A nature-inspired protocol to generate mature hiPSC-derived hepatocytes: Unveiling the role of human intestinal microbiome

The production of hepatocytes derived from human induced pluripotent stem cells (hiPSC-HLC) holds great promise for multiple cell therapies and tissue engineering applications. Nonetheless, the current protocols to generate HLC in vitro are not yet successfully established resulting in low yields of mainly immature cells when compared to the adult counterparts. The major hurdle in recapitulating in vitro the physiological liver maturation process is due to its complexity as it takes approximately 2 years after birth and involves a wide range of biological events (1). Recent findings have been suggesting that liver maturation, that naturally occur during the early postnatal period, can be strongly associated with human intestinal microbiome (2). For example, lithocholic acid and vitamin K2, two intestinal postbiotics, were shown to induce the expression of CYP450 enzymes in HLC and fetal hepatocytes (3). Additionally, studies on germ-free animals reported dissimilar xenobiotic enzyme profiles (4) and an impaired liver regeneration (5) compared to wild type animals. Considering these evidences, we developed a nature-inspired bioprocess to produce relevant numbers of highly functional and mature HLC for application in regenerative medicine. In this study, hiPSC-HLC were generated as 3D cell aggregates in stirred-tank bioreactors according to the integrated bioprocess developed previously by our group (6), and matured with a novel strategy based on human intestinal microbiota’s secretome. The maturation profile of hiPSC-HLC was evaluated at transcriptional and functional levels, and the composition of microbial secretome formulation was also characterized by UPLC-MS/MS, GC-MS and LC–MS/MS technologies. Our results showed an efficient hiPSC differentiation into hepatic lineage with a production of 2.8x106 HLC/mL (~370 million cells in a 200mL ST-BR), displaying a mixture of adult (~80%ALB+ cells) and fetal traits (~30%AFP+ cells and CYP3A7+ cells). Noteworthy, HLC treated with bacterial secretome showed higher ALB expression (87%ALB+ cells), ALB and A1AT secretion, urea synthesis, and basal and inducible CYP3A4 metabolism, when compared to untreated HLC that were cultured in standard hepatocyte maintenance medium. Detailed analytical characterization of the microbial secretome revealed some of the potential biologically active molecules, such as bile acids, short-chain fatty acids and vitamins that could be responsible for HLC in vitro maturation. In conclusion, the protocol developed herein presents high technological relevance due to its efficiency, scalability, and reproducibility, but also unveils the potential role of human intestinal microbiome in hepatic cell maturation. Noteworthy, we also demonstrated that the 3D aggregates of mature hiPSC-HLC were able to adhere and migrate in human hepatic extracellular matrix scaffolds, while maintained their viability and functional features, showing to hold great promise to be used as cell therapy products or as cell ingredients for liver bioengineering applications. This work was funded by Projects EHD16PI02 from CIBERehd and LMP226_18 funded by DGA (Spain) as well as by Fundação para a Ciência e Tecnologia (FCT)-funded projects ERAdicatPH (E-Rare3/0002/2015) and iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344). J.I. A. and P.V. were supported by FCT fellowships SFRH/BD/116780/2016 and SFRH/BD/145767/2019, respectively. [1] Chen C, Soto-Gutierrez A, Baptista PM, Spee B. Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells. Gastroenterology. 2018;154(5):1258–72; [2] Almeida, Joana I., Miguel F. Tenreiro, Lucía Martinez-Santamaria, Aspizua, Sara Guerrero, Javier P. Gisbert, Paula M. Alves, Margarida Serra PMB. Hallmarks of the human intestinal microbiome on liver maturation and function. J Hepatol. 2021; [3] Avior Y, Levy G, Zimerman M, Kitsberg D, Schwartz R, Sadeh R, et al. Microbial-Derived Lithocholic Acid and Vitamin K2 Drive the Metabolic Maturation of Pluripotent Stem Cells-Derived and Fetal Hepatocytes. Hepatology. 2015;62(1):265–78; [4] Selwyn FP, Cheng SL, Bammler TK, Prasad B, Vrana M, Klaassen C, et al. Developmental regulation of drug-processing genes in livers of germ-free mice. Toxicol Sci. 2015;147(1):84–103; [5] Cornell RP, Liljequist BL, Bartizal KF. Depressed liver regeneration after partial hepatectomy of germ‐free, athymic and lipopolysaccharide‐resistant mice. Hepatology. 1990;11(6):916–22; [6] Isidro, I., Vicente, P., Pais, D., Almeida, Joana I., Domingues, M., Abecasis, B., Mertinez-Turrillas, R., Rodriguez-Madoz, Juan R., Aspegren, A., Alves, Paula M., Serra M. On-line monitoring of hiPSC expansion and hepatic differentiation process in a 3D culture system by dielectric spectroscopy. Biotechnol Bioeng

X-chromosome terminal deletion in a female with premature ovarian failure: Haploinsufficiency of X-linked genes as a possible explanation

<p>Abstract</p> <p>Background</p> <p>Premature ovarian failure (POF) has repeatedly been associated to X-chromosome deletions. <it>FMR1 </it>gene premutation allele's carrier women have an increased risk for POF. We intent to determine the cause of POF in a 29 year old female, evaluating both of these situations.</p> <p>Methods</p> <p>Concomitant analysis of <it>FMR1 </it>gene CGG repeat number and karyotype revealed an X-chromosome terminal deletion. Fluorescence <it>in situ </it>further characterized the breakpoint. A methylation assay for <it>FMR1 </it>gene allowed to determine its methylation status, and hence, the methylation status of the normal X-chromosome.</p> <p>Results</p> <p>We report a POF patient with a 46,X,del(X)(q26) karyotype and with skewed X-chromosome inactivation of the structural abnormal X-chromosome.</p> <p>Conclusions</p> <p>Despite the hemizygosity of <it>FMR1 </it>gene, the patient does not present Fragile X syndrome features, since the normal X-chromosome is not subject to methylation. The described deletion supports the hypothesis that haploinsufficiency of X-linked genes can be on the basis of POF, and special attention should be paid to X-linked genes in region Xq28 since they escape inactivation and might have a role in this disorder. A full clinical and cytogenetic characterization of all POF cases is important to highlight a pattern and help to understand which genes are crucial for normal ovarian development.</p

Analysis of long period gratings inscribed by CO2 laser irradiation and estimation of the refractive index modulation

Long period gratings (LPGs) inscribed in single mode fibers (SMFs) using CO2 laser irradiation were modelled numerically using the coupled mode method. The model considers the specifications of the inscription technique, such as the shape of the refractive index modulation that mimics the circularly symmetric point-to-point laser irradiation profile. A simple expression for predicting the resonant wavelength was obtained assuming a two-mode coupling model. However, to explain the spectra of the experimental LPGs, it was necessary to assume a reasonably high refractive index change and a multimode coupling model. Furthermore, using the developed model and a genetic algorithm to fit experimental resonances to simulated ones, we were able to estimate the maximum refractive index change, obtaining a value of 2.2 × 10-3, confirming the high refractive index change. The proposed model also predicts a second order resonance for this high value of refractive index change that was confirmed experimentally. Hence, with this model, we found some significant differences in the LPGs behavior when compared with conventional ones, namely, the emergence of coupling between different cladding modes and the competition of first and second order resonances which change the LPG transmission spectrum.publishe

The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling

The mechanisms underlying the initiation/onset of, and the recovery from, depression are still largely unknown; views that neurogenesis in the hippocampus may be important for the pathogenesis and amelioration of depressive symptoms have gained currency over the years although the original evidence has been challenged. In this study, an unpredictable chronic mild stress protocol was used to induce a depressive-like phenotype in rats. In the last 2 weeks of stress exposure, animals were treated with the antidepressants fluoxetine, imipramine, CP 156,526 or SSR 1494515, alone or combined with methylazoxymethanol, a cytostatic agent used to arrest neurogenesis. We found that antidepressants retain their therapeutic efficacy in reducing both measured indices of depression-like behavior (learned helplessness and anhedonia), even when neurogenesis is blocked. Instead, our experiments suggest re-establishment of neuronal plasticity (dendritic remodeling and synaptic contacts) in the hippocampus and prefrontal cortex, rather than neurogenesis, as the basis for the restoration of behavioral homeostasis by antidepressants.This project used compound(s) provided by the National Cancer Institute’s Chemical Carcinogen Reference Standards Repository (operated under contract N02-CB-07008 by Midwest Research Institute; MAM), Sanofi-Synthelabo (SSR 149415) and Pfizer (CP 156,526). The authors’ work was supported by the Portuguese Foundation for Science and Technology (FCT) (PTDC/SAU-NEU/72699/2006)

Bioprocess optimization for generation of hepatocytes derived from hiPSC and its application in primary hyperoxaluria type 1 disease modelling

Primary hyperoxaluria type 1 (PH1) is a rare metabolic disorder caused by mutations in the hepatic alanine-glyoxylate aminotransferase (AGT). Defective AGT in PH1 patients is characterized by excessive oxalate synthesis, which leads to a broad range of kidney complications including the end-stage renal disease [1]. Combined liver-kidney transplantation remains the only effective treatment; however significant morbidity, mortality and costs encouraged the development of advanced cell- and gene-based therapies for PH1. Thus, our aim was to implement a novel strategy to generate high numbers of functional hepatocyte-like cells (HLC) from PH1 patient derived human induced pluripotent stem cells (PH1.hiPSC), for PH1 disease modelling and further application in drug and therapeutics development. PH1.HLC were differentiated as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion, according to the integrated bioprocess previously developed by our group [2,3]. Briefly, PH1.hiPSC were aggregated and expanded in STB for 4 days preceding the hepatic differentiation. hiPSC to HLC commitment begin by culturing the 3D aggregates in different medium formulations (from Takara BioEurope AB). Two different dissolved oxygen (pO2) conditions were explored: a normoxia (pO2: uncontrolled, 95% air, 5% CO2) throughout the differentiation process (21 days) and a hypoxia with a low oxygen (pO2: 4% O2) environment between day 4 and day 14 of the differentiation. Our results showed that PH1-hiPSC successfully proliferated as 3D aggregates with an expansion factor of 6-fold after 4 days in culture while maintaining their pluripotent phenotype. Low dissolved oxygen concentration during hepatic specification, generate higher yields of HLC and improve gene expression levels of ALB, A1AT and CYP3A4 hepatic markers when compared with HLC differentiated under uncontrolled pO2 conditions. Moreover, Flow cytometry analysis, revealed a higher hepatocyte content of 80% (low pO2) vs 43% (uncontrolled pO2) for albumin, showing a higher process efficiency. Transcriptomic analysis using RNAseq confirmed that hepatocyte differentiation was enhanced in the low dissolved oxygen condition. In addition, these PH1.HLC showed functional characteristics typical of hepatocytes including production of important hepatic proteins (albumin, alpha 1 antitrypsin), urea and bile acids. PH1.HLC also display drug metabolization capacity, CYP450 activity and, by histological assessment, glycogen storage and positive staining for albumin and AFP markers. To further characterize the PH1 disease features, we performed a detailed metabolomic analysis and demonstrated that PH1.HLC show defective AGT activity with significantly higher production and secretion of oxalate for PH1.HLC when compared with HLC generated from healthy counterparts. Overall, controlling the dissolved oxygen concentration at key stages of the hepatic differentiation process improved cell yield and the maturation status of HLC. The bioprocess developed and optimized in this work offers high relevance not only for generation of more accurate in vitro models to study PH1 rare disease, but also towards the development of novel therapies. Acknowledgements & Funding: this study was funded by a grant from ERA-NET E-Rare 3 research program, JTC ERAdicatPH (E-Rare3/0002/2015) and Fundação para a Ciência e Tecnologia project MetaCardio (PTDC/BTM-SAL/32566/2017); iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior, through national funds is acknowledged. P. V., J. I. A. were supported by FCT fellowships SFRH/BD/145767/2019, SFRH/BD/116780/2016 respectively. [1] P. Cochat, N. Engl. J. Med., vol. 369, no. 7, pp. 649–658, 2013. [2] B. Abecasis, J. Biotechnol., vol. 246, pp. 81–93, 2017. [3] I. Isidro, Biotechnol Bioeng, vol. 118, 3610–3617, 2021

Chronic stress and glucocorticoids: from neuronal plasticity to neurodegeneration

Stress and stress hormones, glucocorticoids (GCs), exert widespread actions in central nervous system, ranging from the regulation of gene transcription, cellular signaling, modulation of synaptic structure, and transmission and glial function to behavior. Their actions are mediated by glucocorticoid and mineralocorticoid receptors which are nuclear receptors/transcription factors. While GCs primarily act to maintain homeostasis by inducing physiological and behavioral adaptation, prolonged exposure to stress and elevated GC levels may result in neuro- and psychopathology. There is now ample evidence for cause-effect relationships between prolonged stress, elevated GC levels, and cognitive and mood disorders while the evidence for a link between chronic stress/GC and neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD) diseases is growing. This brief review considers some of the cellular mechanisms through which stress and GC may contribute to the pathogenesis of AD and PD.The work was supported by Grants “PTDC/SAU-NMC/113934/2009,” funded by FCT, Portuguese Foundation for Science and Technology, and project DoIT, Desenvolvimento e Operacionalização da Investigação de Translação (N° do projeto 13853), funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional Fatores de Competitividade (POFC). In addition, this work was also cofinanced by European Union FP7 Project SwitchBox (Nuno Sousa, Osborne F. X. Almeida) and the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER). Sheela Vyas acknowledges grant support from Foundation de France, Physiopathology of Parkinson, France Parkinson and ANR Grant “ParkStrim” N° 13-BSV1-0013-02. Work in FT research group was supported by Agence Nationale de la Recherche (TIMMS and StressPsyco) and Fondation pour la Recherche Médicale, Grant no. DEQ20140329552

An unusual cause of myocardial infarction

Copyright © 2019 European Society of CardiologyBackground: In order to direct the treatment it, is well established that is fundamental to clarify the aetiology of heart failure and the cause of myocardial infarction (MI) with non obstructive coronary artery disease (MINOCA), with CMR being one of the methods of choice in both clinical situations. Case report: A 70 years-old male patient was admitted in our emergency department with complaints of irregular palpitations, progressive dyspnoea and fatigue on exertion, with two weeks of evolution; these symptoms were associated to retrosternal chest pain in the last twelve hours. He had a previous medical history of dyslipidemia, no other cardiovascular risk factors were known. On admission, his heart rate was 130bpm, with an irregularly irregular pulse and the pulmonary auscultation revealed bibasal crackles. The remaining physical examination was unremarkable. The ECG showed an atrial fibrillation rhythm, with a mild ST elevation and T wave inversion in inferior leads. The echocardiogram revealed a diffuse hypokinesia of left ventricle with an ejection fraction of 35-40%. The lab tests documented an elevation of troponin (hs-TnT 210ng/L) and NTproBNP (1945pg/ml). The coronary angiogram showed no lesions.info:eu-repo/semantics/publishedVersio

Female hippocampus vulnerability to environmental stress, a precipitating factor in tau aggregation pathology

Tau-mediated neurodegeneration is a central event in Alzheimer's disease (AD) and other tauopathies. Consistent with suggestions that lifetime stress may be a clinically-relevant precipitant of AD pathology, we previously showed that stress triggers tau hyperphosphorylation and accumulation; however, little is known about the etiopathogenic interaction of chronic stress with other AD risk factors, such as sex and aging. This study focused on how these various factors converge on the cellular mechanisms underlying tau aggregation in the hippocampus of chronically stressed male and female (middle-aged and old) mice expressing the most commonly found disease-associated Tau mutation in humans, P301L-Tau. We report that environmental stress triggers memory impairments in female, but not male, P301L-Tau transgenic mice. Furthermore, stress elevates levels of caspase-3-truncated tau and insoluble tau aggregates exclusively in the female hippocampus while it also alters the expression of the molecular chaperones Hsp90, Hsp70, and Hsp105, thus favoring accumulation of tau aggregates. Our findings provide new insights into the molecular mechanisms through which clinically-relevant precipitating factors contribute to the pathophysiology of AD. Our data point to the exquisite sensitivity of the female hippocampus to stress-triggered tau pathology.This study was supported by the Japanese Soci- ety for the Promotion of Science (IS), the Portuguese Foundation for Science & Technology (IS, AJR, NS), the Max Planck Society (OFXA), and the European Union FP7 Project SwitchBox (OFXA and NS). AT is supported by Research Funding for Longevity Sci- ences (23–39) from National Center for Geriatrics and Gerontology, the Strategic Research Program for Brain Science (“Integrated Research on Neuropsychiatric Disorders”) and a Grant-in-Aid for Scientific Research on Innovative Areas (“Brain Environment”) from the Ministry of Education, Science, Sports and Culture of Japan

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Research Areas: Science & TechnologyAnti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.info:eu-repo/semantics/publishedVersio