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

ヒトiPS細胞由来肺胞スフェロイドの"on-gel培養法"による化合物スクリーニング --I型肺胞上皮細胞の分化を促進するシグナル経路を同定--. 京都大学プレスリリース. 2024-03-29.Constructing "on-gel" alveolar organoids as a new screening platform. 京都大学プレスリリース. 2024-03-29.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

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

iPS細胞を用いてヘルマンスキー・パドラック症候群の肺病態の解析に成功 --研究が困難な遺伝性疾患の治療薬開発の足がかりに--. 京都大学プレスリリース. 2021-11-15.[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

Exfoliated graphene sheets decorated with metal / metal oxide nanoparticles: simple preparation from cation exchanged graphite oxide

We produced carbon hybrid materials of graphene sheets decorated with metal or metal oxide nanoparticles of gold, silver, copper, cobalt, or nickel from cation exchanged graphite oxide. Measurements using powder X-ray diffraction, transmission electron microscopy, and X-ray absorption spectra revealed that the Au and Ag in the materials (Au-Gr and Ag-Gr) existed on graphene sheets as metal nanoparticles, whereas Cu and Co in the materials (Cu-Gr and Co-Gr) existed as a metal oxide. Most Ni particles in Ni-Gr were metal, but the surfaces of large particles were partly oxidized, producing a core-shell structure. The Ag-Gr sample showed a catalytic activity for the oxygen reduction reaction in 1.0 M KOH aq. under an oxygen atmosphere. Ag-Gr is superior as a cathode in alkaline fuel cells, which should not be disturbed by the methanol cross-over problem from the anode. We established an effective approach to prepare a series of graphene-nanoparticle composite materials using heat treatment

Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

iPS細胞を用いて肺胞上皮細胞の分化評価に成功 --肺の障害研究への足がかりに--. 京都大学プレスリリース. 2020-12-14.Alveologenesis is a developmental step involving the expansion of the lung surface area which is essential for gas exchange. The gas exchange process is mediated by alveolar type I (AT1) cells, which are known to be differentiated from alveolar type II (AT2) or bipotent cells. Due to the difficulty of isolating and culturing primary AT1 cells, the mechanism underlying their differentiation is not completely understood. We performed single‐cell RNA sequencing (scRNA‐seq) of fibroblast‐dependent alveolar organoids (FD‐AOs), including human induced pluripotent stem cell (hiPSC)‐derived epithelial cells and fetal lung fibroblasts, and identified hiPSC‐derived AT1 (iAT1) cells. A comparison of the FD‐AOs and fibroblast‐free alveolar organoids showed that iAT1 cells were mainly present in the FD‐AOs. Importantly, the transcriptomes of iAT1 cells were remarkably similar to those of primary AT1 cells. Additionally, XAV‐939, a tankyrase inhibitor, increased iAT1 cells in passaged FD‐AOs, suggesting that these cells were differentiated from hiPSC‐derived AT2 (iAT2) cells through the inhibition of canonical Wnt signaling. Consequently, our scRNA‐seq data allowed us to define iAT1 cells and identify FD‐AOs as a useful model for investigating the mechanism underlying human AT1 cell differentiation from AT2 cells in vitro

Observation of micropores in hard-carbon using Xe-129 NMR porosimetry

The existence of micropores and the change of surface structure in pitch-based hard-carbon in xenon atmosphere were demonstrated using Xe-129 NMR. For high-pressure (4.0 MPa) Xe-129 NMR measurements, the hard-carbon samples in Xe gas showed three peaks at 27, 34 and 210 ppm. The last was attributed to the xenon in micropores (<1 nm) in hard-carbon particles. The NMR spectrum of a sample evacuated at 773 K and exposed to 0.1 MPa Xe gas at 773 K for 24 h showed two peaks at 29 and 128 ppm, which were attributed, respectively, to the xenon atoms adsorbed in the large pores (probably mesopores) and micropores of hard-carbon. With increasing annealing time in Xe gas at 773 K, both peaks shifted and merged into one peak at 50 ppm. The diffusion of adsorbed xenon atoms is very slow, probably because the transfer of molecules or atoms among micropores in hard-carbon does not occur readily. Many micropores are isolated from the outer surface. For that reason, xenon atoms are thought to be adsorbed only by micropores near the surface, which are easily accessible from the surrounding space.</p

iPSC-derived mesenchymal cells that support alveolar organoid development

肺胞オルガノイドをつくることができるヒトiPS細胞由来間葉細胞の作成. 京都大学プレスリリース. 2022-10-12.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

Evolution of mammalian Opn5 as a specialized UV-absorbing pigment by a single amino acid mutation.

Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a Gi-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system

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

iPS細胞を用いて作製した肺胞オルガノイドで間質性肺炎の病態再現に成功 --治療満足度の低い間質性肺炎の治療薬開発に向けて前進--. 京都大学プレスリリース. 2021-11-19.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 αVβ6 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

Effect of suprascapular nerve injury on muscle and regenerated enthesis in a rat rotator cuff tear model

Background Massive rotator cuff tears (RCTs) are complicated by muscle atrophy, fibrosis, and intramuscular fatty degeneration, which are associated with postoperative tendon-to-bone healing failure and poor clinical outcomes. We evaluated muscle and enthesis changes in large tears with or without suprascapular nerve (SN) injury in a rat model. Methods Sixty-two adult Sprague-Dawley rats were divided into SN injury (+) and SN injury (–) groups (n=31 each), comprising tendon (supraspinatus [SSP]/infraspinatus [ISP]) and nerve resection and tendon resection only cases, respectively. Muscle weight measurement, histological evaluation, and biomechanical testing were performed 4, 8, and 12 weeks postoperatively. Ultrastructural analysis with block face imaging was performed 8 weeks postoperatively. Results SSP/ISP muscles in the SN injury (+) group appeared atrophic, with increased fatty tissue and decreased muscle weight, compared to those in the control and SN injury (–) groups. Immunoreactivity was only positive in the SN injury (+) group. Myofibril arrangement irregularity and mitochondrial swelling severity, along with number of fatty cells, were higher in the SN injury (+) group than in the SN injury (–) group. The bone-tendon junction enthesis was firm in the SN injury (–) group; this was atrophic and thinner in the SN injury (+) group, with decreased cell density and immature fibrocartilage. Mechanically, the tendon-bone insertion was significantly weaker in the SN injury (+) group than in the control and SN injury (+) groups. Conclusions In clinical settings, SN injury may cause severe fatty changes and inhibition of postoperative tendon healing in large RCTs. Level of evidence Basic research, controlled laboratory study