RESUMO
INTRODUCTION: Endocrine disruptors are compounds of manmade origin able to interfere with the endocrine system and constitute an important environmental concern. Indeed, detrimental effects on thyroid physiology and functioning have been described. Differences exist in the susceptibility of human sexes to the incidence of thyroid disorders, like autoimmune diseases or cancer. METHODS: To study how different hormonal environments impact the thyroid response to endocrine disruptors, we exposed human embryonic stem cell-derived thyroid organoids to physiological concentrations of sex hormones resembling the serum levels of human females post-ovulation or males of reproductive age for three days. Afterwards, we added 10 µM benzo[a]pyrene or PCB153 for 24 h and analyzed the transcriptome changes via single-cell RNA sequencing with differential gene expression and gene ontology analysis. RESULTS: The sex hormones receptors genes AR, ESR1, ESR2 and PGR were expressed at low levels. Among the thyroid markers, only TG resulted downregulated by benzo[a]pyrene or benzo[a]pyrene with the "male" hormones mix. Both hormone mixtures and benzo[a]pyrene alone upregulated ribosomal genes and genes involved in oxidative phosphorylation, while their combination decreased the expression compared to benzo[a]pyrene alone. The "male" mix and benzo[a]pyrene, alone or in combination, upregulated genes involved in lipid transport and metabolism (APOA1, APOC3, APOA4, FABP1, FABP2, FABP6). The combination of "male" hormones and benzo[a]pyrene induced also genes involved in inflammation and NFkB targets. Benzo[a]pyrene upregulated CYP1A1, CYP1B1 and NQO1 irrespective of the hormonal context. The induction was stronger in the "female" mix. Benzo[a]pyrene alone upregulated genes involved in cell cycle regulation, response to reactive oxygen species and apoptosis. PCB153 had a modest effect in presence of "male" hormones, while we did not observe any changes with the "female" mix. CONCLUSION: This work shows how single cell transcriptomics can be applied to selectively study the in vitro effects of endocrine disrupters and their interaction with different hormonal contexts.
Assuntos
Benzo(a)pireno , Disruptores Endócrinos , Hormônios Esteroides Gonadais , Bifenilos Policlorados , Glândula Tireoide , Transcriptoma , Humanos , Benzo(a)pireno/toxicidade , Bifenilos Policlorados/toxicidade , Disruptores Endócrinos/toxicidade , Transcriptoma/efeitos dos fármacos , Glândula Tireoide/efeitos dos fármacos , Feminino , Masculino , Análise de Célula Única , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismoRESUMO
The convergence of organoid and organ-on-a-chip (OoC) technologies is urgently needed to overcome limitations of current 3D in vitro models. However, integrating organoids in standard OoCs faces several technical challenges, as it is typically laborious, lacks flexibility, and often results in even more complex and less-efficient cell culture protocols. Therefore, specifically adapted and more flexible microfluidic platforms need to be developed to facilitate the incorporation of complex 3D in vitro models. Here, a modular, tubeless fluidic circuit board (FCB) coupled with reversibly sealed cell culture bricks for dynamic culture of embryonic stem cell-derived thyroid follicles is developed. The FCB is fabricated by milling channels in a polycarbonate (PC) plate followed by thermal bonding against another PC plate. LEGO-like fluidic interconnectors allow plug-and-play connection between a variety of cell culture bricks and the FCB. Lock-and-play clamps are integrated in the organoid brick to enable easy (un)loading of organoids. A multiplexed perfusion experiment is conducted with six FCBs, where thyroid organoids are transferred on-chip within minutes and cultured up to 10 d without losing their structure and functionality, thus validating this system as a flexible, easy-to-use platform, capable of synergistically combining organoids with advanced microfluidic platforms.
Assuntos
Organoides , Organoides/citologia , Animais , Camundongos , Dispositivos Lab-On-A-Chip , Cimento de Policarboxilato/química , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Glândula Tireoide/citologia , Microfluídica/métodos , Microfluídica/instrumentação , Células-Tronco Embrionárias/citologiaRESUMO
Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.