|
| Status |
Public on Mar 01, 2006 |
| Title |
Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells |
| Sample type |
RNA |
| |
|
| Source name |
mouse fetal liver
|
| Organism |
Mus musculus |
| Characteristics |
Mouse day 15 fetal liver CD3+ cells
|
| Biomaterial provider |
day 15 mouse fetal liver CD3+ cells, adult splenic CD3+ cells, and fetal liver d15 Gr-1+ cells all from wild-type C57BL/6J mice (Jackson Lab Cat# 000664)
|
| Treatment protocol |
Cultured 3 different cell types
-> Total RNA isolation -> dscDNA -> Biotinylated cRNA -> hybridization to Affymetrix U74Bv2 and U74Cv2 mouse chips
|
| Growth protocol |
FACS sorted cells were cultured for 3 days at a density of 106 cells / ml in IMDM with 10% FBS, and 20 ng/ml SCF, 30 ng/ml Flt - 3 ligand, and 10 ng/ml IL - 6 in a well of a U - bottom 96 - well plate before RNAs were isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using Trizol according to the manufacturer’s recommendations (Life Technologies Inc, Rockville, MD). After quantification by spectrophotometry and 1.2% agarose gel electrophoresis, 15 micro gram of total RNA was used for first strand and second strand cDNA synthesis, followed by in vitro transcription using Ambion T7 MegaScript Kit to produce biotinylated cRNAs.
|
| Label |
phycoerythrin
|
| Label protocol |
Total RNA was extracted from cells using Trizol according to the manufacturer’s recommendations (Life Technologies Inc, Rockville, MD). After quantification by spectrophotometry and 1.2% agarose gel electrophoresis, 15 micro gram of total RNA was used for first strand and second strand cDNA synthesis, followed by in vitro transcription using Ambion T7 MegaScript Kit to produce biotinylated cRNAs.
|
| |
|
| Hybridization protocol |
Standard protocol for Affymetrix gene chips was used. Briefly, biotinylated cRNA was hybridized onto chips in 1X MES hybridization buffer for 16 hr. The fluidics station was then used to wash/stain with streptavidin-phycoerythrin.
|
| Scan protocol |
ScanArray Express HT as scanner, and Microarray Suite as software. Standard protocol.
|
| Description |
Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of critical questions of stem cell biology. Microarray studies showed that the HSC supportive mouse fetal liver CD3+ cells specifically expressed angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). When highly enriched HSCs were cultured in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors for 10 days, a 24 or 30 fold net expansion of long-term HSCs was observed by reconstitution analysis. The coiled-coil domain of Angptl2 mediated the effects of Angptl2 on HSC expansion. Furthermore, angiopoietin-like 5, angiopoietin-like 7, and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.
|
| Data processing |
Microarray data was analyzed by Microarray Suite
|
| |
|
| Submission date |
Nov 10, 2005 |
| Last update date |
Nov 15, 2005 |
| Contact name |
Cheng Cheng Zhang |
| E-mail(s) |
czhang@wi.mit.edu
|
| Organization name |
Whitehead Institute
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL82 |
| Series (1) |
| GSE3609 |
Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells |
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