Comment[GEAAccession]	E-GEAD-951
MAGE-TAB Version	1.1
Investigation Title	Comparing hematopoietic stem cells and early lymphoid progenitors (CBX73)
Experiment Description	Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34-/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura / aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and b lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1- ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC.
Experimental Design	development or differentiation design
Experimental Factor Name	sample_name
Experimental Factor Type	sample_name
Person Last Name	Yokota	Kokame
Person First Name	Takafumi	Koichi
Person Affiliation	National Cardiovascular Center Research Institute	
Person Roles	submitter	
Public Release Date	2025-01-31
Protocol Name	P-GEAD-3898	P-GEAD-3899	P-GEAD-3900	P-GEAD-3901	P-GEAD-3902	P-GEAD-3903	P-GEAD-3904	P-GEAD-3905
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol	growth protocol	treatment protocol
Protocol Description	Fetal livers and cells obtained from adult femurs and tibias of heterozygous Rag1/GFP knock-in mice were harvested and subjected to cell sorting as previously described. In the first step, Rag1 or Rag1 cells were sorted according to levels of GFP expression. The sorted cells were incubated with anti-FcR (2.4G2) before staining with PE-anti-Sca1 and APC-anti-c-kit antibodies, suspended in 7AAD-containing buffer, and subjected to a second round of sorting. Dead cells stained with 7AAD and a few contaminating cells with inappropriate GFP levels were gated out, and the cells were then fractionated according to Sca1 and c-kit to obtain Rag1- c-kitHi Sca1+ (HSC), and Rag1Lo c-kitHi Sca1+ (ELP)., Total RNAs were isolated from the HSC-enriched fraction and the ELP-enriched fraction, both of which were isolated from mouse E14.5 fetal liver., The data were analyzed by a GeneSpring Gx software (Agilent Technologies).	none	From the aliquot of each RNA (24 ng), biotin-labeled cRNA was prepared using GeneChip Two-Cycle Target Labeling and Control Reagents (Affymetrix).	The cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Array (Affymetrix) using a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix).	Fluorescent signals were scanned by GeneChip Scanner 3000 (Affymetrix).	none	Fetal livers and cells obtained from adult femurs and tibias of heterozygous Rag1/GFP knock-in mice were harvested and subjected to cell sorting directly.	none
SDRF File	E-GEAD-951.sdrf.txt
Comment[Number of channel]	single-channel
Comment[Array Design REF]	A-GEAD-156
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX73
Comment[BioProject]	PRJDB20050
Comment[CIBEX Accept Date]	2010-01-01
Comment[CIBEX Public Release Date]	2009-09-11
Comment[Last Update Date]	2025-01-31