**6. Identification of ESAM as a novel HSC marker**

We previously reported that Rag1/GFP- Lin c-kitHigh Sca1+ cells derived from bone marrow or fetal liver of the Rag1/GFP reporter mice reconstituted lympho-hematopoiesis in lethally irradiated recipients, while Rag1/GFP+ Lin c-kitHigh Sca1+ cells only transiently contributed to T and B lymphopoiesis (Igarashi et al., 2002; Yokota et al., 2003). Those data demonstrated that Rag1 expression is useful to distinguish early lymphoid progenitors (ELP) from the long-term HSC. To learn more about the first step of HSC differentiation to the lymphoid lineage, microarray analyses were conducted to search for genes that characterize the initial transition of HSC to ELP. The search brought us a large body of information about genes potentially related to early lymphopoiesis whereas it also identified genes whose expression seemed to correlate with HSC. Among the HSC-related genes, ESAM strongly drew our attention because of its conspicuous expression in the HSC fraction and sharp downregulation on differentiation to ELP.

ESAM was originally identified as an endothelial cell-specific protein (Hirata et al., 2001; Nasdala et al., 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1- c-kitHigh Sca1+ fraction of E14.5 fetal liver could be subdivided into two on the basis of ESAM level (Figure 1A). The subpopulation with the high density of ESAM was enriched for c-kitHigh Sca1High cells, while ones with negative or low levels of ESAM were found in the c-kitHigh Sca1Low subset. In addition, ESAM expression well correlated with hematopoietic stem/progenitor activity (Figure 1B). Cells in the ESAMHigh Rag1 ckitHigh Sca1+ fraction formed more and larger colonies than those in the ESAM-/Low Rag1- ckitHigh Sca1+ fraction. Particularly, majority of CFU-Mix, multi-potent primitive progenitors, were found in the ESAMHigh fraction (Figure 1B and 1C). In limiting dilution stromal cell cocultures, we found that 1 in 2.1 ESAMHigh Rag1- c-kitHigh Sca1+ cells and 1 in 3.5 ESAM-/Low Rag1- c-kitHigh Sca1+ cells gave rise to blood cells. However, 1 in 8 ESAMHigh Rag1- c-kitHigh Sca1+ cells produced CD19+ B lineage cells whereas only 1 in 125 ESAM-/Low Rag1- c-kitHigh Sca1+ cells were lymphopoietic under these conditions. Furthermore, in long-term reconstituting assays, ESAMHigh Rag1- c-kitHigh Sca1+ cells contributed highly to the multilineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM-/Low Rag1- c-kitHigh Sca1+ cells. These results suggested that the long-term multi-lineage HSC in E14.5 fetal liver are exclusively present in the ESAMHigh fraction.
