Micro-patterned culture of iPSC-derived alveolar and airway cells distinguishes SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants necessitated a rapid evaluation system for their pathogenesis. Lung epithelial cells are their entry points; however, in addition to their limited source, the culture of human alveolar epithelial cells is especially complicated. Induced pluripotent stem cells (iPSCs) are an alternative source of human primary stem cells. Here, we report a model for distinguishing SARS-CoV-2 variants at high resolution, using separately induced iPSC-derived alveolar and airway cells in micro-patterned culture plates. The position-specific signals induced the apical-out alveolar type 2 and multiciliated airway cells at the periphery and center of the colonies, respectively. The infection studies in each lineage enabled profiling of the pathogenesis of SARS-CoV-2 variants: infection efficiency, tropism to alveolar and airway lineages, and their responses. These results indicate that this culture system is suitable for predicting the pathogenesis of emergent SARS-CoV-2 variants.

Screening of factors inducing alveolar type 1 epithelial cells using human pluripotent stem cells.

Alveolar type 2 (AT2) epithelial cells are tissue stem cells capable of differentiating into alveolar type 1 (AT1) cells for injury repair and maintenance of lung homeostasis. However, the factors involved in human AT2-to-AT1 cell differentiation are not fully understood. Here, we established SFTPCGFP and AGERmCherry-HiBiT dual-reporter induced pluripotent stem cells (iPSCs), which detected AT2-to-AT1 cell differentiation with high sensitivity and identified factors inducing AT1 cell differentiation from AT2 and their progenitor cells. We also established an "on-gel" alveolar epithelial spheroid culture suitable for medium-throughput screening. Among the 274 chemical compounds, several single compounds, including LATS-IN-1, converted AT1 cells from AT2 and their progenitor cells. Moreover, YAP/TAZ signaling activation and AKT signaling suppression synergistically recapitulated the induction of transcriptomic, morphological, and functionally mature AT1 cells. Our findings provide novel insights into human lung development and lung regenerative medicine.

Practical guide for the diagnosis and management of primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. METHODS: An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. RESULTS: The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. CONCLUSION: This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.

Past and Future of Alveolar Organoids for Lung Regenerative Medicine.

The lung is regarded as having limited regenerative capacity, and there are few treatment options for refractory lung diseases, such as interstitial pneumonia. Lung transplantation is the final option available in some scenarios. Research in this area has been slow owing to the complex structure of the lung for efficient gas exchange between the alveolar spaces and capillaries as well as the difficulty in obtaining specimens from patients with progressive lung disease. However, basic research over the past decade in the field of mouse and human embryology using genetic lineage tracing techniques and stem cell biology using primary and pluripotent stem cell-derived alveolar organoids has begun to clarify the tissue response in various intractable lung diseases and the mechanisms underlying remodeling. Advancement in this area may expand potential therapeutic targets for alveolar regeneration, providing alternatives to lung transplantation, and contribute to the development of effective therapeutic methods that activate or repopulate stem cells in the lung. In this review, we cover research focused on alveolar epithelial cells and discuss methods expected to regenerate lungs that are damaged by diseases.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC.

Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.

Contemporary and emerging technologies for research in children's rare and interstitial lung disease.

Although recent decades have seen the identification, classification and discovery of the genetic basis of many children's interstitial and rare lung disease (chILD) disorders, detailed understanding of pathogenesis and specific therapies are still lacking for most of them. Fortunately, a revolution of technological advancements has created new opportunities to address these critical knowledge gaps. High-throughput sequencing has facilitated analysis of transcription of thousands of genes in thousands of single cells, creating tremendous breakthroughs in understanding normal and diseased cellular biology. Spatial techniques allow analysis of transcriptomes and proteomes at the subcellular level in the context of tissue architecture, in many cases even in formalin-fixed, paraffin-embedded specimens. Gene editing techniques allow creation of "humanized" animal models in a shorter time frame, for improved knowledge and preclinical therapeutic testing. Regenerative medicine approaches and bioengineering advancements facilitate the creation of patient-derived induced pluripotent stem cells and their differentiation into tissue-specific cell types which can be studied in multicellular "organoids" or "organ-on-a-chip" approaches. These technologies, singly and in combination, are already being applied to gain new biological insights into chILD disorders. The time is ripe to systematically apply these technologies to chILD, together with sophisticated data science approaches, to improve both biological understanding and disease-specific therapy.

Characteristic genetic spectrum of primary ciliary dyskinesia in Japanese patients and global ethnic heterogeneity: population-based genomic variation database analysis.

Primary ciliary dyskinesia (PCD) is a hereditary disease caused by pathogenic variants in genes associated with motile cilia. Some variants responsible for PCD are reported to be ethnic-specific or geographical-specific. To identify the responsible PCD variants of Japanese PCD patients, we performed next-generation sequencing of a panel of 32 PCD genes or whole-exome sequencing in 26 newly identified Japanese PCD families. We then combined their genetic data with those from 40 Japanese PCD families reported previously, for an overall analysis of 66 unrelated Japanese PCD families. We conducted Genome Aggregation Database and TogoVar database analyses to reveal the PCD genetic spectrum of the Japanese population and compare with other ethnic groups worldwide. We identified 22 unreported variants among the 31 patients in the 26 newly identified PCD families, including 17 deleterious variants estimated to cause lack of transcription or nonsense-mediated mRNA decay and 5 missense mutations. In all 76 PCD patients from the 66 Japanese families, we identified 53 variants on 141 alleles in total. Copy number variation in DRC1 is the most frequent variant in Japanese PCD patients, followed by DNAH5 c.9018C>T. We found 30 variants specific to the Japanese population, of which 22 are novel. Furthermore, 11 responsible variants in the Japanese PCD patients are common in East Asian populations, while some variants are more frequent in other ethnic groups. In conclusion, PCD is genetically heterogeneous between different ethnicities, and Japanese PCD patients have a characteristic genetic spectrum.

iPSC-derived mesenchymal cells that support alveolar organoid development.

Mesenchymal cells are necessary for organ development. In the lung, distal tip fibroblasts contribute to alveolar and airway epithelial cell differentiation and homeostasis. Here, we report a method for generating human induced pluripotent stem cell (iPSC)-derived mesenchymal cells (iMESs) that can induce human iPSC-derived alveolar and airway epithelial lineages in organoids via epithelial-mesenchymal interaction, without the use of allogenic fetal lung fibroblasts. Through a transcriptome comparison of dermal and lung fibroblasts with their corresponding reprogrammed iPSC-derived iMESs, we found that iMESs had features of lung mesenchyme with the potential to induce alveolar type 2 (AT2) cells. Particularly, RSPO2 and RSPO3 expressed in iMESs directly contributed to AT2 cell induction during organoid formation. We demonstrated that the total iPSC-derived alveolar organoids were useful for characterizing responses to the influenza A (H1N1) virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating their utility for disease modeling.

A Novel Homozygous Variant in GAS2L2 in Two Sisters with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare hereditary disease. We herein report two sisters in their 20s with suspected PCD. They were both born at full term and did not have situs inversus. Chest computed tomography showed similar signs of bronchiectasis in both siblings. Genetic examinations of the family confirmed that the sisters both harbored a homozygous variant in the growth-arrest-specific 2-like 2 (GAS2L2) gene. This is the third report of a family with PCD caused by a GAS2L2 variant.

Mixed tumour of the skin of the lower lip: A case report and review of the literature.

Mixed tumour of the skin or chondroid syringoma (CS) is a rare and mostly benign neoplasm of the sweat glands. Although CS is frequently located on varied parts of the head and neck region, the lower lip is a rarely reported site. The present report describes a case of CS of the lower lip in a 58-year-old male as an expository case to further emphasise the need for proper diagnosis, appropriate treatment and prognostic evaluation. The patient presented with a round, non-tender, slightly hard and mobile mass beneath the mucocutaneous junction of his left lateral side of the lower lip. Radiology revealed a mass measuring 11x11x7 mm3 in size at a depth of ~2 mm. Furthermore, magnetic resonance T1- and T2-weighted images showed slightly low and high signal intensities, respectively. A provisional diagnosis of benign tumour of the lower lip was made, and surgical excision biopsy taken under local anaesthesia, while considering the patient's cosmetic appearance. Histopathology demonstrated features akin to apocrine gland, chondroid and myxoid stroma consistent with the diagnosis of benign CS. No evidence of recurrence or satellites were recorded after a follow-up of nearly 2 years. Although rare, a high index of suspicion for CS among other cutaneous adnexal tumours of the lower lip is necessary. In addition, interprofessional collaboration in the management of such oral tumours could enhance patient satisfaction amid prevailing intraoral and aesthetic concerns.

Perspectives of future lung toxicology studies using human pluripotent stem cells.

The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.

A pediatric case of primary ciliary dyskinesia caused by novel copy number variation in PIH1D3.

An 11-month-old boy with productive cough was referred to our hospital. He had nasal obstruction immediately after birth, and wheezing, wet cough, and rhinorrhea were observed daily after the neonatal period. Clinical and imaging findings revealed secretory otitis media, chronic sinusitis, and bronchiectasis. Primary ciliary dyskinesia was suspected. Transmission electron microscopy of nasal cilia showed defects of the outer and inner dynein arms. Genetic examinations of the family revealed copy number variation in PIH1 domain-containing 3 (PIH1D3) in the proband and mother. This is the first report of a Japanese patient with primary ciliary dyskinesia caused by copy number variation in PIH1D3.

Analysis of the clinical features of Japanese patients with primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a rare hereditary disease. Most reports of PCD in Japan are case reports, and clinical analysis has not been performed. Differences in the causative genes might affect the clinical features in different ethnic groups. The purpose of this study was to clarify the clinical features of Japanese patients with PCD. METHODS: We performed a retrospective chart review of PCD patients seen at Mie University Hospital and patients whose blood samples were sent to us for genetic analysis from 2011 to 2020. Data on the following items were collected and analyzed: age at first visit to the hospital, age at diagnosis of PCD, process of referral to our facility, chief complaint, situs status, PrImary CiliARy DyskinesiA Rule (PICADAR) score, nasal nitric oxide concentration, otoscopic findings, rhinoscopic findings, and paranasal computed tomography scan findings. RESULTS: Sixty-seven patients (24 male, 43 female) were diagnosed with PCD during the study period. Age at diagnosis ranged from 2 months to 69 years (median, 17 years). Respiratory symptoms (77%) were the most common complaint, followed by nasal (15%) and aural (8%) symptoms. Situs inversus was present in 17 (25%) cases. Only 2 cases had congenital cardiac anomalies. The mean PICADAR score was 7.3 (range, 3-14) points. Approximately 50% of tympanic membranes showed retraction, suggesting otitis media with effusion. The mean Lund-Mackay score was 12.8 (range, 7-17) points, suggesting that the radiographic findings were not always severe. There was no significant difference in the total Lund-Mackay score between patients with and without situs inversus (12.7 vs. 12.6, respectively). CONCLUSION: Situs inversus was present in 25% of Japanese PCD patients, which is much lower than observed in other countries. This is a result of differences in the major disease-causing genes. The general rule that "situs inversus is observed in approximately 50% of PCD patients" cannot be applied, at least, in Japanese PCD patients.

Mucoepidermoid carcinoma of the anterior lingual salivary gland: A rare case report.

Mucoepidermoid carcinomas (MECs) are rare head and neck malignant tumours that were originally considered to be benign. It has been estimated that ~20% of MECs in the major salivary glands, such as the parotid gland, and 50% in the several minor salivary glands found in the oral cavity, are malignant. The diagnosis of MECs is mainly based on ancillary and immunohistochemistry testing. However, owing to the difficulty in harvesting adequate material for histological examination, the histopathological diagnosis of intraoral MECs may be particularly challenging. We herein report a rare case of an 82-year-old patient who presented to the Department of Oral and Maxillofacial Surgery of Ryukyu University Hospital with complaints of a progressive swelling and pain in the ventral surface of the apex of the tongue. The patient had previously undergone needle biopsy and the histopathological analysis of the tumour suggested a diagnosis of irritation fibroma. To ensure a more accurate histopathological assessment, an incisional biopsy was performed, in addition to the haematological and radiological assessments. Examination of the obtained surgical specimen confirmed low-grade MEC of the anterior lingual gland. The tumour was surgically excised, the patient healed uneventfully and no recurrence was detected on the regular 3-year follow-up. Although MECs are relatively more common in the minor salivary glands of the oral cavity, they are a rare occurrence in the anterior lingual gland. Therefore, adequate histological material should be surgically harvested to perform a complete evaluation of the morphology and cytology of the tumour and ensure the accuracy of diagnosis.

Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids.

Although alveolar epithelial cells play a critical role in the pathogenesis of pulmonary fibrosis, few practical in vitro models exist to study them. Here, we established a novel in vitro pulmonary fibrosis model using alveolar organoids consisting of human pluripotent stem cell-derived alveolar epithelial cells and primary human lung fibroblasts. In this human model, bleomycin treatment induced phenotypes such as epithelial cell-mediated fibroblast activation, cellular senescence, and presence of alveolar epithelial cells in abnormal differentiation states. Chemical screening performed to target these abnormalities showed that inhibition of ALK5 or blocking of integrin αVß6 ameliorated the fibrogenic changes in the alveolar organoids. Furthermore, organoid contraction and extracellular matrix accumulation in the model recapitulated the pathological changes observed in pulmonary fibrosis. This human model may therefore accelerate the development of highly effective therapeutic agents for otherwise incurable pulmonary fibrosis by targeting alveolar epithelial cells and epithelial-mesenchymal interactions.

Modeling of lung phenotype of Hermansky-Pudlak syndrome type I using patient-specific iPSCs.

BACKGROUND: Somatic cells differentiated from patient-specific human induced pluripotent stem cells (iPSCs) could be a useful tool in human cell-based disease research. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive genetic disorder characterized by oculocutaneous albinism and a platelet dysfunction. HPS patients often suffer from lethal HPS associated interstitial pneumonia (HPSIP). Lung transplantation has been the only treatment for HPSIP. Lysosome-related organelles are impaired in HPS, thereby disrupting alveolar type 2 (AT2) cells with lamellar bodies. HPSIP lungs are characterized by enlarged lamellar bodies. Despite species differences between human and mouse in HPSIP, most studies have been conducted in mice since culturing human AT2 cells is difficult. METHODS: We generated patient-specific iPSCs from patient-derived fibroblasts with the most common bi-allelic variant, c.1472_1487dup16, in HPS1 for modeling severe phenotypes of HPSIP. We then corrected the variant of patient-specific iPSCs using CRISPR-based microhomology-mediated end joining to obtain isogenic controls. The iPSCs were then differentiated into lung epithelial cells using two different lung organoid models, lung bud organoids (LBOs) and alveolar organoids (AOs), and explored the phenotypes contributing to the pathogenesis of HPSIP using transcriptomic and proteomic analyses. RESULTS: The LBOs derived from patient-specific iPSCs successfully recapitulated the abnormalities in morphology and size. Proteomic analysis of AOs involving iPSC-derived AT2 cells and primary lung fibroblasts revealed mitochondrial dysfunction in HPS1 patient-specific alveolar epithelial cells. Further, giant lamellar bodies were recapitulated in patient-specific AT2 cells. CONCLUSIONS: The HPS1 patient-specific iPSCs and their gene-corrected counterparts generated in this study could be a new research tool for understanding the pathogenesis of HPSIP caused by HPS1 deficiency in humans.

The importance of central airway dilatation in patients with bronchiolitis obliterans.

BACKGROUND: Bronchiolitis obliterans (BO) is a clinical syndrome characterised by progressive small airway obstruction, causing significant morbidity and mortality. Central airway dilatation is one of its radiological characteristics, but little is known about the clinical and pathological associations between airway dilatation and BO. METHODS: This retrospective study consecutively included patients who underwent lung transplantation due to BO at Kyoto University Hospital from 2009 to 2019. Demographic and histopathological findings of the resected lungs were compared between patients with and without airway dilatation measured by chest computed tomography (CT) at registration for lung transplantation. RESULTS: Of a total of 38 included patients (median age, 30 years), 34 (89%) had a history of hematopoietic stem-cell transplantation, and 22 (58%) had airway dilatation based on CT. Patients with airway dilatation had a higher frequency of Pseudomonas aeruginosa isolation with greater residual volume than those without airway dilatation. Quantitative CT analysis revealed an increase in lung volume to predictive total lung capacity and a percentage of low attenuation volume <-950 HU at inspiration in association with the extent of airway dilatation. Airway dilatation on CT was associated with an increased number of bronchioles with concentric narrowing of the lumen and thickening of the subepithelium of the walls on histology. CONCLUSIONS: In patients with BO, airway dilatation may reflect increased residual volume or air trapping and pathological extent of obstructive bronchioles, accompanied by a risk of Pseudomonas aeruginosa isolation. More attention should be paid to the development of airway dilatation in the management of BO.