Single-Cell RNA Sequencing for Analyzing the Intestinal Tract in Healthy and Diseased Individuals

Yan, Hua; Ye, Yumeng; Zhao, HanZheng; Zuo, Hongyan; Li, Yang

doi:10.3389/fcell.2022.915654

Cited by 5 publications

(3 citation statements)

References 90 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Single-cell RNA sequencing (scRNA) has significantly expanded our understanding of multiple cellular microenvironments, including the gastrointestinal (GI) mucosal microenvironment that broadly consists of immune, stromal, and epithelial cell subtypes. However, high-fidelity capture of epithelial cells from human biopsy samples for transcriptional profiling remains challenging [1]. Prior scRNA studies of patient-derived colon biopsies have primarily utilized droplet-based scRNA (D-scRNA) platforms, but have well-described high rates of mitochondrial contaminant reads per cell [2, 3], which is a signature of dying or stressed cells that can result in potentially aberrant transcriptional profiles [4].…”

Section: Introductionmentioning

confidence: 99%

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Downie,

Musich,

Geraghty

et al. 2024

Preprint

View full text Add to dashboard Cite

Background & AimsSingle-cell RNA sequencing (scRNA) has empowered many insights into gastrointestinal microenvironments. However, profiling human biopsies using droplet-based scRNA (D-scRNA) is challenging since it requires immediate processing to minimize epithelial cell damage. In contrast, picowell-based (P-scRNA) platforms permit short-term frozen storage before sequencing. We compared P- and D-scRNA platforms on cells derived from human colon biopsies.MethodsEndoscopic rectosigmoid mucosal biopsies were obtained from two adults and conducted D-scRNA (10X Chromium) and P-scRNA (Honeycomb HIVE) in parallel using an individual’s pool of single cells (> 10,000 cells/participant). Three experiments were performed to evaluate 1) P-scRNA with cells under specific storage conditions (immediately processed [fresh], vs. frozen at -20C vs. -80C [2 weeks]); 2) fresh P-scRNA versus fresh D-scRNA; and 3) P-scRNA stored at -80C with fresh D-scRNA.ResultsSignificant recovery of loaded cells was achieved for fresh (80.9%) and -80C (48.5%) P-scRNA and D-scRNA (76.6%), but not -20C P-scRNA (3.7%). However, D-scRNA captures more typeable cells among recovered cells (71.5% vs. 15.8% Fresh and 18.4% -80C P-scRNA), and these cells exhibit higher gene coverage at the expense of higher mitochondrial read fractions across most cell types. Cells profiled using D-scRNA demonstrated more consistent gene expression profiles among the same cell type than those profiled using P-scRNA. Significant intra-cell-type differences were observed in profiled gene classes across platforms.ConclusionsOur results highlight non-overlapping advantages of P-scRNA and D-scRNA and underscore the need for innovation to enable high-fidelity capture of colonic epithelial cells. The platform-specific variation highlights the challenges of maintaining rigor and reproducibility across studies that use different platforms.

show abstract

Section: Introductionmentioning

confidence: 99%

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Downie,

Musich,

Geraghty

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Indeed, scRNA-Seq has been of great significance for in-depth analysis of the occurrence and development mechanisms of intestinal diseases, including identification of novel treatment targets and development of new treatment plans. 6 , 7 However, no previous study has used scRNA-Seq technology to reveal the functions and potential cell molecular mechanisms of different cellular subsets in intestinal tissue during AP-induced intestinal mucosal damage and drug treatment recovery process.…”

Section: Introductionmentioning

confidence: 99%

Single-Cell RNA Sequencing Reveals Roles of Fibroblasts During Intestinal Injury and Repair in Rats with Severe Acute Pancreatitis

Wang,

Xu,

et al. 2023

JIR

View full text Add to dashboard Cite

To explore the molecular mechanisms of intestinal injury and treatment by analyzing changes in cellular heterogeneity and composition in rat ileal tissue during injury and treatment processes. Methods:We constructed a rat model of SAP and evaluated treatment with an injected of monoacylglycerol lipase (MAGL) inhibitor (JZL184) solution using three experimental groups: healthy male Sprague-Dawley (SD) rats injected with vehicle (CON), male SD SAP model rats injected with vehicle (SAP), and male SAP rats injected with JZL184. We obtained and prepared a single-cell suspension of ileal tissue of each rat for single-cell transcriptome sequencing. Results: This project classified changes in cellular heterogeneity and composition in rat ileal tissue during SAP-induced intestinal injury and MAGL treatment. We found that the number of fibroblast clusters was decreased in the SAP group relative to the CON group, and increased after JZL184 treatment. Further analysis of differences in gene expression between cell clusters in each group reveals that fibroblasts had the greatest number of differentially expressed genes. Most notably, expression of genes involved in communication between cells was found to vary during SAP-induced intestinal injury and JZL184 treatment. Among these changes, the degree of difference in expression of genes involved in communication between fibroblasts and other cells was the highest, indicating that fibroblasts in rat ileal tissue affect intestinal injury and repair through cell-to-cell communication. In addition, our results reveal that differentially expressed RNA-binding proteins in fibroblasts may affect their functions in intestinal injury and treatment by affecting the expression of genes regulating communication between cells. Conclusion: These findings emphasize the importance of understanding the interactions between fibroblasts and other cells in the context of intestinal injury, providing valuable insights for further exploring molecular mechanisms and insight for discovering new treatment targets and strategies.

show abstract

“…High-throughput single cell RNA sequencing (scRNA-Seq) is powerful for disclosing the complexity and diversity of cells and relationships among genes involved in tissues, and offers an opportunity to explore unbiased gene expression profiling of cells[ 20 ]. scRNA-Seq also provides insights into specific changes in cell lineages, trajectory inference, and the identification of biomarkers[ 21 , 22 ]. scRNA-seq has been used to study MSCs derived from adipose tissue, bone marrow, endometrium, placenta and dental pulp[ 23 - 27 ].…”

Section: Introductionmentioning

confidence: 99%

Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

Zhang¹,

Li²,

Zeng³

et al. 2023

World J Stem Cells

View full text Add to dashboard Cite

BACKGROUND Accumulating evidence suggests that the maxillary process, to which cranial crest cells migrate, is essential to tooth development. Emerging studies indicate that Cd271 plays an essential role in odontogenesis. However, the underlying mechanisms have yet to be elucidated. AIM To establish the functionally heterogeneous population in the maxillary process, elucidate the effects of Cd271 deficiency on gene expression differences. METHODS p75NTR knockout ( Cd271 -/- ) mice (from American Jackson laboratory) were used to collect the maxillofacial process tissue of p75NTR knockout mice, and the wild-type maxillofacial process of the same pregnant mouse wild was used as control. After single cell suspension, the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system. Finally, the sequencing data in Fastq format were obtained. The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data. The gene expression matrix is read by R software, and Seurat is used to control and standardize the data, reduce the dimension and cluster. We search for marker genes for subgroup annotation by consulting literature and database; explore the effect of p75NTR knockout on mesenchymal stem cells (MSCs) gene expression and cell proportion by cell subgrouping, differential gene analysis, enrichment analysis and protein-protein interaction network analysis; understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis. Last we verified the findings single cell sequencing in vitro . RESULTS We identified 21 cell clusters, and we re-clustered these into three subclusters. Importantly, we revealed the cell–cell communication networks between clusters. We clarified that Cd271 was significantly associated with the regulation of mineralization. CONCLUSION This study provides comprehensive mechanistic insights into the maxillary- process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

show abstract

Single-Cell RNA Sequencing for Analyzing the Intestinal Tract in Healthy and Diseased Individuals

Cited by 5 publications

References 90 publications

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Single-Cell RNA Sequencing Reveals Roles of Fibroblasts During Intestinal Injury and Repair in Rats with Severe Acute Pancreatitis

Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

Contact Info

Product

Resources

About