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.

Successful treatment of COVID-19-related acute respiratory distress syndrome with a rare blood type: A case report.

Extracorporeal membrane oxygenation is indispensable for critically severe COVID-19 patients. However, it would be inapplicable to patients with a rare blood type or blood transfusion refusal. In that case, severely conservative fluid management with the sacrifice of renal functions and hydrocortisone therapy should be considered for better oxygenation.

Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology.

Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multiciliated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.

Core-shell hydrogel microfiber-expanded pluripotent stem cell-derived lung progenitors applicable to lung reconstruction in vivo.

Lung transplantation is the only treatment available for end-stage lung diseases; however, donor shortage is a global issue. The use of human pluripotent stem cells (hPSCs) for organ regeneration is a promising approach. Nevertheless, methods for the expansion of isolated hPSC-derived lung progenitors (hLPs) for transplantation purposes have not yet been reported. Herein, we established an expansion system of hLPs based on their three-dimensional culture in core-shell hydrogel microfibers, that ensures the maintenance of their bipotency for differentiation into alveolar and airway epithelial cells including alveolar type II (AT2) cells. Further, we developed an efficient in vivo transplantation method using an endoscope-assisted transtracheal administration system; the successful engraftment and in vivo differentiation of hLPs into alveolar epithelial cells (incorporated into the alveoli) was observed. Importantly, expanded hLPs in the context of microfibers were successfully transplanted into the murine lungs, opening avenues for cell-based therapies of lung diseases. Therefore, our novel method has potential regenerative medicine applications; additionally, the high-quality hLPs and AT2 cells generated via the microfiber-based technology are valuable for drug discovery purposes.

Complete occlusion of right pulmonary artery in Behçet disease.

When a patient with Behçet disease presents with haemoptysis, pulmonary vascular involvement should be considered.

A method of generating alveolar organoids using human pluripotent stem cells.

The lung consists of branched structures that are anatomically, developmentally and functionally divided into airway and alveolar regions. Each region contributes to lung-specific functions involving a defense system and gas exchange, and their dysfunction can cause fatal lung diseases. In the alveolar region, the cuboidal alveolar type 2 (AT2) cells account for 90% of the alveolar epithelial cells and serve as the tissue stem cells secreting pulmonary surfactant, and flattened alveolar type I (AT1) cells cover most of the alveolar surface directly contributing to gas exchange adjacent to capillary vessels. It has been difficult to culture alveolar epithelial cells in vitro, as the lineage-specific features of those cells are rapidly lost in a conventional two-dimensional culture setting. The culture of alveolar organoids (AOs) is an emerging technique that can help maintain the features of alveolar epithelial cells in vitro, and their application to human disease modeling is eagerly awaited. We herein describe our method of generating and culturing alveolar epithelial cells and AOs derived from human induced pluripotent stem cells (iPSCs). iPSCs derived from lung disease patients, including those with rare genetic diseases, will make help elucidate the disease mechanism and hopefully identify therapeutic targets.

In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids.

It has been challenging to generate in vitro models of alveolar lung diseases, as the stable culture of alveolar type 2 (AT2) cells has been difficult. Methods of generating and expanding AT2 cells derived from induced pluripotent stem cells (iPSCs) have been established and are expected to be applicable to disease modeling. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by dysfunction of lysosome-related organelles, such as lamellar bodies (LBs), in AT2 cells. From an HPS type 2 (HPS2) patient, we established disease-specific iPSCs (HPS2-iPSCs) and their gene-corrected counterparts. By live cell imaging, the LB dynamics were visualized and altered distribution, enlargement, and impaired secretion of LBs were demonstrated in HPS2-iPSC-derived AT2 cells. These findings provide insight into the AT2 dysfunction in HPS patients and support the potential use of human iPSC-derived AT2 cells for future research on alveolar lung diseases.

Long-term expansion of alveolar stem cells derived from human iPS cells in organoids.

The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC+ alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC+ cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells. Single-cell RNA-seq of SFTPC+ cells and their progenitors demonstrated that their differentiation process and cellular heterogeneity resembled those of developing AT2 cells in vivo. AOs were applicable to drug toxicology studies recapitulating AT2-cell-specific phenotypes. Our methods can help scientists overcome the limitations of current approaches to the modeling of human alveoli and should be useful for disease modeling and regenerative medicine.

Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells.

Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a "9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine.

Retrospective efficacy and safety analyses of erlotinib, pemetrexed, and docetaxel in EGFR-mutation-negative patients with previously treated advanced non-squamous non-small-cell lung cancer.

OBJECTIVE: Several guidelines recommend erlotinib, pemetrexed, or docetaxel for second-line chemotherapy in patients with advanced non-squamous non-small-cell lung cancer (NSCLC). The aim of this study was to retrospectively evaluate the efficacy of erlotinib, pemetrexed, and docetaxel in epidermal growth factor receptor (EGFR) mutation-negative patients with previously treated advanced non-squamous NSCLC. MATERIALS AND METHODS: We analyzed the efficacy of these agents in patients with previously treated advanced non-squamous NSCLC who had EGFR wild-type tumors, performance status (PS) of 0, 1, or 2 and received erlotinib, pemetrexed, or docetaxel between December 2007 and September 2011. Variability among patient backgrounds was evaluated using propensity scores to assess comparability. The efficacy of these agents was evaluated in patient subgroups with low variability. RESULTS: The propensity scores showed that the backgrounds of the groups that received second-line therapy with each agent had low variability and were adequate for comparison. Patients were divided into the PS0/1 and PS2 groups for analysis. The median progression-free survival (PFS) in patients treated with erlotinib was 2.8 months in the PS0/1 group, as compared with 1.0 month in the PS0/1/2 group and 0.90 months in the PS2 group. PFS in PS0/1 patients who received erlotinib was comparable to that in PS0/1 patients who received pemetrexed (2.5 months) or docetaxel (1.9 months). Overall survival (OS) in erlotinib-, pemetrexed-, and docetaxel-treated PS0/1 patients was 16.1, 7.4 and 10.0 months, respectively. The study had limited power to detect differences in PFS and OS because of the small sample size. CONCLUSIONS: Erlotinib appears to be a useful second-line option in PS0/1 patients with EGFR mutation-negative advanced non-squamous NSCLC given its mild adverse effects. The results should be carefully interpreted because of the small sample size, limited power, and retrospective nature of the study.

Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells.

No methods for isolating induced alveolar epithelial progenitor cells (AEPCs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs), we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1(+) "ventralized" anterior foregut endoderm cells (VAFECs) in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM(+) cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM(+) cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

A phase II study of pemetrexed in chemotherapy-naive elderly patients aged ≥ 75 years with advanced non-squamous non-small-cell lung cancer (HANSHIN Oncology Group 003).

OBJECTIVE: Pemetrexed has relatively mild toxicity and possibly can be administered long term to patients with non-small-cell lung cancer. We conducted a Phase II trial to evaluate the efficacy and safety of pemetrexed in chemotherapy-naïve elderly patients with advanced non-squamous non-small-cell lung cancer. METHODS: In this multicenter Phase II trial, we recruited elderly patients with non-squamous non-small-cell lung cancer. Patients with previously untreated Stage IIIB or IV non-squamous non-small-cell lung cancer, ≥75 years, Eastern Cooperative Oncology Group performance status 0-1 and adequate organ functions were eligible. Patients received pemetrexed (500 mg/m(2)) intravenously on Day 1 every 3 weeks until disease progression. The primary endpoint was objective response rate. RESULTS: Forty-seven patients were enrolled from August 2009 to July 2011, and 46 patients were eligible. The median age was 79 years (range 75-91 years), 57% were males, 37% had never smoked, 87% had adenocarcinoma, 74% had Stage IV and 33% had epidermal growth factor receptor tyrosine kinase-activating mutation. The median number of cycles was 4 (1-20). The objective response rate was 13.3% (95% confidence interval; 5.1-26.8%), the disease control rate was 66.7% (95% confidence interval 51.0-80.0%), the median progression-free survival was 4.9 months (95% confidence interval 3.0-6.1 months) and the median overall survival was 18.2 months (95% confidence interval 13.2-23.5 months). One Grade 5 infection (pneumonia) was observed. CONCLUSIONS: This study did not meet the primary endpoint. Pemetrexed monotherapy is not recommended in chemotherapy-naïve elderly patients aged ≥75 years with advanced non-squamous non-small-cell lung cancer.

[A case of lung disease and spondylitis due to Mycobacterium intracellulare in a immunocompetent patient].

A 73-year-old immunocompetent woman was diagnosed with pulmonary nontuberculous mycobacterial (NTM) infection and followed up without treatment. She developed lumbago and consulted a physician. Spinal magnetic resonance imaging (MRI) indicated pyogenic spondylitis and she was admitted to the orthopedics department at our hospital 4 months after developing lumbago. Spinal MRI on admission revealed spondylitis in L5/S1 and thus antibiotic agents were administered. However, the antibiotics were ineffective and she underwent surgery. Mycobacterium avium complex (MAC) was identified from cultures of surgical specimens, and the histopathological findings revealed epithelioid cell granuloma with necrosis. Spondylitis due to MAC was diagnosed and the patient was administered with rifampicin, ethambutol, clarithromycin and streptomycin. She was discharged on hospital day 113 with a good outcome. Recently, the number of spondylitis due to NTM infection in immunocompetent patients have been increasing. We should take it into consideration that not only bacteria and Mycobacterium tuberculosis but also NTM infection can cause infectious spondylitis.

How sensitive are epidermal growth factor receptor-tyrosine kinase inhibitors for squamous cell carcinoma of the lung harboring EGFR gene-sensitive mutations?

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutations are found mostly in adenocarcinoma, and rarely in squamous cell carcinoma (SQC). Little is known about SQC harboring EGFR mutations. METHODS: Between April 2006 and October 2010, we investigated the incidence of EGFR activating mutations in SQC of the lung using the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method. The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) was retrospectively evaluated in patients with EGFR-mutated SQC. Further pathologic analyses were performed using immunohistochemistry. RESULTS: Thirty-three of 249 patients with SQC (13.3%) had EGFR mutations, including exon 19 deletion (19 of 33 patients, 58%), L858R point mutation in exon 21 (12 of 33, 36%), and G719S point mutation in exon 18 (2 of 33, 6%). Twenty of these 33 patients received EGFR-TKI therapy, and five of these 20 responded to EGFR-TKIs with a response rate of 25.0% (95% confidence interval [CI], 8.7%-49.1%). The patients' median progression-free survival and median overall survival were 1.4 months (95% CI, 0.7-5.8 months) and 14.6 months (95% CI, 2.9-undeterminable months), respectively. Approximately one third of the EGFR-mutated SQC patients achieved progression-free survival for longer than 6 months. Some of these patients had high carcinoembryonic antigen levels or a history of never smoking, or were positive for thyroid transcription factor-1. CONCLUSIONS: Although EGFR-TKIs seem to be generally less effective in EGFR-mutated SQC than in EGFR-mutated adenocarcinoma, some EGFR-mutated SQC patients can obtain clinical benefit from EGFR-TKIs. To better identify these patients, not only EGFR mutation status, but also clinical factors and pathologic findings should be taken into consideration.

The clinical characteristics of two anti-OJ (anti-isoleucyl-tRNA synthetase) autoantibody-positive interstitial lung disease patients with polymyositis/dermatomyositis.

We herein report the clinical and laboratory characteristics of two anti-OJ (anti-isoleucyl-tRNA synthetase) autoantibody-positive interstitial lung disease patients with polymyositis/dermatomyositis (PM/DM). We compared these characteristics with previously published findings. Previous reports and our present cases show that anti-OJ autoantibody-positive interstitial lung disease (ILD) patients with PM/DM lack the manifestations of Raynaud's phenomenon and sclerodactyly and show good prognoses and responses to glucocorticoid therapy. These results indicate that the presence of anti-OJ autoantibodies may be useful for predicting the prognosis of ILD and its clinical course in PM/DM patients.

Successful treatment of non-small cell lung cancer with gefitinib after severe erlotinib-related hepatotoxicity.

Gefitinib and erlotinib are first-generation small molecular inhibitors of EGFR tyrosine kinase activity. To the best of our knowledge, to date, two reports have stated that patients with NSCLC who develop severe hepatotoxicity secondary to gefitinib treatment can be safely switched to erlotinib. However, the reverse situation has not been reported. Here, we present the first case with non-small cell lung cancer harboring EGFR mutation who developed grade 3/4 hepatotoxicity after initiation of erlotinib, which resolved when therapy was changed to gefitinib. As far as we know, this is the first report showing the efficacy of gefitinib for a non-small cell lung cancer patient who developed severe hepatotoxicity while under erlotinib therapy.

Chemical pneumonitis and acute lung injury caused by inhalation of nickel fumes.

A 50-year-old man with a 30-year occupational history of welding presented with low-grade fever, fatigue and persistent dry cough. Computed tomography (CT) of the chest revealed interlobular septal thickening and bilateral non-segmental patchy ground-glass opacities except in the sub-pleural zone. He revealed that he had inhaled nickel fumes 3 days previously at work. These findings suggested a diagnosis of pneumonitis induced by inhalation of nickel fumes. Fewer reports describe pneumonitis associated with the inhalation of nickel compared with zinc fumes. Although nickel compounds are particularly pernicious among the transition metals and more toxic than zinc compounds, nickel fume inhalation rarely induces lethal acute respiratory distress syndrome. Our patient was successfully treated with corticosteroid.