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| Status |
Public on Sep 26, 2015 |
| Title |
Gene expression profiles of epithelial and mesenchymal cells extracted from heterotypic ex vivo co-cultures or monocultures |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. The goals and objectives of this study were 1.) to characterize and validate the molecular identity of human primary epithelial and stromal/mesenchymal breast cells maintained long-term in novel ex vivo culture conditions in serum free medium. 2.) To analyze changes in gene expression profiles of normal human primary epithelial and stromal/mesenchymal breast cells upon long-term ex vivo co-culture when compared to corresponding monocultures 3.) To study the dynamic reciprocity between normal human primary epithelial and stromal/mesenchymal breast cells. 4.) To identify critical molecular pathways and biomarkers controlling epithelial and/or stromal cell growth and quiescence.
Human primary epithelial progenitor cells and mesenchymal stem cells bearing fluorescent tags were either co-cultured in novel ex vivo culture conditions on ECM coated meshes in serum free medium (M5) or cultured as monocultures in the same conditions for 30 days. The cultures were then dissociated and epithelial and stromal/mesenchymal cells from either co-cultures or monocultures separated by FACS. Gene expression profiling of epithelial or stromal/mesenchymal cells was performed. Clean gene expression profiles from three different epithelial and stromal/mesenchymal cell extracts either grown in co-cultures or monocultures were successfully obtained.
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| Overall design |
Three complete biological replicates of heterotypic co-cultures and corresponding monocultures were set up in parallel. In order to take account of the biological variability tissues from donors in three different age groups (16-25, 26-40, >40) were included in the study. Epithelial progenitor cells were isolated from cleaned epithelial tissue from reduction mammoplasties of tree different donors (MP9, MP13, MP15) immediately infected in suspension with Lentivirus containing an EGFP (Enhanced Green Fluorescent Protein) expression cassette. Adipocyte tissue derived mesenchymal stem cells (hAMSCs) were isolated from cleaned breast fat tissue from reduction mammoplasties of tree different donors (MP22, MP28, MP29) and were immediately infected in suspension with Lentivirus containing a DsRed2 (Red fluorescent Protein) expression cassette. After selection, spheres were dissociated and single cell suspensions prepared. For heterotypic co-cultures, EGFP tagged epithelial progenitor cells were randomly aggregated with DsRed2 tagged hAMSCs (MP9&MP29, MP13&MP22, MP15&MP28). For monocultures the three corresponding epithelial progenitor cell isolates (MP9, MP13, MP15) or hAMSC isolates (MP29, MP22, MP28) were aggregated alone. The aggregates of each condition were then added on top of ECM coated meshes and serum free medium (M5) was regularly supplemented and changed during the whole ex-vivo culture period. After 30 days the cultures were dissociated with mild proteinase/EDTA (HyQtase, Thermo Scientific) treatment for 20 min. Cells remained thereafter on ice and FACS sorts were performed immediately to isolate viable epithelial or stromal cells, based on their fluorescent protein tag. 300 cells of each condition were directly sorted in 100ul TRIZOL in cooled tubes. The samples were immediately frozen on dry ice and stored at -70°C prior to RNA extraction. A total of 12 samples were successfully hybridized on Gene 1.0 AFFYMETRIX human gene chips: Co-culture derived epithelial cells (ESe, Arrays 4, 8, 12), co-culture derived stromal cells (ESs, Arrays 3, 7, 11), monoculture derived epithelial cells (Ee, Arrays 2, 6, 10) and monoculture derived stromal cells (Ss, Arrays 1, 5, 9).
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| Contributor(s) |
Duss S, Cabuy E, Roloff T, Bentires-Alj M |
| Citation(s) |
24916766 |
| Submission date |
Sep 26, 2013 |
| Last update date |
Jan 31, 2023 |
| Contact name |
Tim Roloff |
| E-mail(s) |
tim.roloff@fmi.ch
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| Organization name |
FMI
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| Department |
Functional Genomics
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platforms (1) |
| GPL6244 |
[HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] |
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| Samples (12)
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| GSM1240444 |
hAMSCs_MP29_monoculture_Array1_replicate1 |
| GSM1240445 |
PHBECs_MP9_monoculture_Array2_replicate1 |
| GSM1240446 |
hAMSCs_MP29_coculture_Array3_replicate1 |
| GSM1240447 |
PHBECs_MP9_coculture_Array4_replicate1 |
| GSM1240448 |
hAMSCs_MP22_monoculture_Array5_replicate2 |
| GSM1240449 |
PHBECs_MP13_monoculture_Array6_replicate2 |
| GSM1240450 |
hAMSCs_MP22_coculture_Array7_replicate2 |
| GSM1240451 |
PHBECs_MP13_coculture_Array8_replicate2 |
| GSM1240452 |
hAMSCs_MP28_monoculture_Array9_replicate3 |
| GSM1240453 |
PHBECs_MP15_monoculture_Array10_replicate3 |
| GSM1240454 |
hAMSCs_MP28_coculture_Array11_replicate3 |
| GSM1240455 |
PHBECs_MP15_coculture_Array12_replicate3 |
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| Relations |
| BioProject |
PRJNA221585 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE51201_RAW.tar |
43.1 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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