RESUMEN
In order to perform in vitro cardiotoxicity screening of pharmacological substances, multi-electrode array systems are increasingly used to measure the extracellular field potentials of cell layers of human induced pluripotent stem cell cardiomyocytes. The analysis of the field potentials is usually performed using complex analysis software provided by the hardware manufacturers. In the case of the Cardiac Analysis Tool software from Axion Biosystems, inconsistencies were found in the results, which can significantly influence the cardiotoxicity screening results. In order to obtain more reliable results, a new algorithm was developed and implemented in an easy-to-use software tool, the INCardio Data Analysis Tool, which, due to its high degree of automation, can also be used by inexperienced users. The validation reveals differences in the results of the two tools both in depolarization spike amplitudes and in the time course of the field potential durations. The manual analysis of all signals affected by deviations shows that the results of the newly developed Data Analysis Tool are correct in all cases and can therefore be classified as more accurate and reliable than the reference analysis software.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Cardiotoxicidad , Miocitos Cardíacos , Electrodos , Potenciales de Acción , Células CultivadasRESUMEN
Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3' end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP's genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.
Asunto(s)
Neoplasias , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Vesiculovirus , Animales , Humanos , Ratones , Virus del Papiloma Humano , Ratones Endogámicos C57BL , Neoplasias/terapia , Neoplasias/virología , Infecciones por Papillomavirus/terapia , Vacunas contra Papillomavirus/genética , Vacunas contra Papillomavirus/uso terapéutico , Vacunas Atenuadas , Neoplasias ExperimentalesRESUMEN
Arrhythmogenic Cardiomyopathy (ACM) is a genetically based cardiomyopathy associated with ventricular arrhythmias and fibro-fatty substitution of cardiac tissue. It is characterized by incomplete penetrance. We generated human iPSCs by episomal reprogramming of blood cells from three members of the same family: the proband, affected by ACM and carrying the heterozygous plakophillin2 p.N346Lfs*12 mutation, one asymptomatic carrier of the same mutation and one apparently healthy control. hiPSCs were characterized according to standard protocols including karyotyping, pluripotency marker expression and differentiation towards the three germ layers. These hiPSC lines can be used to study the mechanisms of ACM incomplete penetrance in vitro.
Asunto(s)
Cardiomiopatías , Células Madre Pluripotentes Inducidas , Diferenciación Celular , Heterocigoto , Humanos , MutaciónRESUMEN
The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis.