The adhesion molecule esam1 is a novel hematopoietic stem cell marker - PubMed (original) (raw)

The adhesion molecule esam1 is a novel hematopoietic stem cell marker

A G Lisa Ooi et al. Stem Cells. 2009 Mar.

Abstract

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

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Conflict of interest statement

The other authors have no financial interests to disclose.

Figures

Figure 1

Figure 1. Esam1 is robustly and selectively expressed by HSC

(A) Relative intensity units (left graph) and fold change relative to HSC expression levels (right graph) of Esam1 expression by cDNA microarray analysis of HSC (defined as c-kit+Lin−Sca1+Thy1.1loFlk2− BM cells), ST-HSC (defined as c-kit+Lin−Sca1+Thy1.1loFlk2+ BM cells) and MPP (defined as c-kit+Lin−Sca1+Thy1.1−Flk2+BM cells) [n=6]. (B) Esam1 expression levels in multiple hematopoietic BM populations by RT-qPCR. Data from one representative experiment with triplicate measurements is shown. Error bars indicate the standard deviation. HSC, hematopoietic stem cells; MPP, multipotent progenitors; CMP, common myeloid progenitors; GMP, granulocyte/monocyte progenitor; MEP, megakaryocyte/erythroid progenitor; CLP, common lymphoid progenitor; MacGr, Mac1+/Gr1+ myelomonocytic cells; Ter119, erythroid cells, WBM, unfractionated nucleated bone marrow cells; Brain, unfractionated adult brain cells. (C) Esam1 and β-actin expression levels in human cord blood HSC (CB HSC) (defined as CD34+CD38−Lin−CD90+) as compared to human whole cord blood (WCB) by RT-qPCR, performed in triplicate.

Figure 2

Figure 2. Cell surface Esam1 expression by hematopoietic stem and progenitor cells

(A) Cell surface Esam1 protein expression by HSC, ST-HSC and MPP by flow cytometric analysis using an anti-Esam1 monoclonal antibody. Fluorescence minus one (FMO) without addition of the anti-Esam1 antibody is used as a negative control (grey histogram). (B) Esam1 cell surface expression on HSC compared to putative megakaryocyte progenitor cells (MkPro; c-kit+Lin−Sca1−Slamf1+CD41+ cells) as analyzed by flow cytometric analysis.

Figure 3

Figure 3. Esam1 expression by HSC is age- and strain-independent

(A) Flow cytometric analysis of Esam1 cell surface protein in HSC (defined as c-kit+Lin−Sca1+Flk2−Thy1.1lo cells) from fetal liver (E15.5), young adult bone marrow (8–12 weeks old), and old adult bone marrow (2 years old) of C57BL/6 mice. (B) Correlation between Esam1 expression with Thy1.1 and Flk2 expression within the KLS (c-kit+Lin−Sca1+) population of C57BL/6 mice. Within the KLS population, Esam1+ cells are Thy1.1lo and Flk2−. (C) Esam1 expression on putative stem cells from different mouse strains. A similar proportion of HSC-enriched BM cells (c-kit+Slamf1+Flk2−Lin−) from the C57BL/6, FVB, BALB/c and AKR mouse strains are positive for Esam1 surface expression. Red, Esam1; Blue, FMO (Fluorescence minus one) control.

Figure 4

Figure 4. Esam1 is a viable sort marker for functional HSC

Lin−Sca1+ cells were sorted based on c-kit (blue, “KLS”) and Esam1 (red; “ELS”) expression. CD45.1 mice were transplanted intravenously with 200, 600 and 1800 KLS or ELS cells from congenic CD45.2 mice, together with 2×105 host-type BM cells. The transplanted mice were analyzed for donor-derived mature cells in the peripheral blood every 4 weeks for a period of 16 weeks. Three independent experiments were performed for each cell phenotype and dose, representing at least nine mice per group. (A) ELS (Esam1+Lin−Sca1+) cells give higher total donor contribution as compared to the same numbers of KLS (c-kit+Lin−Sca1+) cells. (*, p<0.03; **, p<0.005; ***, p<0.002; Student’s t test) (B) Donor-derived peripheral cell type composition in mice transplanted with 600 ELS or 600 KLS cells. Over time, ELS cells give rise to a higher proportion of Mac1+ and Mac1+Gr1+ cells and a lower proportion of T cells.

Figure 5

Figure 5. Effects of Esam1 deficiency on hematopoiesis

(A) Esam1-deficient mice have increased frequencies of BM HSC (Lin−c-kit+Sca1+Flk2− cells). The BM cell type composition of wildtype and Esam1-deficient C57BL/6 mice was analyzed by flow cytometry in three independent experiments with three mice in each group and experiment. Open circles indicate HSC frequency in experiment 1; grey circles, experiment 2; black circles, experiment 3; red bars, average HSC frequencies. (B) HSC-enriched BM cells (KLS) from Esam1-deficient mice display increased colony-forming frequency in vitro as compared to wildtype KLS cells. Data from one representative experiment performed in triplicate is shown. MK, megakaryocyte colony; E, erythroid; M, macrophage; G, granulocytic; GM, granulocyte-macrophage; GEMM, granulocyte/erythroid/macrophage/megakaryocyte (mixed). (C) Peripheral blood total donor chimerism in mice transplanted with 100 KLS cells from wildtype (n=10) or Esam1-deficient (n=6) mice. No statistically significant differences in total chimerism were observed. (D) Cell type composition of mice from (C). Over time, Esam1-deficient KLS cells give rise to a disproportionate number of T cells at the expense of B cells (*, p<0.03 at 21 weeks; Student’s t test).

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