Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of PlasmocinTM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days PlasmocinTM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with PlasmocinTM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that PlasmocinTM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.Fil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Riva, Diego Ariel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Videla Guillermo, Richardson. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

AbstractWe studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24–48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P5-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166(low/neg)CD146(low/neg)CD73(+)CD44(low)BMPR1B(+)) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5-6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166(low/neg)BMPR1B(+) putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering.Fil: Wu, Ling. University of California at Los Angeles; Estados UnidosFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of California at Los Angeles; Estados UnidosFil: Kyupelyan, Levon. University of California at Los Angeles; Estados UnidosFil: Latour, Brooke. University of California at Los Angeles; Estados UnidosFil: Gonzalez, Stephanie. University of California at Los Angeles; Estados UnidosFil: Shah, Saumya. University of California at Los Angeles; Estados UnidosFil: Galic, Zoran. University of California at Los Angeles; Estados UnidosFil: Ge, Sundi. University of California at Los Angeles; Estados UnidosFil: Zhu, Yuhua. University of California at Los Angeles; Estados UnidosFil: Petrigliano, Frank A.. University of California at Los Angeles; Estados UnidosFil: Nsair, Ali. University of California at Los Angeles; Estados UnidosFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Li, Xinmin. University of California at Los Angeles; Estados UnidosFil: Lyons, Karen M.. University of California at Los Angeles; Estados UnidosFil: Crooks, Gay M.. University of California at Los Angeles; Estados UnidosFil: McAllister, David R.. University of California at Los Angeles; Estados UnidosFil: Van Handel, Ben. Novogenix Laboratories; Estados UnidosFil: Adams, John S.. University of California at Los Angeles; Estados UnidosFil: Evseenko, Denis. University of California at Los Angeles; Estados Unido

Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells

MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.Fil: Garate, Ximena. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neiman, Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santín Velazque, Natalia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Luzzani, Carlos Daniel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P5-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Novel aspects of parenchymal–mesenchymal interactions: from cell types to molecules and beyond

Mesenchymal stem or stromal cells (MSCs) were initially isolated from the bone marrow and received their name on the basis of their ability to differentiate into multiple lineages such as bone, cartilage, fat and muscle. However, more recent studies suggest that MSCs residing in perivascular compartments of the small and large blood vessels play a regulatory function supporting physiologic and pathologic responses of parenchymal cells, which define the functional representation of an organ or tissue. MSCs secrete or express factors that reach neighbouring parenchymal cells via either a paracrine effect or a direct cell-to-cell interaction promoting functional activity, survival and proliferation of the parenchymal cells. Previous concept of ´epithelial-stromal´ interactions can now be widened. Given that MSC can also support hematopoietic, neuronal and other non-epithelial parenchymal lineages, terms ´parenchymal-stromal´ or ´parenchymal-mesenchymal´ interactions may better describe the supportive or ´trophic´ functions of MSC. Importantly, in many cases, MSCs specifically provide supportive microenvironment for the most primitive stem or progenitor populations and therefore can play a role as ´stem/progenitor niche´ forming cells. So far, regulatory roles of MSCs have been reported in many tissues. In this review article, we summarize the latest studies that focused on the supportive function of MSC. This thread of research leads to a new perspective on the interactions between parenchymal and mesenchymal cells and justifies a principally novel approach for regenerative medicine based on co-application of MSC and parenchymal cell for the most efficient tissue repair.Fil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wu, Ling. University of California at Los Angeles; Estados UnidosFil: Petrigliano, Frank. University of California at Los Angeles; Estados UnidosFil: McAllister, David. University of California at Los Angeles; Estados UnidosFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Evseenko, Denis A.. University of California at Los Angeles; Estados Unido

Leukemia Inhibitory Factor Increases Survival of Pluripotent Stem Cell-Derived Cardiomyocytes

Leukemia inhibitory factor (LIF) is a growth factor with pleiotropic biological functions. It has been reported that LIF acts at different stages during mesoderm development. Also, it has been shown that LIF has a cytoprotective effect on neonatal murine cardiomyocytes (CMs) in culture, but little is known about the role of LIF during human cardiogenesis. Thus, we analyzed the effects of LIF on human pluripotent stem cells (PSC) undergoing cardiac differentiation. We first showed that LIF is expressed in the human heart during early development. We found that the addition of LIF within a precise time window during the in vitro differentiation process significantly increased CMs viability. This finding was associated to a decrease in the expression of pro-apoptotic protein Bax, which coincides with a reduction of the apoptotic rate. Therefore, the addition of LIF may represent a promising strategy for increasing CMs survival derived from PSCs.Fil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Evseenko, Denis. University Of Southern California; Estados UnidosFil: Garate, Ximena. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neiman, Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

2 Role of histone H3 lysine 9 trimethylation during bovine pre-implantation embryonic development

Histones play an important role in DNA’s compaction and organisation into the cellular nucleus. Depending on which histone modification occurs, chromatin can take a conformation of heterochromatin or euchromatin, which are associated with gene repression or expression, respectively. Histone H3 lysine 9 (H3K9) trimethylation (H3K9me3) is associated with gene silencing. At least 3 methyltransferases are able to change the methylation status of H3K9: SUV39H1, SUV39H2, and SETDB1. In several mammalian species, modulation of H3K9 methylation status has been demonstrated to be necessary to achieve a successful pre-implantation embryonic development after IVF or somatic cell NT. The aim of this work was to study the role of H3K9me3 in IVF pre-implantation bovine embryos. For this purpose, immunostaining of H3K9me3 at different pre-implantation stages of development was performed. Further, the relative abundances of the methyltransferases SUV39H1 and SUV39H2 were measured by real-time PCR using luciferase transcript as an exogenous gene for normalization. Finally, to evaluate H3K9me3 involvement during pre-implantation embryonic development, we generated SUV39H1 or SUV39H2 knockout embryos by the CRISPR/Cas9 system. We designed guide RNA targeting SUV39H1 or SUV39H2 and co-injected the presumptive zygote’s cytoplasm 18 h post-fertilization with Cas9 protein. At Day 8 post-fertilization, the number of blastocysts was assessed and embryos were immunostained to evaluate H3K9me3. Results were analysed using Student’s t-test or ANOVA with the post-hoc Tukey test depending on data set (P ≤ 0.05) and reported as means ± standard errors of the mean. Oocytes at germinal vesicle stage and metaphase II as well as embryos at different stages of pre-implantation development (2, 4, and 8 cells, morula, and blastocyst; n = 6) were immunoreactive for H3K9me3. Expression of SUV39H1 and SUV39H2 mRNA decreased significantly as embryonic development progressed, reaching undetectable levels at stages where genome activation had already occurred (morula and blastocyst; P < 0.0001, n = 3). When zygotes were co-injected with the guide RNA targeting SUV39H1/Cas9, embryonic production showed a significant increase compared with the control [42.26% ± 5.03 (28/65) v. 23.86% ± 3.99 (21/88), respectively; P = 0.034, n = 4], and H3K9me3 immunostaining was reduced in treated embryos. Editing efficiency was estimated at 66%. In contrast, no statistical differences were found in embryonic production or H3K9me3 immunostaining in embryos co-injected with the guide RNA targeting SUV39H2/Cas9 (P = 0.57, n = 3). In conclusion, we were able to characterise H3K9me3 and determine transcript levels of methyltransferases SUV39H1 and SUV39H2 in oocytes and different stages of pre-implantation embryonic development. We also demonstrated that SUV39H1 deletion led to an increased embryonic production, suggesting that H3K9me3 removal would allow a greater relaxation of the heterochromatin and consequently a successful activation of embryonic genes. This highlights the essential role of H3K9me3 during bovine pre-implantation embryonic development.Fil: Navarro, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bluguermann, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Von Meyeren, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bariani, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Osycka Salut, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina45th Annual Conference of the IETS (International Embryo Technology Society)Nueva OrleansEstados UnidosInternational Embryo Technology Societ

Evaluation of testicular sperm CRISP2 as a potential target for contraception

Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.Fil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bluguermann, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Busso, Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin