C/EBPα is a critical regulator of early myeloid differentiation. Resent study showed that the loss of C/EBPα led to an augmentation of competitive repopulating activity of hematopoietic stem cells (HSCs). Our previous study using the transgenic (Tg) mice expressing an inducible form of C/EBPα (C/EBPα-ER) also showed that in vivo activation of C/EBPα by 4-hydroxy tamoxifen (4-HT) led to remarkable decrease of HSC population (Abstract #671,

). In order to investigate further the role of C/EBPα in HSC homeostasis, we purified CD34 c-Kithi Sca-1hi lineage cells (CD34KSLs) from the C/EBPα-ER Tg mice and examined the effect of C/EBPα activation on their growth, self-renewal, and differentiation potential. Clonal proliferation assay showed that the growth of about 95 % of CD34KSLs was suppressed in the presence of 4-HT. Single cell colony assay showed that the percentage of CFU-GEMM and CFU-MK remarkably decreased from 80 % to 2.1 % and from 2.1 % to 0 %, respectively by 4-HT treatment. In contrast, percentage of CFU-GEM increased from 0 % to 74 %, indicating that differentiation potential to megakaryocytes (MKs) was selectively lost by C/EBPα activation. To gain more insights into the cell fate determination at clonal level, we performed paired daughter cell colony assay. Sorted CD34KSLs were clonally deposited into 96-well plates and allowed to divide once to generate daughter cell pairs, which were then separated by micromanipulation, and transplanted into control or 4-HT containing methylcellulose. 26 daughter cell pairs out of assessable 41 colony pairs lost MK differentiation capacity in the presence of 4-HT. In contrast, remaining 15 pairs took the same fate of differentiation in control and 4-HT. Surprisingly, while most of the CD34KSLs stopped their growth upon C/EBPα activation, approximately 5 % of the cells proliferated even in the presence of 4-HT. These cells grew from a single cell to generate more than 45 cells and sustained multipotentiality to generate GEMM-colonies even after 8-days of culture. To further examine the effect of C/EBPα activation on self-renewal potential of HSCs, clonally sorted CD34KSLs were allowed to divide once in 4-HT- or control-media, and the fate of each daughter cell was traced in methylcellulose. The result showed that GEMM-colony pairs were generated at similar frequency in control and 4-HT culture, indicating that the self-renewal capacity of CD34KSLs was not affected by C/EBPα. These results indicated that activation of C/EBPα caused growth suppression and impaired differentiation to MKs in the majority of CD34KSLs. However, small fraction does not respond to C/EBPα activation, indicating CD34KSLs could be classified into two subpopulations by their sensitivity to C/EBPα.

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