. A control group of non-operated corneas included 10 aged-matched normal eyes. The study was conducted at University Hospital Ramón y Cajal and Vissum Hospital, Madrid, Spain. Corneal densitometry readings and postoperative best-corrected visual acuity in subjects with primary and secondary DSAEK were recorded 6 months after the surgery using the Pentacam Scheimpflug system (Oculus, inc.,Wetzlar, Germany). Results: In primary DSAEK median densitometry values (range) were statistically significantly higher (p < 0.05) than normal subjects for the full thickness, posterior and anterior part of the paracentral cornea; and the anterior part of the central cornea. In secondary DSAEK, median densitometry values were statistically significantly higher than normal subjects at all levels of the central and paracentral cornea. In secondary DSAEK, median densitometry values (range) were statistically significantly higher than in primary DSAEK in the full-thickness, anterior part and interface of the central cornea and in the full-thickness and posterior part of the paracentral cornea. Median visual acuity between groups (p = 0.47) was statistically better for the primary DSAEK group, which also had a higher percentage of patients achieving BCVA of ≥ 20/40 and ≥20/25 than the secondary DSAEK group (100 % vs. 62 % and 60 % vs. 0 % respectively). Conclusions: There is an increase in central corneal light scattering after secondary DSAEK performed after a failed DMEK as compared to primary DSAEK. This has a negative impact on final visual acuity that needs to be considered in each patient when starting DMEK surgery. © 2013 Springer-Verlag Berlin Heidelberg.


Nistal M.,Autonomous University of Madrid | Gonzalez-Peramato P.,Autonomous University of Madrid | De Miguel M.P.,La Paz Hospital Research Institute
Reproductive Toxicology | Year: 2013

Studies over the last years show an increase in testicular cancer, hypospadias and cryptorchidism in industrial countries, leading to the concept of testicular dysgenesis syndrome (TDS). It is hypothesized that TDS is caused by estrogen and antiandrogen exposure during fetal life, accompanied by incomplete maturation of testicular Sertoli cells (SC). However, it is not known if SC disruption is a primary cause or a response to fetal Leydig cell testosterone production changes.To determine if SC differentiation is directly affected by estrogens, we compared SC maturation between adult gender reassignment cases exposed to estrogen and antiandrogen therapy, and those of typical TDS in adult cryptorchidism.We found similar expression of immature SC markers M2A antigen, inhibin bodies and Anti Mullerian Hormone, and the absence of maturation marker androgen receptor in SC of both types of patients. These data supports the occurrence of true SC dedifferentiation caused by estrogen exposure in adult humans. Our data also suggests that SC maturation is directly disrupted in TDS. © 2013 Elsevier Inc.


de Miguel M.P.,La Paz Hospital Research Institute | Fuentes-Julian S.,La Paz Hospital Research Institute | Blazquez-Martinez A.,La Paz Hospital Research Institute | Pascual C.Y.,Infanta Sofia Hospital | And 3 more authors.
Current Molecular Medicine | Year: 2012

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-β, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory. © 2012 Bentham Science Publishers.


Lopez-Iglesias P.,La Paz Hospital Research Institute | Alcaina Y.,La Paz Hospital Research Institute | Tapia N.,Max Planck Institute for Molecular Biomedicine | Sabour D.,Max Planck Institute for Molecular Biomedicine | And 11 more authors.
Antioxidants and Redox Signaling | Year: 2015

To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). Results: We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified was the Yamanaka genes but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. Hypoxia induced a glycolytic program in PGCs in detriment of mitochondrial oxidative phosphorylation. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGLs), which differentiate into cells of the three germ layers in vitro and contribute to the internal cell mass of the blastocyst in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1α stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to the lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signaling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. Innovation and Conclusion: Our data suggest that the two main properties of stemness, pluripotency and self-renewal, are differentially regulated in PGC reprogramming induced by hypoxia. Antioxid. Redox Signal. 22, 205-223. © 2015 Mary Ann Liebert, Inc.


de Miguel M.P.,La Paz Hospital Research Institute | Fuentes-Julian S.,La Paz Hospital Research Institute | Alcaina Y.,La Paz Hospital Research Institute
Stem Cell Reviews and Reports | Year: 2010

Pluripotency is defined as the potential of a cell to differentiate into cells of the three germ layers: endoderm, mesoderm and ectoderm. In vivo, the presence of pluripotent stem cells is transient during the very early embryo. However, immortal cell lines with the same properties can be obtained in vitro and grown indefinitely in laboratories under specific conditions. These cells can be induced to differentiate into all the cell types of the organism through different assays, thereby proving their functional pluripotency. This review focuses on the pluripotent stem cells of mammals, giving special attention to the comparison between mouse and human. In particular, embryonic stem cells, epiblast-derived stem cells, primordial germ cells, embryonic germ cells, very small embryonic-like cells and induced pluripotent stem cells will be compared in terms of the following: in vivo specification and location; surface and intracellular markers; in vitro dependence on growth factors; signal transduction pathways; epigenetic characteristics; and pluripotency genes and functional assays. © 2010 Springer Science+Business Media, LLC.


De Miguel M.P.,La Paz Hospital Research Institute | Alcaina Y.,La Paz Hospital Research Institute | De Sainz La Maza D.,La Paz Hospital Research Institute | Lopez-Iglesias P.,La Paz Hospital Research Institute
Current Molecular Medicine | Year: 2015

Hypoxia is defined as a reduction in oxygen supply to a tissue below physiological levels. However, physiological hypoxic conditions occur during early embryonic development; and in adult organisms, many cells such as bone marrow stem cells are located within hypoxic niches. Thus, certain processes take place in hypoxia, and recent studies highlight the relevance of hypoxia in stem cell cancer physiology. Cellular response to hypoxia depends on hypoxia-inducible factors (HIFs), which are stabilized under low oxygen conditions. In a hypoxic context, various inducible HIF alpha subunits are able to form dimers with constant beta subunits and bind the hypoxia response elements (HRE) in the genome, acting as transcription factors, inducing a wide variety of gene expression. Typically, the HIF pathway has been shown to enhance vascular endothelial growth factor (VEGF) expression, which would be responsible for angiogenesis and, therefore, re-oxygenation of the hypoxic sites. Embryonic stem cells inhibit a severely hypoxic environment, which dictates their glycolytic metabolism, whereas differentiated cells shift toward the more efficient aerobic respiration for their metabolic demands. Accordingly, low oxygen tension levels have been reported to enhance induced pluripotent stem cell (iPS) generation. HIFs have also been shown to enhance pluripotency-related gene expression, including Oct4 (Octamer-binding transcription factor 4), Nanog and Wnt. Therefore, cell metabolism might play a role in stemness maintenance, proliferation and cell reprogramming. Moreover, in the hypoxic microenvironment of cancer cells, metabolism shifts from oxidative phosphorylation to anaerobic glycolysis, a process known as the Warburg effect, which is involved in cancer progression and malignancy. © 2015, Current Cancer Drug Targets, All rights Reserved.


Nistal M.,La Paz Hospital Research Institute | Pastrian L.G.,La Paz Hospital Research Institute | Gonzalez-Peramato P.,La Paz Hospital Research Institute | De Miguel M.P.,La Paz Hospital Research Institute
Histopathology | Year: 2011

Aims: To provide a marker for immature and dysgenetic Sertoli cells which allows easy identification in patients in which Sertoli cell maturation does not take place properly, such as those consulting for cryptorchidism, testicular tumours and infertility. Methods and results: We performed immunohistochemistry against inhibin-α subunit and the endoplasmic reticulum marker Grp78 in normal human testes from fetal life to adulthood, and in several testicular lesions where Sertoli cell maturation is abnormal. We describe a pattern of inhibin immunostain (inhibin bodies of 2-9μm in diameter at the Sertoli cells cytoplasm apical pole) in immature and dysgenetic Sertoli cells that facilitates their identification. Inhibin bodies were found in tubules with either no germ cells or only spermatogonia or carcinoma in situ (CIS) and seminoma cells, but not in tubules containing more advanced germ cells. Conclusions: Our data provide a new marker of immature and dysgenetic Sertoli cells. In addition, our data suggest that inhibin bodies represent a slower transit of inhibin through the endoplasmic reticulum, as inhibin bodies were associated with Grp78. © 2011 Blackwell Publishing Limited.


PubMed | La Paz Hospital Research Institute
Type: Journal Article | Journal: Current molecular medicine | Year: 2012

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor- and interferon- might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohns disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.


PubMed | La Paz Hospital Research Institute, IdiPAZ and Ramon y Cajal Hospital Research Institute
Type: Journal Article | Journal: PloS one | Year: 2015

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.

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