Tips and Tricks for Successfully Culturing and Adapting Human Induced Pluripotent Stem Cells [Protocol]

Protocol[Abstract] Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications.We thank the laboratory staff from INIBIC-CHUAC, the Radio Physics department from the Oncology Center of Galicia and the Servicio de Xenética (CHUAC) for their assistance. We also thank the staff from Oza University Library (University of A Coruña) for their collaboration. We also thank our funding agencies: Instituto de Salud Carlos III-General Subdirection of Assessment and Promotion of the Research—European Regional Development Fund (FEDER) “A way of making Europe” (PI17/02197 and PI20/00933); Rede Galega deTerapia Celular and Grupos con Potencial de Crecemento, Xunta de Galicia (R2016/036, R2014/050, CN2012/142, ED431B 2020/55, and GPC2014/048); the University of A Coruña; M.P.-R. and S.R.-F. are granted by a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund) and C.S.-R. was beneficiary of a postdoctoral fellowship from Xunta de GaliciaXunta de Galicia; R2016/036Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; ED431B 2020/55Xunta de Galicia; GPC2014/04

Osteoprotegerin CGA Haplotype Protection against Cerebrovascular Complications in Anti-CCP Negative Patients with Rheumatoid Arthritis

INTRODUCTION: Rheumatoid arthritis is an inflammatory disease with high incidence of cardiovascular disease due to accelerated atherosclerosis. Osteoprotegerin (OPG) has been associated with increased risk of atherosclerotic disease in the general population. Several polymorphisms in the OPG gene with functional effects on cardiovascular disease in non-rheumatic individuals have been described. Therefore, we aimed to analyze the effect of three of these functional OPG polymorphisms on the risk of cardiovascular disease in a large and well-characterized cohort of Spanish patients with rheumatoid arthritis. METHODS: Three OPG gene variants (rs3134063, rs2073618 and rs3134069) were genotyped by TaqMan assays in 2027 Spanish patients with rheumatoid arthritis. Anti-cyclic citrullinated peptide (anti-CCP) antibody testing was positive in 997 of 1714 tested. Also, 18.3% of the whole series had experienced cardiovascular events, including 5.4% with cerebrovascular accidents. The relationship between OPG variants and cardiovascular events was assessed using Cox regression. RESULTS: No association between OPG gene variants and cardiovascular disease was observed in the whole group of rheumatoid arthritis patients or in anti-CCP positive patients. Nevertheless, a protective effect of CGA haplotype on the risk of cardiovascular disease in general, and specifically in the risk of cerebrovascular complications after adjusting for sex, age at disease diagnosis and traditional cardiovascular risk factors was disclosed in anti-CCP negative patients (HR = 0.54; 95%CI: 0.31-0.95; p = 0.032 and HR = 0.17; 95%CI: 0.04-0.78; p = 0.022, respectively). CONCLUSION: Our results indicate a protective effect of the OPG CGA haplotype on cardiovascular risk, mainly due to a protective effect against cerebrovascular events in anti-CCP negative rheumatoid arthritis patients

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case–control study

[Abstract] INTRODUCTION: Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti-cyclic citrullinated peptide-positive (anti-CCP+) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies. METHODS: A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described. RESULTS: Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term--the originally reported finding--or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP+ patients or with any of the other methods. CONCLUSIONS: None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists.Instituto de Salud Carlos III; 11/01048Instituto de Salud Carlos III; 12/01909Instituto de Salud Carlos III; RD12/0009/000

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case-control study

Introduction: Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti-cyclic citrullinated peptide-positive (anti-CCP+) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies. Methods: A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described. Results: Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term the originally reported finding or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP+ patients or with any of the other methods. Conclusions: None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists

Influence of elevated-CRP level-related polymorphisms in non-rheumatic Caucasians on the risk of subclinical atherosclerosis and cardiovascular disease in rheumatoid arthritis

Association between elevated C-reactive protein (CRP) serum levels and subclinical atherosclerosis and cardiovascular (CV) events was described in rheumatoid arthritis (RA). CRP, HNF1A, LEPR, GCKR, NLRP3, IL1F10, PPP1R3B, ASCL1, HNF4A and SALL1 exert an influence on elevated CRP serum levels in non-rheumatic Caucasians. Consequently, we evaluated the potential role of these genes in the development of CV events and subclinical atherosclerosis in RA patients. Three tag CRP polymorphisms and HNF1A, LEPR, GCKR, NLRP3, IL1F10, PPP1R3B, ASCL1, HNF4A and SALL1 were genotyped in 2,313 Spanish patients by TaqMan. Subclinical atherosclerosis was determined in 1,298 of them by carotid ultrasonography (by assessment of carotid intima-media thickness-cIMT-and presence/absence of carotid plaques). CRP serum levels at diagnosis and at the time of carotid ultrasonography were measured in 1,662 and 1,193 patients, respectively, by immunoturbidimetry. Interestingly, a relationship between CRP and CRP serum levels at diagnosis and at the time of the carotid ultrasonography was disclosed. However, no statistically significant differences were found when CRP, HNF1A, LEPR, GCKR, NLRP3, IL1F10, PPP1R3B, ASCL1, HNF4A and SALL1 were evaluated according to the presence/absence of CV events, carotid plaques and cIMT after adjustment. Our results do not confirm an association between these genes and CV disease in RA

SMAD3 rs17228212 Gene Polymorphism Is Associated with Reduced Risk to Cerebrovascular Accidents and Subclinical Atherosclerosis in Anti-CCP Negative Spanish Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis and increased risk of cardiovascular (CV) disease. Previous genome-wide association studies have described SMAD3 rs17228212 polymorphism as an important signal associated with CV events. The aim of the present study was to evaluate for the first time the relationship between this gene polymorphism and the susceptibility to CV manifestations and its potential association with the presence of subclinical atherosclerosis assessed by the evaluation of carotid intima-media thickness (cIMT) in patients with RA

Ingeniería Forestal y ambiental en medios insulares

Las Islas Canarias a pesar de su reducida extensión y del relativo poco peso específico a nivel mundial, no es ajena a los problemas globales detectados en la conservación de bosques y en la importancia que éstos tienen para obtener beneficios económicos, socioculturales y ambientales. La gestión forestal sostenible es en este sentido esencial para asegurar y compatibilizar los diversos beneficios del bosque. El papel específico de los bosques y su gestión son sin embargo temas aún por conocer en nuestras islas, por lo que el Año Internacional de los Bosques ha representado una oportunidad única para dar a conocer el mundo forestal y acercarlo a nuestra sociedad. El presente libro consta de 25 capítulos donde se ha contemplado la mayoría de los aspectos a tener en cuenta en la planificación y gestión del medio forestal y natural. Desde la historia forestal del archipiélago, hasta el uso y técnicas de manejo de los recursos naturales, incluyendo el agua, la energía en forma de biomasa y la selvicultura

Intermediate Molecular Phenotypes to Identify Genetic Markers of Anthracycline-Induced Cardiotoxicity Risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice (n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.J.P.L.’s lab is sponsored by Grant PID2020-118527RB-I00 funded by MCIN/AEI/10.13039/ 501100011039; Grant PDC2021-121735-I00 funded by MCIN/AEI/10.13039/501100011039 and by the “European Union Next Generation EU/PRTR”, the Regional Government of Castile and León (CSI144P20). J.P.L. and P.L.S. are supported by the Carlos III Health Institute (PIE14/00066). AGN laboratory and human patients’ studies are supported by an ISCIII project grant (PI18/01242). The Human Genotyping unit is a member of CeGen, PRB3, and is supported by grant PT17/0019 of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. SCLl is supported by MINECO/FEDER research grants (RTI2018-094130-B-100). CH was supported by the Department of Defense (DoD) BCRP, No. BC190820; and the National Cancer Institute (NCI) at the National Institutes of Health (NIH), No. R01CA184476. Lawrence Berkeley National Laboratory (LBNL) is a multi-program national laboratory operated by the University of California for the DOE under contract DE AC02-05CH11231. The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023 of the PE I + D +i, 2017–2020, funded by ISCIII and FEDER. RCC is funded by fellowships from the Spanish Regional Government of Castile and León. NGS is a recipient of an FPU fellowship (MINECO/FEDER). hiPSC-CM studies were funded in part by the “la Caixa” Banking Foundation under the project code HR18-00304 and a Severo Ochoa CNIC Intramural Project (Exp. 12-2016 IGP) to J.J.S

Urine metabolome profiling of immune-mediated inflammatory diseases

Background: Immune-mediated inflammatory diseases (IMIDs) are a group of complex and prevalent diseases where disease diagnostic and activity monitoring is highly challenging. The determination of the metabolite profiles of biological samples is becoming a powerful approach to identify new biomarkers of clinical utility. In order to identify new metabolite biomarkers of diagnosis and disease activity, we have performed the first large-scale profiling of the urine metabolome of the six most prevalent IMIDs: rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic lupus erythematosus, Crohn?s disease, and ulcerative colitis. Methods: Using nuclear magnetic resonance, we analyzed the urine metabolome in a discovery cohort of 1210 patients and 100 controls. Within each IMID, two patient subgroups were recruited representing extreme disease activity (very high vs. very low). Metabolite association analysis with disease diagnosis and disease activity was performed using multivariate linear regression in order to control for the effects of clinical, epidemiological, or technical variability. After multiple test correction, the most significant metabolite biomarkers were validated in an independent cohort of 1200 patients and 200 controls. Results: In the discovery cohort, we identified 28 significant associations between urine metabolite levels and disease diagnosis and three significant metabolite associations with disease activity (PFDR < 0.05). Using the validation cohort, we validated 26 of the diagnostic associations and all three metabolite associations with disease activity (PFDR < 0.05). Combining all diagnostic biomarkers using multivariate classifiers we obtained a good disease prediction accuracy in all IMIDs and particularly high in inflammatory bowel diseases. Several of the associated metabolites were found to be commonly altered in multiple IMIDs, some of which can be considered as hub biomarkers. The analysis of the metabolic reactions connecting the IMID-associated metabolites showed an overrepresentation of citric acid cycle, phenylalanine, and glycine-serine metabolism pathways. Conclusions: This study shows that urine is a source of biomarkers of clinical utility in IMIDs. We have found that IMIDs show similar metabolic changes, particularly between clinically similar diseases and we have found, for the first time, the presence of hub metabolites. These findings represent an important step in the development of more efficient and less invasive diagnostic and disease monitoring methods in IMIDs

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

Background: The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. Methods: We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero ([removed]2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. Findings: 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16–0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26–0·57; p[removed]11 página