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

Advancing manufacture of hiPSC-derived hepatocytes with improved functionality: A nature-inspired protocol

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Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers

Monitor educador

Tres juegos para el comedor: 1.-La limpieza de los dientes. 2.-Mamá cocinera y el tragatodo. 3.-Grupos de comida.MadridES

Data from: Bone-to-body biometric relationships for Owens and Lahontan tui chubs and their hybrids in California

Regression parameters for the length of several bony structures against fish body length, and for body length against body weight, were determined for Owens tui chub (Siphateles bicolor snyderi), Lahontan tui chub (S. b. obesa) and hybrid swarm deriving from the two species. A total of 211 individuals from 16 localities from the Owens River and neighboring basins along the border between California and Nevada were used for regression analyses. The coefficient of determination of linear regressions for scales, pharyngeal arches, dentaries, cleithra, and opercula against body length were consistently high (r2 埲.9). Differences between subspecies were mainly with reference to the intercept parameter in comparisons involving Lahontan tui chub. Coefficients of determination from log-linear length-weight regressions were also high (r2 埲.9) for individual taxa and for the pooled data set combining both Lahontan and hybrid species. The length-weight relation ship did not differ between subspecies. Estimates of the length-weight relationship using data pooling both Lahontan and hybrid tui chub suggest a weak allometric growth effect (P<0.05). The bone-length to body-length and body-length to body-weight relationships presented here will be useful tools for future dietary studies of tui chub predators as well as for archaeological and paleontological studies on tui chub remains

Table A1 - Length, weight, and bonelength measurement values of the studied Owens tui chub Siphateles bicolor snyderi, Lahontan tui chub S. b. obesa and hybrid specimens derived from the two species collected in 2001 and 2002 from the Owens River and neighboring basins along the border between California and Nevada

Table A1 - Length, weight, and bonelength measurement values of the studied Owens tui chub Siphateles bicolor snyderi, Lahontan tui chub S. b. obesa and hybrid specimens derived from the two species collected in 2001 and 2002 from the Owens River and neighboring basins along the border between California and Nevad

Burgos (Provincia). Mapas generales. 1868

En el margen superior: Diccionario geográfico-estadístico-histórico ; Atlas de España y sus posesiones de ultramarCopia digital. Valladolid : Junta de Castilla y León. Consejería de Cultura y Turismo, 2009-2010Inserta: Información general sobre la provincia ; planos de: Burgos, Contornos de Burgos, Miranda de Ebro, Briviesca, Pancorbo, Aranda de Duero, Belorado, Medina de Pomar, Castrogeriz, Roa y Lerm

Segovia (Provincia). Mapas generales. 1849

En el margen superior: Diccionario geográfico-estadístico-histórico ; Atlas de España y sus posesiones de ultramarCopia digital. Valladolid : Junta de Castilla y León. Consejería de Cultura y Turismo, 2009-2010Inserta: Información general sobre la provincia ; planos de: Segovia, Contornos de Segovia, La Granja, Contornos de Sta. María la Real de Nieva, Contornos de Sepúlved