'Ex vivo' gene correction of PRPF31 c.165G>A mutation causing retinitis pigmentosa

Motivation: Retinitis pigmentosa (RP) is the most common form of retinal dystrophy, a group of blinding diseases characterized by progressive photoreceptor death, with a prevalence of 1 in 4000. RP is highly-heterogeneous, with 15% of autosomal dominant cases caused by mutations in the pre-mRNA processing factors (PRPFs), components of the spliceosome.To date, there are no effective treatments for RP. Gene editing is a rapidly evolving field that may in the future, allow the repair of a mutated endogenous locus. CRISPR/Cas9 system has a mechanism of action based on nucleotide recognition of target DNA by engineered single-guide RNA (sgRNA) and Cas9 endonuclease activity. Genomic edition of patient-derived induced pluripotent stem cells (iPSCs) would allow autologous transplantation of repaired cells, once differentiated to retinal cell types.Methods: iPSCs obtained from a RP patient with a PRPF31 c.165G>A mutation were the starting biological material. Pluripotency of the iPSCs was checked by inmunofluorescence (IF) analysis.Disease phenotyping of the cell line was performed by IF for PRPF31 and for the ciliary protein ARL13B, as PRPF31 mutations have been previously described to affect cilia.sgRNAs directed to the mutation were designed using the web crispor.tefor.net. The best sgRNA and a ssODN template, covering the mutation site, were synthesized by IDT. The sgRNA-CRISPR/Cas9 complex was assembled and co-transfected with the ssODN into the iPSCs. FACs was used to measure the efficiency and to select transfected cells. A bulk transfected cell population was analyzed by Sanger sequencing to check for HR-mediated knock-in. Selection of individual iPSC clones and genotyping is being performed to search  for corrected clones.Results: Positive labeling for OCT4, NANOG, SSEA3, SSEA4 and TRA-1-81 showed pluripotency of the iPSC line. PRPF31 immunolocalization and quantificacion have been used to phenotype the iPSC line compared to a healthy control. Sanger sequencing of the genomic DNA showed successful editing of the mutation in the bulk population of transfected cells. Different culture conditions were tested for iPSC clonal selection. Best conditions provided a 0.8 % of efficiency. The CRISPR/Cas9-corrected iPSC clone will be differentiated to retinal pigment epithelium (RPE) and photoreceptors, in parallel with uncorrected PRPF31-iPSCs, to establish if in situ gene editing restores key celular and functional phenotypes associated with this type of RP

Draft genome sequence of three antibiotic-resistant Leuconostoc mesenteroides strains of dairy origin

Leuconostoc mesenteroides is a lactic acid bacterium (LAB) commonly associated with fermented foods. Here, we report the genome sequence of three selected dairy strains, showing atypical antibiotic resistances (AR). Genome analysis provided a better understanding of the genetic bases of AR in Leuconostoc and its potential transferability among foodborne bacteria.Peer Reviewe

Generation of a human iPS cell line from a patient with retinitis pigmentosa due to EYS mutation

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease. Mutations in EYS have been associated with autosomal recessive RP. The human iPS cell line, CABi002-A, derived from peripheral blood mononuclear cells from a patient carrying a heterozygous double mutation in EYS gene was generated by non-integrative reprogramming technology, using hOCT3/4, hSOX2, hc-MYC and hKLF4 reprogramming factors. Pluripotency and differentiation capacity were assessed by immunocytochemistry and RT-PCR. This iPSC line can be further differentiated towards the affected cells to understand the pathophysiology of the disease and test new therapeutic strategies.Cellex FoundationFundación Progreso y Salu

Optimization of brain organoids as models for the study of neurodegenerative diseases

ABSTRACTMotivation: One of the current challenges faced by neuroscience is the limited availability of in vitro models of neurodegenerative diseases, as there are notable anatomical and molecular differences among murine and human brain. As a result, significant efforts have been made towards the development of new models based on human cells, with cerebral organoids standing out as a particularly promising approach. Brain organoids are 3D models usually developed from induced pluripotent stem cells (iPSCs) that simulate the composition and cytoarchitecture of different regions of the human brain. They may allow us to obtain in vitro information about the human brain, which makes them a valuable model for investigating neurodegenerative diseases. Nevertheless, they present disadvantages associated with the absence of essential components for their development and functionality. Therefore, we will investigate the effect of incorporating an extracellular matrix (ECM) from human and pig brain into the culture of these organoids, as it contains specific combinations of components that play a role in multiple neuronal processes. On the other hand, in order to find the optimal model for generating this organoids, two protocols, Lancaster (1) and Rosebrock (2), have been compared. Lancaster’s protocol is the most cited for brain organoids, but following it, other embryonic layers are developed. Rosebrock’s protocol, is a modification of the Lancaster’s protocol, in which SMAD pathway inhibitors are used to avoid the formation of non-ectodermal layers.Methods: We generated brain organoids from iPSC, following two protocols: Lancaster (1) and Rosebrock (2). Two ECM conditions were used during cultures: Matrigel versus ECM obtained from pig brain. Subsequently, the addition of human ECM will be tested. Finally, organoids are being characterized using different techniques such as immunofluorescence, RT-PCR and expression arrays.Results: We have found molecular differences among brain organoids obtained following the different protocols, as those obtained following Rosebrock’s protocol express fewer endodermal and mesodermal markers than those obtained with Lancaster's protocol. As well, we have identified different features between those organoids matured with ECM and those matured only with Matrigel, the former being larger than the latter. Further studies will discern whether there are other relevant differences between the models

Molecular Identification and Quantification of Tetracycline and Erythromycin Resistance Genes in Spanish and Italian Retail Cheeses

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18 log10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.The study was partially supported by a Spain-Italy bilateral collaboration program (Ref. IT2009-0080 and IT105MD12L). Financial support was further provided by projects from CICYT (Ref. AGL2011-24300-ALI) and INIA (Ref. RM2011-00005-00-00). A. B. Flórez and S. Delgado were supported by research contracts under Juan de la Cierva Program (Ref. JCI-2010-07457 and JCI-2008-02391, resp.). A. Alegría was awarded a scholarship of the Severo Ochoa program from FICYT (Ref. BP08-053).Peer Reviewe

Whole genome analysis as a tool for the safety assessment of antibiotic resistance in food-processing bacteria

Trabajo presentado en la 2nd EFSA Scientific Conference, celebrado en Milán, Italia, del 14 al 16 de octubre de 2015Acquisition of antibiotic resistances (AR) by pathogens leads ultimately to a failure of antibiotic therapy. The food chain is considered a key player in the transmission of AR determinants to pathogens from reservoirs in commensal and beneficial bacteria. Therefore, the absence of transmissible AR genes in bacteria used as starter and adjunct cultures for food and feed processing is considered to be critical (EFSA, 2012; EFSA Journal, 10:2740). Genome sequencing allows the inspection of the whole genetic makeup of bacteria in the search for the basis of desirable and undesirable traits, including that of AR. Thus, in silico sequence analysis and comparison against databases can be used as a tool for the safety assessment of microorganisms intended to be used in food systems. This communication reports on the genome analysis of three Leuconostoc mesenteroides strains of dairy origin showing atypical resistances to tetracycline (LbT16), erythromycin and clindamycin (LbE15), and kanamycin, streptomycin, tetracycline and virginiamycin (LbE16). Genes encoding for erythromycin [erm(B)] and tetracycline [tet(S)] resistance had already been detected by PCR. Genome analysis confirmed the presence of these genes and identified others which encode uncommon AR in lactic acid bacteria. Analysis of the genes and their flanking regions revealed a potential of some to be horizontally transferred to other bacteria. This study demonstrates the effectiveness of combining genome sequencing and bioinformatics analysis as an affordable tool for the safety assessment of food bacteria. This innovative approach could become a novel paradigm in the selection programs of starters for the food industry.Peer Reviewe

Preclinical evaluation of the safety and potency of neural stem cells from the germinal zone (Gz-NSC) for the treatment of intraventricular hemorrhage (IVH) consequences.

Motivation: Intraventricular hemorrhage (IVH) is a common cause of morbidity and mortality in premature infants with no available treatment. After IVH, there is a rupture of the germinal zone into the ventricles that entail the loss of neural stem cells (Gz-NSC). These Gz-NSC can be retrieved from the cerebrospinal fluid (CSF) of IVH patients, obtained after the therapeutic neuroendoscopic lavage performed in these patients to decrease intracranial pressure and that is usually discarded. We have found that Gz-NSC have the potential to differentiate into neuroblasts, oligodendrocyte precursors and few astrocytes when grafted into human brain organoids from iPSCs and mouse brains (1,2). We are evaluating the safety and efficacy profile of CSF-derived Gz-NSC in order to develop a cell therapy for IVH patients.Methods: To examine the differentiation potency of Gz-NSC, we used immunofluorescence assays, fluorescence microspy techniques and computer analysis (ImageJ) to expand previous data, increase sample size, and quantify cell differentiation of grafted Gz-NSC cells in mouse brains and human brain organoids derived from iPSCs.To study the safety profile, flow cytometry assays were carried on to analyze Gz-NSC cell proliferation (Ki67) and immunogenicity (CD80,CD86,CD40, major histocompatibility complex class II (MHC-II)).Results: Based on the immunofluorescence assays, we have found less cells expressing doublecortin, an immature neural protein, and more cells expressing parvalbumin, an interneuron marker, in human brain organoids compared to animal models, suggesting that host can influence cell fate.On the other hand, in order to study the immunogenicity of the Gz-NSC (safety profile), we have analyzed the expression of MHC-II and co-stimulatory molecules (CD80, CD86, CD40) in Gz-NSC before and after in vitro differentiation. Flow cytometry assays revealed Gz-NSC do not express co-stimulatory molecules and express different levels of MHC-II that are reduced when differentiated in vitro, which decreases the probability of an immune response in a future Gz-NSC based cell therapyConclusions: Taking into account that Gz-NSC have the potency to differentiate to a wide range of cerebral cell linages in both, human organoids and animal models, and are weakly immunogenic, an autologous Gz-NSC cell therapy could be a promising opportunity for IVH patients to overcome some of the neurocognitive problems associated to their condition

Programa Intergeneracional de coaprendizaje: transmisión de valores en la etapa infantil a través de varias generaciones

Memoria ID12-0210. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2012-2013