ヒト多能性幹細胞由来の肺胞オルガノイドを用いた肺線維症の疾患モデリング

京都大学新制・論文博士博士(医学)乙第13502号論医博第2261号(主査)教授 村川 泰裕, 教授 柳田 素子, 教授 長船 健二学位規則第4条第2項該当Doctor of Medical ScienceKyoto UniversityDFA

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

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

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

Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

Stress signals cause abnormal proteins to accumulate in the endoplasmic reticulum (ER). Such stress is known as ER stress, which has been suggested to be involved in neurodegenerative diseases, diabetes, obesity and cancer. ER stress activates the unfolded protein response (UPR) to reduce levels of abnormal proteins by inducing the production of chaperon proteins such as GRP78, and to attenuate translation through the phosphorylation of eIF2α. However, excessive stress leads to apoptosis by generating transcription factors such as CHOP. Casein kinase 2 (CK2) is a serine/threonine kinase involved in regulating neoplasia, cell survival and viral infections. In the present study, we investigated a possible linkage between CK2 and ER stress using mouse primary cultured glial cells. 4,5,6,7-tetrabromobenzotriazole (TBB), a CK2-specific inhibitor, attenuated ER stress-induced XBP-1 splicing and subsequent induction of GRP78 expression, but was ineffective against ER stress-induced eIF2α phosphorylation and CHOP expression. Similar results were obtained when endogenous CK2 expression was knocked-down by siRNA. Immunohistochemical analysis suggested that CK2 was present at the ER. These results indicate CK2 to be linked with UPR and to resist ER stress by activating the XBP-1-GRP78 arm of UPR

Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress

Abstract Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti‐inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress‐induced leptin resistance, characterized by insensitivity to the actions of the anti‐obesity hormone leptin. This result was further supported by flurbiprofen attenuating high‐fat diet‐induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti‐obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity

A CK2 inhibitor inhibited ER stress-induced GRP78 expression.

<p>4,5,6,7-tetrabromobenzotriazole (TBB) inhibited GRP78 expression at the mRNA and protein levels. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. RT-PCR was performed using specific primers for each mRNA. n = 4/group *<i>p</i><0.05 compared with ER stress (Tm, Tg) alone. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h, and subjected to Western blotting. n = 3/group *<i>p</i><0.05 compared with ER stress (Tm) alone. Results are expressed as the means ± S.E.</p

CK2 inhibitor did not affect ER stress-induced ATF6 processing.

<p>(A) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 18 h and Western blotting analysis was performed. TBB inhibited ER stress-induced GRP78 expression. *<i>p</i><0.05 (B) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 4 h and Western blotting analysis was performed using ATF6 antibody. (C) Densitometric analysis of p90ATF6. (D) Densitometric analysis of p50 ATF6. TBB did not affect ER stress-induced ATF6 cleavage. n = 2–3/group. Results are expressed as the means ± S.E.</p