Project description:The rupture of ovarian follicles during ovulation is a crucial and intricate process essential for procreation, yet the molecular mechanisms behind this process are not fully understood. Here, we use high-resolution spatial transcriptomics to reveal the spatiotemporal regulation of cell-type-specific molecular programs driving follicle maturation and rupture during hormone-induced ovulation.
Project description:Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Project description:Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Project description:Tissue function relies on the precise spatial organization of cells characterized by distinct molecular profiles. Single-cell RNA-Seq captures molecular profiles but not spatial organization. Conversely, spatial profiling assays to date have lacked global transcriptome information, throughput or single-cell resolution. Here, we develop High-Density Spatial Transcriptomics (HDST), a method for RNA-Seq at high spatial resolution. Spatially barcoded reverse transcription oligonucleotides are coupled to beads that are randomly deposited into tightly packed individual microsized wells on a slide. The position of each bead is decoded with sequential hybridization using complementary oligonucleotides providing a unique bead-specific spatial address. We then capture, and spatially in situ barcode, RNA from the histological tissue sections placed on the HDST array. HDST recovers hundreds of thousands of transcript-coupled spatial barcodes per experiment at 2 μm resolution. We demonstrate HDST in the mouse brain, use it to resolve spatial expression patterns and cell types, and show how to combine it with histological stains to relate expression patterns to tissue architecture and anatomy. HDST opens the way to spatial analysis of tissues at high resolution.
Project description:Cryopreservation is an important technique for preserving fertility potential through ovarian tissue banking. Slow freezing has traditionally been the standard protocol used for ovarian tissue cryopreservation. However, vitrification, as an emerging method, is considered to be an alternative to slow freezing, thus generating some debate in the field. This study utilizes high-resolution spatial transcriptomics to comprehensively profile and compare the molecular impacts of two cryopreservation methods - slow freezing and vitrification - on human ovarian tissues at an unprecedented resolution. Over 135,000 spots were profiled from Fresh and cryopreserved ovarian tissue slices, identifying 8 major cell types and 25 subtypes through marker expression. Detailed spatial localization patterns revealed heterogeneous stromal cell subpopulations and ovarian structures. Transcriptional profiling showed that both cryopreservation methods significantly repressed gene expression, indicative of tissue damage. However, slow freezing induced a broader pro-survival inflammatory and tissue remodeling response compared to vitrification. Gene set enrichment analysis identified elevated ribosomal processes, inflammatory signaling pathways like TNF-?/NF-?B and IL-6/STAT3, as well as extracellular matrix (ECM) organization and collagen synthesis as dominant response themes in slow frozen tissue, which were observed to a lesser degree in vitrified tissue. Key response drivers included MYC, TIMP1, and DCN, with stromal cells located in the cortical regions showing the strongest pathway enrichment. Ribosomal gene expression displayed spatial gradients and supported stromal responses after freezing. We finally demonstrated signaling pathway differences between the two cryopreservation methods, noting TGF-? as a highly possible pathway responsible for coordinating with certain endothelial subgroups to enhance ECM deposition. In summary, this study provides a comprehensive atlas of cell types and their spatial structures in human ovary. It reveals that slow freezing elicits a strong but balanced inflammatory and tissue regenerative state compared to the vitrification. It also offers insights on cryoinjury to ovarian stroma, which may guide optimization of ovarian tissue cryopreservation protocols for clinical applications.