Gene expression data of murine hematopoietic stem cells after in vitro culture and anemic stress

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. It has been unknown whether HSC respond to acute anemic stress. We compared frequency and differentiation potential of HSC in steady-state vs acute anemic mice, and discovered that HSC rapidly expand upon the anemia induction and change their lineage balance to more erythrocytic. CD244- CD48- c-Kit+ Sca-I+ Lineage- (CD244-CD48-KSL) cells were sorted from bone marrow of 10 weeks old C57BL/6JRj mice. A part of the cells were cultured in Stemspan SFEM medium supplemented with 100 ng/ml of murine stem cell factor (mSCF) and 100 ng/ml of human thrombopoietin (hTPO), and 7-days later CD244- and CD244+CD48-KSL fractions were re-sorted. In another setting, 10 weeks old mice were injected with PBS or 60 mg/kg of phenylhydrazine, or phlebotomized, and 24 hours later CD150+CD48-KSL cells were isolated from the bone marrow.