In this study, we generate neural progenitor cells (NPCs) and neurons from induced pluripotent stem cells (iPSCs) from patients with PTHS to analyze the diseased cellular phenotypes under relevant genomic context. Importantly, we also derive patterned brain organoids, which have been successfully used to model cellular pathology during early neurodevelopment in several disorders33,34,35,36. Our data show that PTHS organoids are aberrant in size and structure, containing a higher percentage of NPCs and fewer neurons. PTHS NPCs exhibit reduced proliferation and impaired ability to differentiate into neurons. Importantly, we identify reduced canonical Wnt/β-catenin signaling and expression of SOX transcription factors as dysregulated events that mechanistically result in the PTHS cellular abnormalities. We also pharmacologically manipulate Wnt signaling and genetically correct TCF4 expression, which rescue the PTHS neural characteristics. Taken together, our data reveal novel cellular and molecular phenotypes in human cells with clinically relevant TCF4 mutations and show that these aberrations are reversible, providing routes for therapeutic intervention in individuals carrying genetic diseases associated with this gene.
Previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs [38, 41, 42]. However, scoring schemes for acute murine pneumonia existing to date are very superficial, addressing only a few, rather unspecific parameters [45, 49, 50]. More importantly, they hardly allow for a differentiating perspective between distinct pathogens or for group comparisons, e.g., infections of wild type versus genetically modified mice. Clearly, there is a strong need for more precise and pathogen- as well as model-specific parameters to allow for an accurate description and semiquantification of the inflammatory phenotype for reliable and reproducible comparisons between experimental groups within each model. Therefore, we have recently adapted more specific scoring criteria for S. pneumoniae and S. aureus-induced pneumonia [38, 42]. However, such pathogen-specific scoring criteria have not been employed for other lung pathogens in mice.
Transnasal infection of mice with S. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. Specific for this model, lesions widely expanded down to the periphery of the lung lobes (Fig 1A) with inflammation closely surrounding the airways and blood vessels. Pneumococcal spread led to an early immune response which was mainly characterized by predominantly intrabronchial (Fig 1B) and intraalveolar (Fig 1C) infiltrations of neutrophils provoking a lobular, suppurative bronchopneumonia with consolidation of affected lung areas. Large areas of coagulation and liquefaction necrosis (Fig 1D, arrowhead) as indicated by cellular fragmentation, decay, and loss of cellular details, accumulation of cellular and karyorrhectic debris as well as karyorrhexis, karyopyknosis, and karyolysis with consecutive hemorrhage were also present. The perivascular interstitium was widely expanded by edema due to vascular leakage  with massive extravasation of neutrophils recruited into perivascular spaces (Fig 1E). Furthermore, suppurative and necrotizing vasculitis accompanied by hyaline thrombi within small-sized blood vessels were occasionally present, indicating early histological evidence of incipient septicemia. Increased pulmonary vascular permeability  also led to expanded areas of protein-rich alveolar edema which presented as homogenous, lightly pink material within the alveolar spaces in the HE stain (Fig 1F, asterisk). A distinctive histopathological feature of pneumococcal pneumonia was the occurrence of massive suppurative to necrotizing pleuritis (Fig 1G, arrowhead) and steatitis (Fig 1H) with widespread dispersion of bacteria into the thoracic cavity, likely accounting for the painful and morbid clinical behavior with rapid progression in affected mice. Myriads of pneumococci were clearly visible as bluish to purple dots of approximately 1 μm in size in the standard HE stain, mostly located on the pleural surface, in the mediastinal adipose tissue or within perivascular spaces in the lungs. 2b1af7f3a8