BioProject

Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. More...

Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. However those cell surface phenotypes do not represent functional HSC after in vitro culture. We compared gene expression profiling of phenotypic HSC (CD48-KSL cells) before and after in vitro culture, and discovered that cultured HSC express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSC can be divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. Induction of CD244 may partially be triggered by induction of endoplasmic reticulum (ER) stress, as chemically induced ER stress signal increased CD244+ subpopulation while ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which were correlated to the output of reconstitution assay. These data suggest CD244 positivity is a potent marker to exclude non-functional HSC after in vitro culture thereby useful to elucidate mechanism of functional decline of HSC during ex vivo treatment. Overall design: CD48- c-Kit+ Sca-I+ Lineage- (CD48-KSL) cells were sorted from bone marrow of 10 weeks old or 18 months old C57BL/6 SJL mice. A part of young HSCs were then cultured in Stemspan SFEM medium supplemented with 100 ng/ml of murine stem cell factor (mSCF) and 100 ng/ml of human thrombopoietin (hTPO) with or without 100 μM of tauroursodeoxycholic acid (TUDCA) or 100 μM of tauroursodeoxycholic acid (TUDCA) , and 14-days later CD48-KSL fraction was re-sorted. Less...