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

High-Resolution Heteronuclear Multidimensional NMR of Proteins in Living Insect Cells Using a Baculovirus Protein Expression System

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D ¹H–¹⁵N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the <i>Streptococcus</i> protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned <i>ab initio</i>, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D ¹⁵N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution[S]

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry