protocol-for-single-cell-dissociation-rna-sequencing-isolation

Protocol for Single-Cell Dissociation, RNA Sequencing, and Isolation of Myoepithelial Cells and Fibroblasts from the Aged Lacrimal and Salivary Glands

Aging of the lacrimal gland (LG) is associated with reduced tear secretion and chronic inflammation, contributing to dry eye disease. Age-related LG ...

optimized-preparation-whole-murine-tumor-bearing-lung-tissue-for-flow

Optimized Preparation of Whole Murine Tumor-Bearing Lung Tissue for Flow Cytometry and Single-Cell RNA-Sequencing

NSCLC accounts for approximately 85% of lung cancers, and Kirsten rat sarcoma (KRAS) is the most commonly mutated gene in NSCLC. Although management of ...

Scripps Research Institute

The Scripps Research Institute

The lacrimal gland (LG) secretes aqueous tears. Previous studies have provided insights into the cell lineage relationships during tissue morphogenesis. However, little is known about the cell types composing the adult LG and their progenitors. Using scRNAseq, we established the first comprehensive cell atlas of the adult mouse LG to investigate the cell hierarchy, its secretory repertoire, and the sex differences. Our analysis uncovered the complexity of the stromal landscape. Epithelium subclustering revealed myoepithelial cells, acinar subsets, and two novel acinar subpopulations:  and  cells. The ductal compartment contained  multilayered ducts and an  cluster formed by luminal and intercalated duct cells.  progenitors were identified as:  basal ductal cells,  cells of  ducts, and + cells of the  acinar and  epithelial clusters. Lineage tracing experiments revealed that the + adult populations contribute to the myoepithelial, acinar, and ductal lineages. Using scRNAseq data, we found that the postnatally developing LG epithelium harbored key features of putative adult progenitors. Finally, we showed that acinar cells produce most of the sex-biased lipocalins and secretoglobins detected in mouse tears. Our study provides a wealth of new data on LG maintenance and identifies the cellular origin of sex-biased tear components.

The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice.

Myoepithelial cells (MECs) are a unique subset of epithelial cells that possess several smooth muscle cell characteristics, such as a high number of actin-myosin filaments and the ability to contract. These cells are primarily located around the secretory cells of exocrine glands, including the salivary, mammary, lacrimal, and sweat glands. Their primary functions involve the construction of the basement membrane and help with secretion of gland products through contraction. So far, no comparative analysis of MECs in different exocrine glands had ever evaluated their differences. In this review, we took advantage of the various publicly available scRNAseq data from mouse exocrine glands to identify their shared and unique characteristics. The aim of this review is to compare the role of MECs in maintaining healthy glandular function, their involvement in disease states, and their regenerative capacity, with a particular emphasis on the latest research findings in these areas.

A closer look into the cellular and molecular biology of myoepithelial cells across various exocrine glands.

The lacrimal gland (LG) develops age-related alterations. This study investigated the lipid content of the accumulated secretions within the murine aged LG.

Aging Affects Lipid Homeostasis in Lacrimal Glands by Upregulating Lipogenesis and Changing Its Specificity.

NSCLC accounts for approximately 85% of lung cancers, and Kirsten rat sarcoma (KRAS) is the most commonly mutated gene in NSCLC. Although management of NSCLC has advanced markedly in recent years, its heterogeneity and plasticity remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) has become an essential tool for dissecting cellular heterogeneity in complex tissues such as the lung. However, generating high-quality single-cell suspensions from tumor-bearing lung tissue is challenging, particularly during tumor formation when the tissue becomes fibrotic and heterogeneous. A key requirement for successful scRNA-seq is the preparation of viable single cells with minimal processing-induced stress or damage. Here, we present a detailed step-by-step protocol for isolating high-quality single-cell suspensions from both healthy and tumor-bearing mouse lung tissue in a Kras; tdTomato reporter mouse model. The procedure includes tissue perfusion, controlled enzymatic digestion, gentle mechanical dissociation, red blood cell lysis, filtration, and viability assessment, and achieves >85% viable cells prior to scRNA-seq. Finally, the tdTomato tumor cells are efficiently isolated by FACS and can be detected as distinct clusters by scRNA-seq using this protocol.

Optimized protocol for processing murine tumor-bearing lung tissue for flow cytometry and single cell RNA-sequencing.

To establish an in vitro model of smoking-associated oxidative injury to the corneal endothelium and evaluate an injury-mitigating factor.

A Cross-Species Hydroquinone Model Replicates Smoking-Related Corneal Endothelial Oxidative Injury and Enables Therapeutic Screening of FGF10.

Tissue and organ regeneration is a remarkable ability observed in many animal species, which has been significantly reduced or lost in several vertebrate lineages, partly due to the evolutionary loss of regeneration-associated genes. For example, many genes involved in limb and skin regeneration in anamniotes (fish and amphibians) have been lost in amniotes (reptiles, birds, and mammals) following their transition to terrestrial life. This raises the intriguing question of whether reintroducing such lost genes could partially restore regenerative abilities. Here, we investigated whether the ag1 gene, which plays a key role in regeneration in fish and amphibians and was lost in amniotes, could enhance skin wound healing in mice. We generated transgenic mice with inducible expression of the  ag1 gene in the skin and compared wound healing dynamics between induced and non-induced groups. Induction of ag1 resulted in approximately 20% faster wound closure. Transcriptomic analysis revealed enhanced activation of multiple pathways involved in wound repair and, notably, the upregulation of a subset of genes typically associated with scarless healing in amphibians and mammalian fetuses. Altogether, our findings demonstrate that a gene lost more than 300 million years ago can still stimulate reparative processes in mammalian tissues, highlighting the potential of ancient gene reactivation to enhance tissue repair in modern vertebrates.

ArticleTotal : 2	Year
Publication title Cited by 1	2026
Publication title	2026

Article	Year
The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice. Cells\| PubMed ID: 37408269	2023
A closer look into the cellular and molecular biology of myoepithelial cells across various exocrine glands. The ocular surface\| PubMed ID: 38141817	2024
Aging Affects Lipid Homeostasis in Lacrimal Glands by Upregulating Lipogenesis and Changing Its Specificity. Investigative ophthalmology & visual science\| PubMed ID: 41031741	2025
Optimized protocol for processing murine tumor-bearing lung tissue for flow cytometry and single cell RNA-sequencing. bioRxiv : the preprint server for biology\| PubMed ID: 41394615	2025
A Cross-Species Hydroquinone Model Replicates Smoking-Related Corneal Endothelial Oxidative Injury and Enables Therapeutic Screening of FGF10. Investigative ophthalmology & visual science\| PubMed ID: 41789845	2026
Restoring , an ancient regeneration gene lost in amniotes, accelerates skin healing in mice. Frontiers in cell and developmental biology\| PubMed ID: 41799438	2026

Anastasiia Ivanova

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