Abstract 1020 Inclusive living subcellular sequencing clarifies physical physiological and human pathological features in osteoimmune diversity

Recent single-cell transcriptomic studies have clarified the cellular diversity within cell populations. Spatial transcriptomics using fixed tissues connects transcriptomics with positional information on histological specimens. However, these technologies cannot decode the dynamic localization of mRNA inside living cells. To decode intracellular gene expression in living cells, we developed intra-single cell sequencing (iSCseq), a new approach combining confocal imaging, picking up any cellular and subcellular components inside both osteoclasts (OCs) and ordinary small cells using the Single CellomeTM System SS2000 (Yokogawa, Japan), and SMART-seq single cell library for next-generation sequencing. This study aimed to establish a new technology, iSCseq, to examine subcellular transcriptomic heterogeneity in living OCs and small cells. Matching the data points obtained by iSCseq with conventional scRNA-seq datasets has clarified the physiological and pathological mechanisms of bone resorption in humans. The number of genes detected with a 3 μm probe was equivalent to that of genes with conventional fluid-based chromium (10x genomics, U.S.), and genes with a 10 μm probe were more than twice those detected with chromium. iSCseq demonstrated subcellular heterogeneity of gene expression in a single cell. Multinucleated cells showed multiple stages of differentiation within a cell. The physical responses and physiological distribution of mitochondria and calcium can be explained by gene expression. Inclusive iSCseq integrated with similar scRNA-seq datasets has elucidated the physiological mysteries in the context of osteoimmune diversity, including hematopoietic stem and mesenchymal cells. Furthermore, we clarified the human skeletal pathogenesis of bone resorption in the tumor microenvironment. The iSCseq approach has the potential to enhance cellular physiology and provide clues for identifying therapeutic targets for human diseases. (Ref. of iSCseq; Okada, bioRxiv 2022). JSPS KAKENHI grant numbers JP 20K22973, 21K16703. 22KK0272, 23K15736 (HO), JP 21K19589 (HH and HO), JP 21K19883 (UC, HH and HO), JP 22K10212 (SK, HH and HO), JP 16H06279 (HO, PAGS: Platform for Advanced Genome Science) and 22H04925 (HO, PAGS ver.2) AMED grant number JP21zf0127002 (HH and HO) and JP21bm0704071 (HH, MS and HO).