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 ...

Scripps Research Institute

The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future.

Origin and Lineage Plasticity of Endogenous Lacrimal Gland Epithelial Stem/Progenitor Cells.

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1, alias Thbs1) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca] with VIP and UTP was significantly smaller in MECs from TSP1 compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1 mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1 compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1 mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1 compared with WT mice.

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

Aqueous deficiency dry eye (ADDE) is a chronic condition affecting millions, with symptoms ranging from a dry itchiness to blurred vision and accompanied by an increased risk of eye infections. ADDE typically arises from disorders of the lacrimal gland that produces tears necessary for eye lubrication. Cannabis users frequently report dry eye, but the basis for this is unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of tearing.

THC Regulates Tearing via Cannabinoid CB1 Receptors.

Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10:R26-tdTomato mouse, we show that FGF10 cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10 cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10 cells shows that the epithelial FGF10 population express the hallmarks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10 cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2b ductal and myoepithelial cells.

A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands.

The lacrimal gland (LG) is an exocrine gland that produces the watery part of the tear film that lubricates the ocular surface. Chronic inflammation, such as Sjögren's syndrome (SS), is one of the leading causes of aqueous-deficiency dry eye (ADDE) disease worldwide. In this study we analyzed the chronic inflammation in the LGs of the NOD.B10Sn-H2b/J () mice, a mouse model of SS, utilizing bulk RNAseq and Visium spatial gene expression. With Seurat we performed unsupervised clustering and analyzed the spatial cell distribution and gene expression changes in all cell clusters within the LG sections. Moreover, for the first time, we analyzed and validated specific pathways defined by bulk RNAseq using Visium technology to determine activation of these pathways within the LG sections. This analysis suggests that altered metabolism and the hallmarks of inflammatory responses from both epithelial and immune cells drive inflammation. The most significant pathway enriched in upregulated DEGs was the "TYROBP Causal Network", that has not been described previously in SS. We also noted a significant decrease in lipid metabolism in the LG of the  mice. Our data suggests that modulation of these pathways can provide a therapeutic strategy to treat ADDE.

Spatial transcriptomics of the lacrimal gland features macrophage activity and epithelium metabolism as key alterations during chronic inflammation.

Aging is a complex biological process in which many organs are pathologically affected. We previously reported that aged C57BL/6J had increased lacrimal gland (LG) lymphoid infiltrates that suggest ectopic lymphoid structures. However, these ectopic lymphoid structures have not been fully investigated. Using C57BL/6J mice of different ages, we analyzed the transcriptome of aged murine LGs and characterized the B and T cell populations. Age-related changes in the LG include increased differentially expressed genes associated with B and T cell activation, germinal center formation, and infiltration by marginal zone-like B cells. We also identified an age-related increase in B1 cells and CD19B220 cells. B220CD19 cells were GL7 (germinal center-like) and marginal zone-like and progressively increased with age. There was an upregulation of transcripts related to T follicular helper cells, and the number of these cells also increased as mice aged. Compared to a mouse model of Sjögren syndrome, aged LGs have similar transcriptome responses but also unique ones. And lastly, the ectopic lymphoid structures in aged LGs are not exclusive to a specific mouse background as aged diverse outbred mice also have immune infiltration. Altogether, this study identifies a profound change in the immune landscape of aged LGs where B cells become predominant. Further studies are necessary to investigate the specific function of these B cells during the aged LGs.

Ectopic lymphoid structures in the aged lacrimal glands.

Lacrimal gland inflammation triggers dry eye disease through impaired tear secretion by the epithelium. As aberrant inflammasome activation occurs in autoimmune disorders including Sjögren's syndrome, we analyzed the inflammasome pathway during acute and chronic inflammation and investigated its potential regulators. Bacterial infection was mimicked by the intraglandular injection of lipopolysaccharide (LPS) and nigericin, known to activate the NLRP3 inflammasome. Acute injury of the lacrimal gland was induced by interleukin (IL)-1α injection. Chronic inflammation was studied using two Sjögren's syndrome models: diseased  compared to healthy  mice and Thrombospondin-1-null (TSP-1) compared to TSP-1 mice. Inflammasome activation was investigated by immunostaining using the  reporter mouse, by Western blotting, and by RNAseq. LPS/Nigericin, IL-1α and chronic inflammation induced inflammasomes in lacrimal gland epithelial cells. Acute and chronic inflammation of the lacrimal gland upregulated multiple inflammasome sensors, caspases 1/4, and interleukins  and . We also found increased IL-1β maturation in Sjögren's syndrome models compared with healthy control lacrimal glands. Using RNA-seq data of regenerating lacrimal glands, we found that lipogenic genes were upregulated during the resolution of inflammation following acute injury. In chronically inflamed  lacrimal glands, an altered lipid metabolism was associated with disease progression: genes for cholesterol metabolism were upregulated, while genes involved in mitochondrial metabolism and fatty acid synthesis were downregulated, including peroxisome proliferator-activated receptor alpha (PPARα)/sterol regulatory element-binding 1 (SREBP-1)-dependent signaling. We conclude that epithelial cells can promote immune responses by forming inflammasomes, and that sustained inflammasome activation, together with an altered lipid metabolism, are key players of Sjögren's syndrome-like pathogenesis in the  mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism.

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.

Primary Sjögren's syndrome (pSS) is an autoimmune disease that mainly attacks the lacrimal glands causing severe aqueous-deficient dry eye. Clinical evidence indicates the DNA sensing mechanism in the pathogenesis of pSS. The purpose of the present study is to determine the pro-inflammatory effect of self-genomic DNA (gDNA) on myoepithelial cells (MECs), which along with acinar and ductal cells is a major cell type of the lacrimal gland.

Genomic DNA activates the AIM2 inflammasome and STING pathways to induce inflammation in lacrimal gland myoepithelial cells.

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.

Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease for which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken centre stage, as these cells are believed to drive structural remodelling and lung function impairment. While aMYFs are now widely believed to originate from alveolar fibroblasts, the heterogeneity and ultimate fate of aMYFs during fibrosis resolution remain elusive. We have shown previously that aMYF dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution.

Evidence for a lipofibroblast-to-  myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice.

Advanced age is one of the most recognizable risk factors for dry eye. Dry eye disease affects millions worldwide and can result from age-related lacrimal gland dysfunction, which correlates with a decline in lacrimal gland secretory cell function and chronic inflammation. This study investigated the potential of calorie restriction to maintain lacrimal gland and ocular surface health. Adult female C57BL/6 J mice were subjected to a 40% calorie restriction for 4 months, starting at 6-7 months and continuing until 10-11 months. These mice were compared to controls fed ad libitum. Bulk RNA sequencing of lacrimal glands, conjunctiva, and cornea subjected to calorie restriction compared to ad libitum revealed significant differentially expressed genes (DEGs). Pathways enriched in the upregulated DEGs indicate enhanced circadian rhythm, secretory functions, and lipid metabolism. These findings were confirmed using individual qRT-PCR and western blotting. In contrast, pathways enriched in the downregulated DEGs were associated with immune cell activation, adaptive immune responses, extracellular matrix remodeling, and metalloproteinase activity. Histological sections of calorie-restricted lacrimal glands revealed reduced mononuclear cell infiltration and fewer positive cells for CD4, CD19, and MHC II than in ad libitum lacrimal glands. Calorie restriction also prevented age-related corneal barrier dysfunction and mitigated age-related conjunctival goblet cell loss, hallmarks of dry eye disease. These findings suggest that calorie restriction supports lacrimal gland and ocular surface health by reducing inflammation and extracellular matrix remodeling and by enhancing the lacrimal gland's secretory function.

Article	Year
Origin and Lineage Plasticity of Endogenous Lacrimal Gland Epithelial Stem/Progenitor Cells. iScience\| PubMed ID: 32559730	2020
Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease. The American journal of pathology\| PubMed ID: 32679229	2020
THC Regulates Tearing via Cannabinoid CB1 Receptors. Investigative ophthalmology & visual science\| PubMed ID: 32852544	2020
A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands. Cell reports\| PubMed ID: 35417692	2022
Spatial transcriptomics of the lacrimal gland features macrophage activity and epithelium metabolism as key alterations during chronic inflammation. Frontiers in immunology\| PubMed ID: 36341342	2022
Ectopic lymphoid structures in the aged lacrimal glands. Clinical immunology (Orlando, Fla.)\| PubMed ID: 36740002	2023
Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism. International journal of molecular sciences\| PubMed ID: 36901740	2023
The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice. Cells\| PubMed ID: 37408269	2023
Genomic DNA activates the AIM2 inflammasome and STING pathways to induce inflammation in lacrimal gland myoepithelial cells. The ocular surface\| PubMed ID: 37769964	2023
A closer look into the cellular and molecular biology of myoepithelial cells across various exocrine glands. The ocular surface\| PubMed ID: 38141817	2024
Evidence for a lipofibroblast-to- myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice. The European respiratory journal\| PubMed ID: 39401861	2025
Exploring the transformative effects of calorie restriction on the lacrimal gland in adult mice. GeroScience\| PubMed ID: 40580246	2025

Vanessa Delcroix

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