|
| Status |
Public on Jan 03, 2017 |
| Title |
BM-MSCs_co-culture_rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow
|
| Treatment protocol |
After 10 days of culture, cells were collected and sorted by a FACSAria II flow cytometer (Becton Dickinson). Flow cytometric data were analyzed using FACSDiva software (Becton Dickinson). MSCs were identified on the basis of CD73, CD90, CD105 co-expression and lack of CD45. CB-CD34+ cells were identified on the basis of CD45 and CD34 co-expression. At least 3x10^5 gated events were acquired for each sample. Purity of FACS-isolated cells was greater than 95%, as assessed by flow cytometric analysis of post-sorting samples.
|
| Growth protocol |
BM-MSCs were thawed at 37°C and 5x10^5 cells were plated in T75 flasks. Cells were cultured in complete ISCOVE’s modified Dulbecco’s medium supplemented with 10% FBS for a week until 80% confluence. CB-CD34+ cells were seeded directly onto the MSCs layer at a final concentration of 3x10^5 cells/flask. Cells were maintained in serum-free medium (StemSpan H3000, StemCell Technologies) supplemented with 100 ng/ml Stem Cell Factor (SCF) (Peprotech), Thrombopoietin (TPO) (Peprotech) and Granulocyte Colony Stimulating Factor (G-CSF) (Italfarmaco) for 10 days at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated using Qiagen RNeasy kit according to manufactures’ instructions (Qiagen, Hilden, Germany).
|
| Label |
Biotin
|
| Label protocol |
Biotinylated aRNA were prepared according to the standard Affymetrix protocol from 50 ng total RNA using the GeneChip 3’ IVT Expression kit (Affymetrix)
|
| |
|
| Hybridization protocol |
Labelled RNA were fragmented and hybridized on Affymetrix HG-U133A PLUS 2.0 GeneChip arrays that were subsequently washed and stained using the GeneChip Hybridization, Wash and Stain Kit (Affymetrix)
|
| Scan protocol |
The GeneChip was scanned and data extracted using GeneChip scanner 3000 7G (Affimetrix, SantanClara, CA).
|
| Data processing |
The data were analyzed with Affymetrix® Expression Console™ Software and normalyzed with RMA algorithm. Subsequently analyses for differentially expressed genes cathegorization were performed with Partek® Genomics Suite software version 6.6 (Partek Inc., St. Louis, MO). The analysis of molecular pathways was carried out through the DAVID Functional Annotation Bioinformatics Microarray Analysis. Broad Institute Gene Set Enrichment Analysis (GSEA) software was performed to identify significant enrichments in differentially expressed genes signatures, in case of nominal P values corrected for False Discovery Rate (FDR) for enrichment scores minor than 0.05. The network analyses were generated through the use of Ingenuity Pathway Analysis® (IPA®, Qiagen).
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| |
|
| Submission date |
Dec 07, 2016 |
| Last update date |
Jan 03, 2017 |
| Contact name |
Simone Perucca |
| E-mail(s) |
[email protected]
|
| Phone |
+39 0303998464
|
| Organization name |
University of Brescia
|
| Department |
Department of Clinical and experimental sciences
|
| Lab |
Chair of Hematology
|
| Street address |
Viale Europa
|
| City |
Brescia |
| ZIP/Postal code |
25123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE90970 |
Mesenchymal Stromal Cells Induce Ex Vivo Proliferation and Erythroid Commitment of Cord Blood Haematopoietic Stem Cells |
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