Repopulating Activity of Ex Vivo-Expanded Murine Hematopoietic Stem Cells Resides in the CD48^-c-Kit⁺Sca-1⁺Lineage Marker^- Cell Population

¹ Subteam for Manipulation of Cell Fate, RIKEN BioResource Center, Tsukuba, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
² Subteam for Manipulation of Cell Fate, RIKEN BioResource Center, Tsukuba, Japan
³ Cancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan

	Abstract

A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor (SCF), thrombopoietin (TPO), fibroblast growth factor-1 (FGF-1), and insulin-like growth factor-2 (IGF-2), single CD34^-/lowc-Kit⁺Sca-1⁺lineage marker^- (CD34^-KSL) cells gave rise to varying numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48^-KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48^-KSL cells (cycling HSCs) and CD48⁺KSL cells (cycling non-HSCs) compared with freshly isolated CD34^-KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to be a better understanding of ex vivo-expanded HSCs.