|
| Status |
Public on May 22, 2019 |
| Title |
CMT1A hiPSC 5165 Schwann cells - biological repeat 1 |
| Sample type |
RNA |
| |
|
| Source name |
CMT1A hiPSC 5165
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: CMT1A
fibroblast donor age: 51
cell type: hiPSC
differentiated cell type: Schwann cell
|
| Treatment protocol |
Human pluripotent stem cells were plated and differentiated into putative Schwann cells. Briefly, colonies of pluripotent cells were rendered into single cells and plated on Geltrex, an artificial basement membrane-like matrix, in accordance with previous protocols9. They were then treated with a combination of small molecules to induce neuronal and Schwann cell differentiation. On day 0, to initiate differentiation, aspirate the medium and add KSR medium containing 10 μM SB-431542 and 500 nM LDN-193189. On day 2 change medium to KSR medium containing 10 μM SB-431542, 500 nM LDN-193189, 3 μM CHIR 99021, 10 μM DAPT. On day 4 change medium to KSR/NB (3:1) medium containing 3 μM CHIR 99021 and 10 μM DAPT (final concentrations are for the combined KSR/NB mixture). On day 6, change medium to KSR/NB (1:1) medium containing 3 μM CHIR 99021 and 10 μM DAPT. On day 8, change medium to KSR/NB (1:3) medium containing 3 μM CHIR 99021 and 10 μM DAPT. On day 10, 14, and 18 change medium to NB medium containing 200 μM dibutyrl cAMP and 200 μM sodium L-ascorbate. Maintain in this manner until day 21-23 of differentiation, when the cells are ready for purification. Schwann cells were purified with Fluorescence Activated Cell Sorting (FACS) after 21-23 days of differentiation using a PE-conjugated antibody to the alpha4 integrin, CD49d (R&D Systems, FAB1354P), and then utilized for gene expression profiling.
|
| Growth protocol |
hiPSCs and hESCs were maintained on MEF feeder layers through weekly passaging for up to 50 passages. Daily media change was performed with human ESC media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol then QIAGEN RNeasy kit
|
| Label |
biotin
|
| Label protocol |
NuGEN Ovation RNA amplification System v2 kit following manufacturer's protocol
|
| |
|
| Hybridization protocol |
18hrs at 45° C with rotation (60rpm) as described by Affymetrix in their GeneChip Expression Analysis Technical Manual
|
| Scan protocol |
Using Affymetrix’ GeneChip Scanner 3000 7G and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual
|
| Description |
gene expression data from hiPSCs differentiated into Schwann cells for 21 days
BMuk-CMT1A-B52_5165-1a-PrimeView_(PrimeView).CEL
|
| Data processing |
Affymetrix CEL files were extracted and their data normalized with the Partek GS 6.6 platform. RMA normalization was used to create quantile-normalized log2 transcript signal values, which were used in subsequent ANOVA analyses.
|
| |
|
| Submission date |
Jun 18, 2015 |
| Last update date |
May 22, 2019 |
| Contact name |
Gabsang Lee |
| E-mail(s) |
glee48@jhmi.edu
|
| Phone |
443-287-8631
|
| Organization name |
The Johns Hopkins University
|
| Department |
School of Medicine
|
| Lab |
Institute for Cell Engineering
|
| Street address |
733 North Broadway
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL15207 |
| Series (1) |
| GSE69988 |
Modeling CMT1A with human induced pluripotent stem cell derived Schwann cells |
|
