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

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

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

The antitumor activity of xanthohumol

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells

ヒドロシキプロピルシクロデキストリンは、アミオダロンが誘導する肺胞上皮細胞の脂質異常を改善する

京都大学新制・論文博士博士(医学)乙第13481号論医博第2256号新制||医||1059(附属図書館)(主査)教授 平井 豊博, 教授 岩田 想, 教授 秋山 芳展学位規則第4条第2項該当Doctor of Medical ScienceKyoto UniversityDFA

Synthesis and Assignment of the Absolute Configuration of Indenotryptoline Bisindole Alkaloid BE-54017

Synthesis of the indenotryptoline bisindole alkaloid, BE-54017, was accomplished using osmium-promoted <i>cis</i>-dihydroxylation of maleimide as a key step. After optical resolution, the absolute configuration of this molecule was determined by comparing its optical rotation and HPLC profile to those obtained for BE-54017 derived from enantiopure cladoniamide A, whose stereochemistry has been reported previously. BE-54017 with the correct absolute stereochemistry induced apoptosis of epidermal growth factor (EGF)-stimulated EGF receptor overexpressing A431 cells and inhibited vacuolar-type H⁺-ATPase (V-ATPase)

Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

Summary: 5′-Adenosine monophosphate-activated protein kinase (AMPK) is a potential therapeutic target for various medical conditions. We here identify a small-molecule compound (RX-375) that activates AMPK and inhibits fatty acid synthesis in cultured human hepatocytes. RX-375 does not bind to AMPK but interacts with prohibitins (PHB1 and PHB2), which were found to form a complex with AMPK. RX-375 induced dissociation of this complex, and PHBs knockdown resulted in AMPK activation, in the cultured cells. Administration of RX-375 to obese mice activated AMPK and ameliorated steatosis in the liver. High-throughput screening based on disruption of the AMPK-PHB interaction identified a second small-molecule compound that activates AMPK, confirming the importance of this interaction in the regulation of AMPK. Our results thus indicate that PHBs are previously unrecognized negative regulators of AMPK, and that compounds that prevent the AMPK-PHB interaction constitute a class of AMPK activator