**7. ESAM marks HSC in different developmental stages and in different species**

Hematopoietic cells arise from mesoderm precursors at different sites and stages of development (de Bruijn et al., 2000; Oberlin et al., 2002). We previously determined that, while myelo-erythroid progenitors emerge from the yolk sac, hematopoietic progenitors with lymphopoietic potential first develop in the paraaortic splanchnopleura (pSp) /AGM region (Yokota et al., 2006). ESAM+ cells in the AGM were found to co-express c-kit and endothelial antigens, Tie2, CD34 and CD31/PECAM-1 that are known as a marker set for emerging HSC. However, the earlier hematopoietic progenitors in the yolk sac that have limited life span and little lymphopoietic activity were harbored in the ESAMLow Tie2Low ckitHigh fraction (Figure 2).

or fetal liver of the Rag1/GFP reporter mice reconstituted lympho-hematopoiesis in lethally

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 down-

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-

kitHigh 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

**7. ESAM marks HSC in different developmental stages and in different** 

Hematopoietic cells arise from mesoderm precursors at different sites and stages of development (de Bruijn et al., 2000; Oberlin et al., 2002). We previously determined that, while myelo-erythroid progenitors emerge from the yolk sac, hematopoietic progenitors with lymphopoietic potential first develop in the paraaortic splanchnopleura (pSp) /AGM region (Yokota et al., 2006). ESAM+ cells in the AGM were found to co-express c-kit and endothelial antigens, Tie2, CD34 and CD31/PECAM-1 that are known as a marker set for emerging HSC. However, the earlier hematopoietic progenitors in the yolk sac that have limited life span and little lymphopoietic activity were harbored in the ESAMLow Tie2Low c-

c-kitHigh Sca1+ cells derived from bone marrow

c-kitHigh Sca1+ cells only transiently contributed

c-

Lin-

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

We previously reported that Rag1/GFP-

regulation on differentiation to ELP.

fraction.

**species** 

kitHigh fraction (Figure 2).

irradiated recipients, while Rag1/GFP+ Lin-

(A) Flow cytometry analysis was performed for mouse E14.5 fetal liver cells using anti-c-kit, anti- Sca1, and anti-ESAM Abs. ESAM-/Lo or ESAMHi cells of the Rag1/GFP ckitHi Sca1+ fraction were sorted and subjected to methylcellulose colony formation assay. Numbers of CFUs (B) and morphology of the colonies (C) are shown. (Modified from reference Yokota et al., 2009)

Fig. 1. ESAM expression on the HSC-enriched population of mouse fetal liver

Markers for Hematopoietic Stem Cells: Histories and Recent Achievements 85

ESAM expression was also detected on HSC within the Lin- c-kitHigh Sca1+ fraction in adult bone marrow. Interestingly, while the expression level was slightly decreased in the adolescent period, it was up-regulated again in aged mice. In addition, Ooi et al showed that the ESAM+

2009). Furthermore, by using a rabbit anti-human polyclonal ESAM antibody and flow cytometry, we also detected ESAM expression on human cord blood CD34+ cells (Figure 3). The intensity of ESAM expression, however, was similar between CD34+ CD38- and CD34+ CD38+ cells, suggesting that the ESAM+ gate covers committed as well as non-committed hematopoietic progenitors. ESAM expression might serve as an alternative marker to CD34 for the selection of hematopoietic stem/progenitor cells in human. It is noteworthy that, although majority of human cord blood CD34- CD38+ fraction were negative for the ESAM staining, the fraction contains a small ESAM+ population. Further study is necessary to

CD34+ cells were firstly enriched from cord blood mononuclear cells by magnetic beads conjugated with an anti-human CD34 antibody, and then stained with anti-CD34, anti-CD38, and anti-ESAM antibodies. The left panel shows CD34 and CD38 expression profile of the CD34+ enriched population. The middle and right panels indicate ESAM expression (red tinted) on CD34+ CD38+ or CD34+ CD38 cells, respectively. Dot lines show background staining levels with control IgG for an anti-ESAM

In this chapter, we summarized 2 decades achievements for the identification of HSC and introduced our recent discovery of human ESAM as a new HSC marker. Although it is possible in mouse to purify the long-term multi-lineage HSC with high efficiency, characterization of human HSC has lagged behind partly due to insufficient information about their cell surface antigens. As a new tool, ESAM expression might contribute to improve the purification strategy of human HSC, not only from human hematopoietic

ESAM transcripts in human cord blood CD34+ CD38-

Fig. 3. ESAM expression on human cord blood CD34+ cells

characterize those ESAM+ CD34- cells.

 Sca1+ gating could more effectively enrich adult bone marrow for the long-term reconstituting HSC than the conventional LSK gating, and that ESAM expression in HSC is conserved between different mouse strains (Ooi et al., 2009). Based on these observations, we conclude that ESAM serves as an effective and durable marker for HSC throughout life in mice. The importance of ESAM as a HSC marker has been further enhanced by the findings that its expression in HSC is conserved between mouse and man. Ooi et al detected abundant

Lin-

Thy1/CD90+ cells (Ooi et al.,

Lin-

antibody.

**8. Conclusion** 

Yolk sac or the caudal half of embryo proper were obtained and pooled from E9.5 embryos of wild type C57B6 mice. The obtained cells were stained with the anti-ESAM Ab followed by goat anti-rat IgG-FITC, anti-c-kit-APC, anti-Tie2-PE, and 7AAD. (A) The profiles of Tie2 and c-kit expression are shown in the left panels. In the right panels, ESAM expression in each gate is shown in histograms. The sorted fractions were labeled with "a" to "f". The sorted cells were subjected to methylcellulose colony formation assay (B) and tested in the MS5 co-culture system (C). (Modified from reference Yokota et al., 2009)

Fig. 2. Yolk sac hematopoietic cells differ from those in the embryo proper with respect to ESAM expression and lymphopoietic activity.

ESAM expression was also detected on HSC within the Lin- c-kitHigh Sca1+ fraction in adult bone marrow. Interestingly, while the expression level was slightly decreased in the adolescent period, it was up-regulated again in aged mice. In addition, Ooi et al showed that the ESAM+ Lin- Sca1+ gating could more effectively enrich adult bone marrow for the long-term reconstituting HSC than the conventional LSK gating, and that ESAM expression in HSC is conserved between different mouse strains (Ooi et al., 2009). Based on these observations, we conclude that ESAM serves as an effective and durable marker for HSC throughout life in mice.

The importance of ESAM as a HSC marker has been further enhanced by the findings that its expression in HSC is conserved between mouse and man. Ooi et al detected abundant ESAM transcripts in human cord blood CD34+ CD38- Lin- Thy1/CD90+ cells (Ooi et al., 2009). Furthermore, by using a rabbit anti-human polyclonal ESAM antibody and flow cytometry, we also detected ESAM expression on human cord blood CD34+ cells (Figure 3). The intensity of ESAM expression, however, was similar between CD34+ CD38- and CD34+ CD38+ cells, suggesting that the ESAM+ gate covers committed as well as non-committed hematopoietic progenitors. ESAM expression might serve as an alternative marker to CD34 for the selection of hematopoietic stem/progenitor cells in human. It is noteworthy that, although majority of human cord blood CD34- CD38+ fraction were negative for the ESAM staining, the fraction contains a small ESAM+ population. Further study is necessary to characterize those ESAM+ CD34- cells.

CD34+ cells were firstly enriched from cord blood mononuclear cells by magnetic beads conjugated with an anti-human CD34 antibody, and then stained with anti-CD34, anti-CD38, and anti-ESAM antibodies. The left panel shows CD34 and CD38 expression profile of the CD34+ enriched population. The middle and right panels indicate ESAM expression (red tinted) on CD34+ CD38+ or CD34+ CD38 cells, respectively. Dot lines show background staining levels with control IgG for an anti-ESAM antibody.

Fig. 3. ESAM expression on human cord blood CD34+ cells

#### **8. Conclusion**

84 Advances in Hematopoietic Stem Cell Research

Yolk sac or the caudal half of embryo proper were obtained and pooled from E9.5 embryos of wild type C57B6 mice. The obtained cells were stained with the anti-ESAM Ab followed by goat anti-rat IgG-FITC, anti-c-kit-APC, anti-Tie2-PE, and 7AAD. (A) The profiles of Tie2 and c-kit expression are shown in the left panels. In the right panels, ESAM expression in each gate is shown in histograms. The sorted fractions were labeled with "a" to "f". The sorted cells were subjected to methylcellulose colony formation assay (B) and tested in the MS5 co-culture system (C). (Modified from reference Yokota et al.,

Fig. 2. Yolk sac hematopoietic cells differ from those in the embryo proper with respect to

2009)

ESAM expression and lymphopoietic activity.

In this chapter, we summarized 2 decades achievements for the identification of HSC and introduced our recent discovery of human ESAM as a new HSC marker. Although it is possible in mouse to purify the long-term multi-lineage HSC with high efficiency, characterization of human HSC has lagged behind partly due to insufficient information about their cell surface antigens. As a new tool, ESAM expression might contribute to improve the purification strategy of human HSC, not only from human hematopoietic

Markers for Hematopoietic Stem Cells: Histories and Recent Achievements 87

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#### **9. References**


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**Part 2** 

**Regulation of Hematopoietic Stem Cells** 

