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Background <p>The gastroesophageal tract, including the esophagus, gastroesophageal junction, and stomach, plays a central role in digestive health. Disruptions in its cellular and molecular composition are implicated in various diseases, including gastroesophageal reflux disease, infection-associated inflammation, Barrett’s esophagus, and cancers. A deeper understanding of tissue development and homeostasis is essential to uncover the mechanisms underlying these conditions.</p> Methods <p>We developed a high-quality, integrated protocol for isolating single cells from mouse gastroesophageal tissues at different developmental stages, from embryonic to adult. This protocol enables high-resolution single-cell RNA sequencing (scRNA-seq) to explore cellular heterogeneity, lineage trajectories, and gene expression dynamics. We also describe a method to generate 3D organoids from adult epithelial stem cells, faithfully replicating native architecture. Additionally, we present a spatial analysis workflow that combines single-molecule RNA in situ hybridization with immunofluorescence for simultaneous detection of transcripts and proteins in tissue sections.</p> Results <p>The protocol supports robust single-cell capture and transcriptomic profiling across developmental stages, revealing dynamic changes in cell populations and transcriptional programs. The generated 3D organoids recapitulate in vivo epithelial structures and provide a tractable model system for functional studies. Spatial RNA-protein mapping enables the identification of cell-type-specific expression patterns and their localization in the tissue context.</p> Discussion <p>This integrative workflow provides a powerful tool for studying epithelial development, spatial organization, and disease-related alterations in the gastroesophageal tract. With optimized approaches for tissue dissociation and imaging, this protocol will significantly advance investigations in developmental biology and gastroenterology.</p>

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Integrated protocol for single-cell RNA sequencing, organoid culture, and RNA-protein spatial analysis in gastroesophageal tissues

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月7日

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  • Naveen Kumar,
  • Pon Ganish Prakash,
  • Rajendra Kumar Gurumurthy,
  • Cindrilla Chumduri

摘要

Background

The gastroesophageal tract, including the esophagus, gastroesophageal junction, and stomach, plays a central role in digestive health. Disruptions in its cellular and molecular composition are implicated in various diseases, including gastroesophageal reflux disease, infection-associated inflammation, Barrett’s esophagus, and cancers. A deeper understanding of tissue development and homeostasis is essential to uncover the mechanisms underlying these conditions.

Methods

We developed a high-quality, integrated protocol for isolating single cells from mouse gastroesophageal tissues at different developmental stages, from embryonic to adult. This protocol enables high-resolution single-cell RNA sequencing (scRNA-seq) to explore cellular heterogeneity, lineage trajectories, and gene expression dynamics. We also describe a method to generate 3D organoids from adult epithelial stem cells, faithfully replicating native architecture. Additionally, we present a spatial analysis workflow that combines single-molecule RNA in situ hybridization with immunofluorescence for simultaneous detection of transcripts and proteins in tissue sections.

Results

The protocol supports robust single-cell capture and transcriptomic profiling across developmental stages, revealing dynamic changes in cell populations and transcriptional programs. The generated 3D organoids recapitulate in vivo epithelial structures and provide a tractable model system for functional studies. Spatial RNA-protein mapping enables the identification of cell-type-specific expression patterns and their localization in the tissue context.

Discussion

This integrative workflow provides a powerful tool for studying epithelial development, spatial organization, and disease-related alterations in the gastroesophageal tract. With optimized approaches for tissue dissociation and imaging, this protocol will significantly advance investigations in developmental biology and gastroenterology.