RESUMO
Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.
Assuntos
Diferenciação Celular/genética , Cílios/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mucosa Respiratória/citologia , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos , Infecções Respiratórias/genética , Infecções Respiratórias/fisiopatologiaRESUMO
Whole-organ engineering-based on the functional repopulation of acellular whole-organ scaffolds derived from perfusion-based in toto decellularization of the specific organ system-is one of the most promising fields in tissue engineering. However, to date, we still have hardly any insights into the process of perfusion-based scaffold generation itself, with human-scale scaffolds usually obtained by adoption of small animal decellularization models, although those organs are of decreased biomass and potentially different biological characteristics. Therefore, in this study we analyzed perfusion-based human-scale whole-heart decellularization by evaluating and comparing the dynamics of biomass discharge and its kinetic characteristics during in toto decellularization of ovine and rodent hearts, while introducing a theoretical model of biomass depletion during perfusion-based whole-heart decellularization. Our results suggest highly varying process characteristics for the in toto decellularization of individual human-scale organs, such as protein discharge kinetics or time-dependent viscoelasticity of formed debris, despite seemingly consistent inter-sample decellularization efficacy, as evaluated by conventional disruptive analysis of obtained ECM scaffolds. Hence, the here exposed insights into the mechanistics of whole-heart decellularization as well as the introduced non-disruptive process accompanying tools may help to monitor and further optimize the decellularization process, especially with regards to human-scale scaffold production.
Assuntos
Coração/fisiologia , Ovinos/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Biomassa , DNA/química , Elasticidade , Matriz Extracelular , Humanos , Masculino , Modelos Animais , Modelos Teóricos , Perfusão , Ratos , Reologia , ViscosidadeRESUMO
The most common in vitro technology to generate human antibodies is phage display. This technology is a key technology to select recombinant antibodies for the use as research tools, in diagnostic tests, and for the development of therapeutics.In this review, the high-throughput compatible selection of antibodies (scFv) in microtiter plates is described. The given detailed protocols allow the antibody selection ("panning"), screening and identification of monoclonal antibodies in less than 1 week.
Assuntos
Clonagem Molecular/métodos , Biblioteca Gênica , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Humanos , Anticorpos de Cadeia Única/imunologiaRESUMO
Whole-organ engineering is an innovative field of regenerative medicine with growing translational perspectives. Recent reports suggest the feasibility of decellularization and repopulation of entire human size hearts. However, little is known about the susceptibility of epicardial adipose tissue (EAT) to decellularization. In this study, human size hearts of ovine donors were subjected to perfusion-based decellularization using detergent solutions. Upon basic histological evaluation and total DNA measurement myocardial regions prove largely decellularized while EAT demonstrated cellular remnants, further confirmed by transmission electron microscopy. Western blot analysis showed a significant reduction in lipid-associated and cardiac proteins. However, gas chromatography revealed unchanged proportional composition of fatty acids in EAT of decellularized whole hearts. Finally, cell culture medium conditioned with EAT from decellularized whole hearts had a significant deleterious effect on cardiac fibroblasts. These data suggest that perfusion decellularization of human size whole hearts provides inconsistent efficacy regarding donor material removal from myocardial regions as opposed to EAT.