Factors that influence the quality of metabolomics data in in vitro cell toxicity studies: a systematic survey

REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) is a global strategy and regulation policy of the EU that aims to improve the protection of human health and the environment through the better and earlier identification of the intrinsic properties of chemical substances. It entered into force on 1st June 2007 (EC 1907/2006). REACH and EU policies plead for the use of robust high-throughput 'omic' techniques for the in vitro investigation of the toxicity of chemicals that can provide an estimation of their hazards as well as information regarding the underlying mechanisms of toxicity. In agreement with the 3R's principles, cultured cells are nowadays widely used for this purpose, where metabolomics can provide a real-time picture of the metabolic effects caused by exposure of cells to xenobiotics, enabling the estimations about their toxicological hazards. High quality and robust metabolomics data sets are essential for precise and accurate hazard predictions. Currently, the acquisition of consistent and representative metabolomic data is hampered by experimental drawbacks that hinder reproducibility and difficult robust hazard interpretation. Using the differentiated human liver HepG2 cells as model system, and incubating with hepatotoxic (acetaminophen and valproic acid) and non-hepatotoxic compounds (citric acid), we evaluated in-depth the impact of several key experimental factors (namely, cell passage, processing day and storage time, and compound treatment) and instrumental factors (batch effect) on the outcome of an UPLC-MS metabolomic analysis data set. Results showed that processing day and storage time had a significant impact on the retrieved cell's metabolome, while the effect of cell passage was minor. Meta-analysis of results from pathway analysis showed that batch effect corrections and quality control (QC) measures are critical to enable consistent and meaningful estimations of the effects caused by compounds on cells. The quantitative analysis of the changes in metabolic pathways upon bioactive compound treatment remained consistent despite the concurrent causes of metabolomic data variation. Thus, upon appropriate data retrieval and correction and by an innovative metabolic pathway analysis, the metabolic alteration predictions remained conclusive despite the acknowledged sources of variability

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

Reprogramación directa de células somáticas de pacientes a hepatocitos: aplicación en el estudio de la hepatotoxicidad por fármacos y en las metabolopatías hepáticas

ACIF/2019/145El escenario ideal para poder estudiar metabolopatías hepáticas y fenómenos de hepatotoxicidad idiosincrásica sería disponer de las células propias del hígado del paciente. Sin embargo, obtener tejido con calidad y cantidad como para poder aislar hepatocitos y plantear un estudio mecanístico, es ética y técnicamente imposible. El empleo de líneas celulares hepáticas tampoco se considera una opción adecuada, ya que no reproducen todas las peculiaridades genéticas de un individuo particular. Por ello, una alternativa factible es la reprogramación hepática, esto es la generación de hepatocitos a partir de células somáticas de pacientes, adentrándose en el campo de la medicina personalizada. A diferencia de la reprogramación hepática basada en células troncales pluripotentes inducidas (iPSC), la reprogramación hepática directa es capaz de generar células de tipo hepático (HLC) sin necesidad de pasar por un estadio intermedio de pluripotencia. Mediante esta estrategia directa, lograda con la expresión lentiviral de factores hepáticos, se consigue una reducción considerable del tiempo y coste del proceso de la reprogramación, sin repercutir en el fenotipo alcanzado. En este trabajo, hemos mejorado significativamente y aportado nuevos matices a la reprogramación hepática directa. La estrategia desarrollada consiste en el empleo de un vector policistrónico inducible que contiene 3 factores hepáticos básicos de reprogramación a HLC (HNF1A, HNF4A y FOXA3), junto con un método de inmortalización celular basado en la expresión de la telomerasa humana. Esta combinación nos permite obtener una línea celular de fibroblastos humanos inmortalizados, con un crecimiento continuo y capaz de reprogramarse en 12 días a HLC mediante la adición de doxiciclina al medio de cultivo. Las HLC obtenidas realizan funciones típicas de los hepatocitos humanos adultos, son fenotípicamente homogéneas a lo largo del tiempo, de bajo coste y compatibles con las técnicas de screening clásicas de estudios farmacotoxicológicos. La aplicación de esta estrategia en pacientes con metabolopatías hepáticas nos ha permitido modelizar la deficiencia en α1-antitripsina y la glucogenosis de tipo IXa, reproduciendo in vitro el fenotipo alterado del hepatocito. En el caso particular de la glucogenosis, el hecho de aislar y secuenciar el mRNA de las HLC permitió un diagnóstico genético del paciente, identificando una nueva mutación en el gen PHKA2 que generaba un error de splicing. Este diagnóstico genético personalizado permitió localizar la misma mutación en la hermana del afectado, incluyéndola en un programa de consejo genético. La capacidad que muestra la reprogramación directa de reproducir in vitro estos fenotipos patológicos nos permite ser optimistas para afrontar un futuro reto, el estudio del daño hepático inducido por fármacos de carácter idiosincrásico (iDILI). Como prueba de concepto, hemos reprogramado células de pacientes con iDILI y comprobado que alcanzan un fenotipo hepático razonable, que incluye la expresión de genes implicados en el metabolismo de xenobióticos. Con ello pretendemos llegar a reproducir in vitro el episodio de toxicidad acontecido a un paciente, identificando con ayuda de la metabolómica el fármaco causal (diagnóstico) y el mecanismo por el que ejerce la toxicidad.FEDER - Fondos Europeos - Generalitat Valencian

Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis

The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or alterations in intron sequences. Notwithstanding, elucidation of the pathogenicity of a specific variant might be critical for an accurate diagnosis. In this study, we described a novel intronic variant c.2597 + 5G > T in the donor splice sequence of the PHKA2 gene. To investigate PHKA2 mRNA splicing, as well as the functional consequences on glycogen metabolism, we generated hepatocyte-like cells from a proband’s fibroblasts by direct reprogramming. We demonstrated an aberrant splicing of PHKA2, resulting in the incorporation of a 27 bp upstream of intron 23 into exon 23, which leads to an immediate premature STOP codon. The truncated protein was unable to phosphorylate the PYGL protein, causing a 4-fold increase in the accumulation of glycogen in hepatocyte-like cells. Collectively, the generation of personalized hepatocyte-like cells enabled an unequivocal molecular diagnosis and qualified the sister’s proband, a carrier of the same mutation, as a candidate for a preimplantation genetic diagnosis. Additionally, our direct reprogramming strategy allows for an unlimited source of “diseased” hepatocyte-like cells compatible with high-throughput platforms

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

Abstract Background High-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable. Methods We have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions. Results Clonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks. Conclusion Our strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality

Derivation of healthy hepatocyte-like cells from a female patient with ornithine transcarbamylase deficiency through X-inactivation selection

Autologous cell replacement therapy for inherited metabolic disorders requires the correction of the underlying genetic mutation in patient's cells. An unexplored alternative for females affected from X-linked diseases is the clonal selection of cells randomly silencing the X-chromosome containing the mutant allele, without in vivo or ex vivo genome editing. In this report, we have isolated dermal fibroblasts from a female patient affected of ornithine transcarbamylase deficiency and obtained clones based on inactivation status of either maternally or paternally inherited X chromosome, followed by differentiation to hepatocytes. Hepatocyte-like cells derived from these clones display indistinct features characteristic of hepatocytes, but express either the mutant or wild type OTC allele depending on X-inactivation pattern. When clonally derived hepatocyte-like cells were transplanted into FRG® KO mice, they were able to colonize the liver and recapitulate OTC-dependent phenotype conditioned by X-chromosome inactivation pattern. This approach opens new strategies for cell therapy of X-linked metabolic diseases and experimental in vitro models for drug development for such diseases.Published versionFunding was provided by Ministerio de Ciencia e Innovacion (Grant No. SAF-51991R) and Generalitat Valenciana (Grant Nos. PROMETEO/2019/060, ACIF/2019/145)