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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 30, 2023 |
| Title |
Single-Cell RNA Sequencing of Mouse Fibroblasts Grown as Spheroids Reveals Presence of Mesenchymal Stem-Like Cells |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Compared to conventional monolayer cell culture, three-dimensional (or spheroid) cultures are more reflective of the in vivo environment and represent a better means of representing normal tissue. Therefore, understanding the biology of spheroid-derived cells is important for a more complete appreciation of in vivo tissue function. Although it has been shown that culture conditions such as nutrients, oxygen, cell passage etc. alter gene expression patterns of the cells, the gene expression profile of spheroid as compared to conventional monolayer culture in primary cells has not been studied. To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids. After quality control, a total of 11,515 cells (8,022 spheroid cells and 3,493 cells of monolayer fibroblasts) were analyzed using Uniform Manifold Approximation and Projection (UMAP) which identified 2 largely separated libraries. These libraries contained cells with 2,491 and 8722 detected genes (nFeature_RNA) for monolayer and spheroid cells respectively. UMAP clustering of the integrated datasets identified 8 significant cell clusters (resolution=0.3) in which clusters 0, 1, 2, 4 and 7 had a higher number of cells in the spheroid library. Differential gene expression analyses revealed that each cluster was characterized by a specific transcriptional profile. Among cell clusters, cluster 4 exhibited significantly increased expression of collagen family genes, including Col1a2, Col3a1, Col1a1, Col5a2, Col4a, Col4a2, Col5a3, Col5a1, Col6a1, Col12a1 and Col15a1. Furthermore, expression of mesenchymal stem cell genes [Sca-1 (Ly6a), CD29 (Itgb1), CD44, CD90.1 (Thy1)] as well as a group of genes important for stem cell self-renewal including Notch2, Sox4, Sox9 Klf2, and Foxp1 were significantly increased in cluster 4. Immunofluorescence (IF) staining was performed to validate the protein expression of selected genes. Our work provides a significant advance in characterizing the global gene expression profile of spheroid culture of mouse TEFs compared to monolayer culture. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with mesenchymal stem-like cells.
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| Overall design |
To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids.
To establish tail/ear fibroblasts (TEFs), the tails and ears from adult mice (8-12 week-old C57BL/6 or FVB) were peeled, minced into 1 cm pieces, placed on culture dishes, and incubated in DMEM/F-12 containing 1% Penicillin and Streptomycin and 20% heat inactivated fetal bovine serum for 7 days. Cells that migrated out of the graft pieces were transferred to new plates and maintained in DMEM/F-12 supplemented with 10% FBS (passage 2). TEFs from passage 3 were used for all experiments.
For monalayer, cells were plated on pretreated cell culture plate using DMEM/F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng/ml). For the suspension culture, cells were plated at 100-300 cells per well in ULA 96 well round-bottom plates (Corning® Costar® Ultra-Low Attachment Multiple Well Plate, Cat#7007) in 200 µl DMEM/F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng/ml) plus 10 µM ROCKi. Cells were maintained in 5% CO2, 21% O2 and 74% N2 at 37oC for 7 days. Half of the culture medium was removed and replaced every other day.
We then performed single cell RNA sequencing (scRNAseq) on mouse dermal fibroblasts cultured as monolayers on cell culture dishes or grown as spheroids. Between 70,000-100,000 mouse adherence-cultured fibroblasts (monolayer) and dissociated spheroid cells (single cells) were freshly prepared and their gene expression profile analyzed using single-cell 3' RNA-sequencing.
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| Citation(s) |
37887352 |
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| Submission date |
Aug 29, 2023 |
| Last update date |
Oct 31, 2023 |
| Organization |
Ottawa Hospital Research Institute |
| Phone |
(613) 737-8899 -73255
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| Department |
Cellular and Molecular Medicine
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| Lab |
Ottawa Bioinformatics Core Facility
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| Street address |
501 Smyth Rd.
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| City |
Ottawa |
| State/province |
ON |
| ZIP/Postal code |
K1H 8L6 |
| Country |
Canada |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (2) |
| GSM7744082 |
Mouse dermal fibroblasts cultured as monolayers |
| GSM7744083 |
Mouse dermal fibroblasts cultured as spheroids |
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| Relations |
| BioProject |
PRJNA1010165 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE241814_RAW.tar |
41.4 Mb |
(http)(custom) |
TAR (of H5) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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