To systematically characterize the loss of tissue integrity and organ dysfunction resulting from aging, we produced an in-depth spatial transcriptomic profile of nine tissues in male mice during aging. We showed that senescence-sensitive spots (SSSs) colocalized with elevated entropy in organizational structure and that the aggregation of immunoglobulin-expressing cells is a characteristic feature of the microenvironment surrounding SSSs. Immunoglobulin G (IgG) accumulated across the aged tissues in both male and female mice, and a similar phenomenon was observed in human tissues, suggesting the potential of the abnormal elevation of immunoglobulins as an evolutionarily conserved feature in aging. Furthermore, we observed that IgG could induce a pro-senescent state in macrophages and microglia, thereby exacerbating tissue aging, and that targeted reduction of IgG mitigated aging across various tissues in male mice. This study provides a high-resolution spatial depiction of aging and indicates the pivotal role of immunoglobulin-associated senescence during the aging process.

Recent discoveries about the molecular heterogeneity of the cerebellar cortex suggest the existence of functionally divergent subclasses of anatomically defined cell types. Using spatial transcriptome and single-nucleus RNA-seq analysis, we mapped 3D transcriptomic atlases of the whole cerebellum of mice, marmosets, and macaques at the single-cell resolution. Comparative analysis revealed specific cell types, cell localizations, and intra-cerebellum molecular heterogeneity across species. A comprehensive database generated from this study will expand the acknowledgment of the mammalian cerebellum.

" The molecular and cellular mechanisms underlying the function of the cochlear nucleus (CN) remain to be fully elucidated. Using single-nucleus RNA sequencing and single-cell spatial transcriptome analyses, we identified transcriptome-defined cell types, as well as their spatial distribution and marker genes. These data also allowed us to acquire a new definition of CN subregions. By comparing transcriptomic profiles between normal mice and mutant mice with congenital hearing loss due to hair cell malfunction, we further identified glutamatergic Spp1+-bushy cell as a primary cell type exhibiting hearing loss-induced alteration in gene expression. Among highly affected genes in the bushy cell, we found that deletion of a developmentally regulated osteopontin-encoding gene Spp1 affected CN processing of auditory signals. Together, our study provides the most comprehensive molecular and cellular atlas of CN to date and identifies critical hearing loss-related cell types and specific genes that may serve as potential therapeutic targets. "

Cholestatic injuries, characterized by regional damage around the periportal region, lack curative therapies and cause considerable mortality. In this study, we generated a high-definition spatiotemporal atlas during cholestatic injury and repair by Stereo-seq and single-cell transcriptomics. We uncovered that cholangiocytes function as a periportal hub (cholangio-hub) by integrating multiple signals with neighboring cells. Feedback between cholangiocytes and lipid-associated macrophages (LAM) was detected in the cholangio-hub, which is related to the differentiation of LAM, a recently identified subpopulation of macrophages crucial in tissue injury. Moreover, the cholangio-hub highly expressed TGFβ, which is associated with cholangiocyte conversion of liver progenitor-like cells during injury and dampened proliferation of periportal hepatocytes during recovery. Importantly, spatiotemporal analysis revealed a key inhibitory rheostat for hepatocyte proliferation. Our data provide a comprehensive resource for demarcating regional cholestatic injuries.

Optimization and application of clustering algorithms in integrated analysis of multiomics dataset

The developing brain has a complex and well-organized anatomical structure comprising different types of neural and non-neural cells. Stem cells, progenitors, and newborn neurons tightly interact with their neighbouring cells and tissue microenvironment, and this intricate interplay ultimately shapes the output of neurogenesis. Given the relevance of spatial cues during brain development, we acknowledge the necessity for a transcriptomics atlas within the tissue context accessible to the neurodevelopmental community. To fulfil this need, we offer an open-access spatial gene expression browser of the embryonic mouse brain at the peak of neurogenesis. Using 10x Visium technology, we generated spatially-resolved RNAseq data from E13.5 embryonic brain sections. Unsupervised clustering reliably defined specific cell type populations of diverse lineages and maturational states. Differential expression analysis revealed unique transcriptional signatures across specific embryonic brain areas, uncovering novel features inherent to particular anatomical domains. Furthermore, we integrated single-cell RNAseq data from E13.5 mouse brains into our Spatial Transcriptomics data, adding tissue context to single-cell resolution. In summary, we provide a valuable tool that enables the exploration and discovery of unforeseen molecular players involved in neurogenesis, particularly in the crosstalk between different cell types.

The basic analysis steps of spatial transcriptomics involves obtaining gene expression information from both space and cells. This process requires a set of tools to be completed, and existing tools face performance issues when dealing with large data sets. These issues include computationally intensive spatial localization, RNA genome alignment, and excessive memory usage in large chip scenarios. These problems affect the applicability and efficiency of the process. To address these issues, a high-performance and accurate spatial transcriptomics data analysis workflow called Stereo-Seq Analysis Workflow (SAW) has been developed for the Stereo-Seq technology developed by BGI. This workflow includes mRNA spatial position reconstruction, genome alignment, gene expression matrix generation and clustering, and generate results files in a universal format for subsequent personalized analysis. The excutation time for the entire analysis process is ~148 minutes on 1G reads 1*1 cm chip test data, 1.8 times faster than unoptimized workflow.

Here, we investigated the effects of Rhynchophylline (RHY) on the mouse brain spatial transcriptome. More precisely, we injected male and female mice intraperitoneally with either saline (NaCl) or RHY, either at Zeitgeber time (ZT referring to time in hour after light onset) 0 or ZT0 and ZT11. Brains were sampled at ZT4 or ZT14, respectively, and immediately frozen embedded in OCT. Brains were treated and libraries were prepared according to 10x Genomics protocols for Visium Spatial Gene Expression. Sequencing was conducted by Genome Quebec (Montreal, Quebec, Canada). Findings reveal molecular routes by which RHY acts on the brain in a sleep-relevant context. Please cite the original paper when using these data (Ballester Roig et al., Biol Direct, 2023) and see also the submission GSE217058.

Here, we investigated the effects of Rhynchophylline (RHY) on the mouse brain spatial transcriptome. More precisely, we injected male and female mice intraperitoneally with either saline (NaCl) or RHY, either at Zeitgeber time (ZT referring to time in hour after light onset) 0 or at ZT0 and ZT11. Brains were sampled at ZT4 or ZT14, respectively, and immediately frozen embedded in OCT. Brains were treated and libraries were prepared according to 10x Genomics protocols for Visium Spatial Gene Expression. Sequencing was conducted by Genome Quebec (Montreal, Quebec, Canada). Findings reveal molecular routes by which RHY acts on the brain in a sleep-relevant context. Please cite the original paper when using these data (Ballester Roig et al., Biol Direct, 2023).

Megakaryocytes are bone marrow (BM) resident cells that derive from hematopoietic stem cells. A pivotal function of megakaryocytes is the generation of platelets through the release of long protrusions called proplatelets into sinusoidal vessels. single-cell RNA-sequencing on murine BM megakaryocytes has previously revealed transcriptional heterogeneity with segmentation into four distinct categories. These studies postulated functions beyond platelet production with evidence for immunoregulatory and stem cell niche supporting subtypes, as well as a cycling population. The spatial context and transcriptional heterogeneity of megakaryocytes is of great interest as localization of for instance the vasculature is a necessity for platelet production. For single-cell RNA sequencing this spatial orientation is however lost due to the dissociation of tissues. Recent technological advances have enabled the interrogation of gene expression profiles of tissues in situ. This enables the integration of morphological, situational and transcriptional information to classify cells in the context of their microenvironment. In the following we present, for the first time, the application of this technology to BM megakaryocytes at a single cell level.