ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias

Accepted Manuscript ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias Tomohiko Ishibashi, Takafumi Yokota, Hirokazu Tanaka, Michiko Ichii, Takao Sudo, Yusuke Satoh, Yukiko Doi, Tomoaki Ueda, Akira Tanimura, Yuri Hamanaka, Sachiko Ezoe, Hirohiko Shibayama, Kenji Oritani, Yuzuru Kanakura PII:

S0301-472X(16)00005-9

DOI:

10.1016/j.exphem.2015.12.010

Reference:

EXPHEM 3351

To appear in:

Experimental Hematology

Received Date: 14 January 2015 Revised Date:

24 December 2015

Accepted Date: 28 December 2015

Please cite this article as: Ishibashi T, Yokota T, Tanaka H, Ichii M, Sudo T, Satoh Y, Doi Y, Ueda T, Tanimura A, Hamanaka Y, Ezoe S, Shibayama H, Oritani K, Kanakura Y, ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias, Experimental Hematology (2016), doi: 10.1016/j.exphem.2015.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

ESAM is a novel human hematopoietic stem cell marker associated with a subset of

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human leukemias

3 Authors

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Tomohiko Ishibashi1, Takafumi Yokota1, Hirokazu Tanaka2, Michiko Ichii1,

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Takao Sudo1, Yusuke Satoh1,3, Yukiko Doi1, Tomoaki Ueda1, Akira Tanimura1,

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Yuri Hamanaka1, Sachiko Ezoe1, Hirohiko Shibayama1, Kenji Oritani1, Yuzuru Kanakura1

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8 Affiliations

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Suita, Osaka 565-0871, Japan

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Osakasayama, Osaka 589-8511, Japan

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Department of Hematology and Oncology, Osaka University Graduate School of Medicine,

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Department of Hematology and Rheumatology, Kinki University Faculty of Medicine,

Department of Lifestyle Studies, Kobe Shoin Women’s University, Kobe 657-0015, Japan

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Takafumi Yokota, Department of Hematology and Oncology, Osaka University Graduate

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School of Medicine, C9, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; E-mail:

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[email protected]

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Category for the Table of Contents

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Stem Cells (hematopoietic, mesenchymal, embryonic and induced pluripotent stem cells)

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Word Count: 3,530 words

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Conflict of interest

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The authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT Abstract

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Reliable markers are essential to increase our understanding of the biological features of

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human hematopoietic stem cells and to facilitate the application of hematopoietic stem cells

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in the field of transplantation and regenerative medicine. We previously identified endothelial

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cell-selective adhesion molecule (ESAM) as a novel functional marker of hematopoietic stem

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cells in mice. Here, we found that ESAM can also be used to purify human hematopoietic

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stem cells from all the currently available sources (adult bone marrow, mobilized peripheral

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blood, and cord blood). Multipotent colony-forming units and long-term

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hematopoietic-reconstituting cells in immunodeficient mice were exclusively found in the

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ESAMHigh fraction of CD34+ CD38– cells. The CD34+ CD38– fraction of cord blood and

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collagenase-treated bone marrow contained cells showing extremely high expression of

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ESAM; these cells are likely to be related to the endothelial lineage. Leukemia cell lines of

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erythroid and megakaryocyte origin, but not those of myeloid or lymphoid descent, were

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ESAM-positive. However, high ESAM expression was observed in some primary acute

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myeloid leukemia cells. Furthermore, KG-1a myeloid leukemia cells switched from

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ESAM-negative to ESAM-positive with repeated leukemia reconstitution in vivo. Thus,

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ESAM is a useful marker for studying both human hematopoietic stem cells and leukemia

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cells.

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Introduction

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Lifelong hematopoiesis is maintained by hematopoietic stem cells (HSCs), which have self-renewal capacities and can differentiate into all types of blood cells. HSCs are

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essential for transplantation and regenerative therapies designed to cure patients with

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hematological diseases. The successful outcome of therapy requires a reliable method for

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identifying authentic HSCs. Therefore, it is important to identify cell surface markers that

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distinguish HSCs from other cells.

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Since a previous study showed that a set of cell surface proteins can be used to enrich multipotent hematopoietic progenitors in the mouse bone marrow (BM) [1], numerous

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studies have examined murine HSC markers (reviewed by Yokota et al. [2]). Murine HSCs

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can be efficiently isolated from BM as Lin– Sca-1+ c-kit+ (LSK) CD90/Thy1Low CD34–/Low

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CD150+ CD48– cells. Indeed, ~50% of cells with the LSK CD150+ CD48– phenotype exhibit

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the potential for long-term multi-lineage reconstitution [3]. In humans, however, the same

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method cannot be applied because of critical differences between the murine and human HSC

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phenotypes. Human HSCs express neither CD150 nor Sca-1 [4, 5]. In addition, human HSCs

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are enriched in the CD34+ CD38– fraction [6, 7], whereas many studies have located murine

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HSCs in the CD34– CD38+ fraction [8-10]. These discrepancies have limited the translation

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of the findings from mice to humans. Therefore, the identification of common HSC antigens

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between the two species would be a significant advancement in translational studies of HSC

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biology.

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We previously identified endothelial cell-selective adhesion molecule (ESAM) as a

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novel marker for murine HSCs [11]. The functional importance of ESAM is underscored by

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the finding that ESAM-deficient mice suffer from severe and prolonged BM suppression after

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treatment with the cytotoxic agent 5-fluorouracil (5-FU) [12]. The detection of ESAM

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ACCEPTED MANUSCRIPT transcripts in human cord blood (CB) CD34+ CD38– Lin– CD90/Thy1+ cells has increased

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interest in ESAM as an HSC marker [13]. However, it is unclear whether its expression

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pattern changes in human hematopoietic stem/progenitor cells with the differentiation status,

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or in a manner that depends on the tissue source. In addition, the expression of ESAM in a

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broad panel of human leukemia cells has not been evaluated.

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In this study, we examined whether ESAM expression is a potential marker of human HSCs from diverse sources. Interestingly, in addition to adult BM (ABM), most

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CD34+ CD38– cells in granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral

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blood (GMPB) expressed ESAM. We also identified a previously uncharacterized subset of

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CB CD34+ CD38– cells that expressed extremely high levels of ESAM. Furthermore, we

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observed oscillations in ESAM expression on leukemia cells depending on the surrounding

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environment.

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Materials and methods

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Human samples

BM and GMPB were obtained from related HSC transplantation donors. CB was

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obtained from healthy full-term newborns. BM was also obtained from patients of acute

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leukemia, and the femoral head of patients who underwent hip replacement surgery. The

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Institutional Review Board at the Osaka University Hospital approved all protocols, and

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written informed consent was obtained from all the participants. To prepare BM cells

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adjacent to bone tissues, trabecular tissues of the femora were treated with 2 mg/mL

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collagenase IV and DNase and gently agitated for 1 h at 37°C.

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Additional methods are described in detail in the Supplementary methods.

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Results

99 High ESAM expression indicates primitive hematopoietic progenitors in human CD34+

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CD38– cells

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Our previous study demonstrated that sorting for high ESAM expression enriches

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the proportion of authentic HSCs in the LSK fraction of murine fetal liver and ABM. In this

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study, we examined whether this was also true for human cells. Using flow cytometry, we

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analyzed ESAM expression on BM mononuclear cells (MNCs). A large number of human

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BM CD34+ cells were found to express ESAM, and most CD34+ CD38– cells, which

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included the human HSC-enriched fraction, expressed ESAM (Figure 1A). We found that in

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both ABM and GMPB, more than 80% of CD34+ CD38– cells expressed high levels of

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ESAM (Figure 1B, C). The addition of anti-CD90 and CD45RA antibodies can reportedly

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divide the CD34+ CD38– fraction into three subpopulations, including CD90+ CD45RA– HSC,

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CD90– CD45RA– multipotent progenitors, and CD90– CD45RA+ multi-lymphoid progenitors

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(MLP) [14] (Figure 1D, left). We found that most HSCs expressed high levels of ESAM,

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whereas many MLPs lost ESAM expression (Figure 1D, middle). Furthermore, estimation of

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the mean fluorescence intensity indicated that ESAM expression markedly declined as the

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cells progressively differentiated from HSCs into MLPs (Figure 1D, right).

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Next, we compared the differentiation and growth potential of ESAM–/Low and

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ESAMHigh cells. We purified CD34+ CD38– ESAM–/Low and CD34+ CD38– ESAMHigh cells

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from ABM, and subjected the cells to a methylcellulose assay or grew the cells in MS5

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stromal cell co-culture. While both cell types produced hematopoietic colonies with high

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frequency, only ESAMHigh cells included lineage-mixed colony-forming units (CFU-Mix)

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ACCEPTED MANUSCRIPT with both myeloid and erythroid potential (Figure 2A). Under the MS5 co-culture conditions,

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the ESAM–/Low cells generated CD45+ hematopoietic cells more effectively than the

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ESAMHigh cells during the first week (Figure 2B, left); however, the output of CD45+ cells

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did not increase to the same extent as that from ESAMHigh cells during the second week and

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thereafter (Figure 2B, right). Indeed, hematopoietic cells rapidly expanded from the

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ESAMHigh fraction after the second week, and this was maintained throughout the culture

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period (Figure 2B). In addition, the ESAMHigh fraction gave rise to a substantial number of

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CD19+ B-lineage cells (Figure 2C). Similar results were obtained when GMPB-derived cells

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were used (data not shown). These observations indicate that while both the ESAM–/Low and

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ESAMHigh cells in CD34+ CD38– included hematopoietic progenitors, higher ESAM

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expression distinguished the more primitive and multipotent cells.

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High ESAM expression reveals a new subpopulation of human CB CD34+ CD38– cells

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We also analyzed ESAM expression on human CB MNCs. Similarly to ABM and

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GMPB, most cells in the CB CD34+ CD38– fraction expressed ESAM. However, we detected

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a new sub-fraction of cells showing very high ESAM expression, referred to as ESAMBright,

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which was not observed in the ABM and GMPB CD34+ CD38– fractions. The ESAMBright

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fraction showed approximately 10-fold higher expression of ESAM compared to the

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ESAMHigh fraction. According to the ESAM expression level, we subdivided the CB CD34+

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CD38– fraction into three populations, including ESAM–/Low, ESAMHigh, and ESAMBright

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(Figure 3A). While the percentage of ESAM–/Low cells among the total CD34+ CD38– cells

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was consistently low in the tested CB samples, the frequencies of ESAMHigh and ESAMBright

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cells varied widely among samples (Figure 3B).

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ESAMHigh, but not ESAMBright, indicate authentic HSCs in CB Next, we performed functional assessment of the subpopulations based on ESAM expression levels. In some cases, ESAM–/Low cells were omitted from the assessment because

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their frequency in the CB CD34+ CD38– fraction was too low for accurate evaluation. We

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predicted that ESAMBright cells would be enriched for more primitive progenitors; however,

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the opposite was observed. In methylcellulose assays, the ESAMHigh fraction produced

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significantly more CFUs than the ESAMBright fraction (Figure 4A). Furthermore, only the

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ESAMHigh fraction was capable of colony formation in the CFU-mix, which contained

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primitive multipotent progenitors.

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To determine the frequency of primitive progenitors with both lymphoid and myeloid potential, limiting dilution analyses were performed in the MS5 co-culture system.

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While 1 in 12 ESAMHigh cells produced both CD19+ lymphoid lineage and CD33+ myeloid

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lineage cells, only 1 in 63 ESAMBright cells produced both lineage cells (Figure 4B). These

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results suggest that primitive myelo-lymphoid progenitors are enriched in the ESAMHigh

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fraction, but not in the ESAMBright population, of human CB CD34+ CD38– cells. To evaluate the long-term reconstitution activity, we first transplanted ESAMHigh or

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ESAMBright cells from the CB CD34+ CD38– fraction into 2.2 Gy-irradiated NOD/Shi-scid,

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IL-2γ-null (NOG) mice. While all five recipients of the ESAMBright cells died within 1 month

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after transplantation, all five recipients of the ESAMHigh cells survived. Surviving

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ESAMHigh-transplanted mice were sacrificed 7 months after transplantation, and BM was

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evaluated. We found that the ESAMHigh cells effectively reconstituted human hematopoiesis

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in all recipients (data not shown). Next, we performed serial transplantation assays to

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compare ESAM–/Low, ESAMHigh, and ESAMBright cells purified from the CB CD34+ CD38–

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fraction. The number of transplanted cells in each group was determined according to their

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ESAMBright-transplanted mice died before assessment in the first experiment, we reduced the

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irradiation dose to 1.8 Gy. All recipients survived and were analyzed 3 months after

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transplantation. Human hematopoiesis was only observed in mice transplanted with

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ESAMHigh cells (Figure 4C). Furthermore, transplanted cells contributed substantially to the

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myeloid, B-lymphoid, and T-lymphoid lineages (Figure 4D). In the secondary transplantation,

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whole BM MNCs from the recipients of ESAMHigh cells were transplanted into sub-lethally

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irradiated NOG mice. All mice were sacrificed 4 months after transplantation, and chimerism

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of human cells in the BM was evaluated. Long-term human hematopoiesis was again

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detected in the three lineages (Figure 4E and 4F). Thus, the ESAMHigh fraction, but not the

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ESAM–/Low and ESAMBright fractions, contains long-term reconstituting human HSCs.

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CD34+ CD38– ESAMBright fraction consists of non-hematopoietic lineage cells

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The unexpected results of our functional evaluation of CB CD34+ CD38– ESAMBright cells prompted us to characterize these cells more precisely. Morphologically,

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ESAMBright cells were larger in size and had less basophilic and more abundant cytoplasm

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compared with ESAM–/Low and ESAMHigh cells (Figure 5A). While ESAM–/Low and

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ESAMHigh cells were of uniform size and morphology, ESAMBright cells were polymorphic,

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suggesting that they are heterogeneous.

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With respect to the cell surface phenotype, ESAMBright cells did not express CD45

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(Figure 5B, left). In addition, while ESAMHigh cells expressed CD133, another marker of

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human HSCs [15, 16], this marker was completely absent in ESAMBright cells (Figure 5B,

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right). Back plotting of the ESAMHigh and ESAMBright cells onto the CD34 and CD38 profile

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showed that CD34 expression levels were much higher on ESAMBright cells (Figure 5C).

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ACCEPTED MANUSCRIPT Furthermore, these cells expressed vascular endothelial (VE)-cadherin and vascular

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endothelial growth factor receptor-2 (VEGFR-2), which are endothelial lineage-related

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molecules (Figure 5D). ESAMBright cells also expressed CD146/S-endo, and a small subset

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was positive for the expression of CD118/leukemia inhibitory factor receptor (data not

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shown). These results suggest that the main population of CB CD34+ CD38– ESAMBright cells

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was not hematopoietic but rather comprised of endothelial lineage and other mesodermal

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cells.

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Based on the above findings, we assumed that a similar ESAMBright cell population existed in BM. Since aspirated BM cells did not contain ESAMBright cells (Figure 1B), we

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treated bone samples with collagenase to collect mesenchymal cells that are adherent to bone

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tissues. This treatment yielded a substantial CD34+ CD38– ESAMBright population from ABM

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(Figure 5E), the phenotype of which was similar to its CB counterpart. We then performed a

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microarray experiment to compare the gene expression profiles of the CD34+ CD38–

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ESAMHigh and CD34+ CD38– ESAMBright populations. The former cells expressed

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HSC-related genes, whereas the latter showed more endothelial-related profiles [17] (Figure

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5F; the data have been deposited in Gene Expression Omnibus under accession number

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GSE63877). Indeed, CD34+ CD38– ESAMBright cells from collagenase-treated bone produced

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CD31+ cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells in the

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presence of VEGF, stromal cell-derived factor, and interleukin (IL)-16 (Figure 5G). These

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observations indicate that the CD34+ CD38– fraction, which is conventionally considered the

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human HSC fraction, may also contain a substantial number of non-hematopoietic cells. Thus,

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the inclusion of ESAM or other markers appears to provide a more accurate estimation of

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HSC numbers.

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Some types of human leukemia cells express ESAM Cell surface antigens on normal hematopoietic stem/progenitor cells are often observed on leukemia cells. Since some of these antigens are useful for determining leukemia

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lineage and have utility as prognostic indicators [18, 19], we examined whether ESAM would

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be a valuable addition to this antigen panel. First, we examined human leukemia cell lines

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derived from patients with acute myeloid leukemia (AML); all of these cell lines were

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uniformly negative for ESAM expression (Figure 6A). KG-1a cells showed a contrasting

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profile for CD34 and ESAM. Lymphoid lineage cell lines were also negative for ESAM. In

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contrast, the erythroid leukemia cell line HEL and the megakaryocytic leukemia cell line

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CMK showed high ESAM expression. Additionally, K562 cells that originated from a

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chronic myeloid leukemia that subsequently transformed into acute erythroleukemia also

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expressed low levels of ESAM. These results indicate that ESAM expression segregates

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leukemia cells with erythroid or megakaryocyte lineage.

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We then evaluated ESAM expression on primary leukemia cells isolated from patients with AML (n = 15) and acute lymphoblastic leukemia (ALL, n = 3) at the time of

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diagnosis. Interestingly, while ALL cases were nearly negative for ESAM, 10 of 15 AML

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cases were clearly ESAM-positive (Figure 6B). Notably, the ESAM expression pattern was

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completely different in the same AML category (Figure 6B). Thus, AML cells may change

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their ESAM expression levels according to the cell-intrinsic features and/or the surrounding

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environment.

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KG-1a leukemic cells express ESAM concomitant with the acquisition of a more

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aggressive phenotype

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The discrepancy between AML cell lines and primary AML cells with respect to

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ACCEPTED MANUSCRIPT ESAM expression indicates that this antigen is differentially regulated in vivo. Therefore, we

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inoculated ESAM– KG-1a cell lines into NOD/SCID mice and harvested reconstituted KG-1a

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(rKG-1a) cells 10 weeks after inoculation. The cells were then cultured in vitro for 3 weeks

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and inoculated again into NOD/SCID mice (Figure 7A). These rKG-1a cells also showed

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high leukemic activity (Figure 7B). Flow cytometry analyses revealed that although parental

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KG-1a cells were ESAM-negative, rKG-1a cells showed high ESAM expression (Figure 7C).

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To investigate the significance of ESAM up-regulation in rKG-1a cells, ESAM– and ESAM+

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rKG-1a cells were purified and inoculated into sub-lethally irradiated NOD/SCID mice.

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Three months after transplantation, all mice were sacrificed, and the BM cells were analyzed.

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Flow cytometry analyses revealed that ESAM– rKG-1a cells did not engraft, whereas ESAM+

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rKG-1a cells engrafted (Figure 7D and 7E). These results indicate that leukemia cells

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fluctuate in their surface phenotype according to surrounding environment and that ESAM

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expression is related to the affinity of leukemia cells to BM stromal cells.

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Discussion Precisely defining the immunophenotype of HSCs is critical for the successful outcome of transplantation therapy for hematological malignancies. In addition, recent

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progress in regenerative therapy using induced pluripotent stem cells demands that reliable

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cell surface antigens are available to distinguish authentic HSCs from lineage-specific

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progenitors [20, 21]. However, it is more difficult to characterize human HSCs and their

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proximate progenitors compared with murine HSCs, in part because of the limited

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information available regarding human HSC surface antigens [2, 22]. The developmental

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process of HSCs with respect to ontogeny and transcription factors is thought to be similar

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between mice and humans. Therefore, identifying common HSC antigens shared by the two

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species will greatly facilitate basic and clinical studies of HSC biology. In this study, we

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found that ESAM is a robust and cross-species HSC marker. Furthermore, the molecule is

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functional on HSCs and is associated with specific subsets of leukemia cells.

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Using strict phenotypic definitions of human HSCs and their proximate progenitors

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in the CD34+ CD38– fraction [14], we found that the CD90+ CD45RA– HSCs expressed high

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levels of ESAM, and that this expression decreased during their differentiation into lymphoid

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progenitors. In our culture and transplantation experiments, we also showed that high ESAM

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expression segregates with multi-lineage potential and SCID-repopulating HSCs in humans;

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these data are consistent with our previous findings in mice [11]. In addition, and similar to

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the case for murine HSCs, ESAM was evidently a robust marker of human HSCs regardless

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of changes to their functional properties and expression of other cell surface antigens. We

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also assessed the long-term reconstitution capacity of ESAMHigh CD34+ CD38– cells from

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various tissues (BM, GMPB, and CB) simultaneously and observed comparable results (data

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not shown).

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ACCEPTED MANUSCRIPT Our previous study highlighted the functional importance of ESAM in the

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proliferation and differentiation of HSCs. ESAM expression is markedly increased on murine

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HSCs following cytotoxic insult [12]. Additionally, ESAM-deficient mice are less tolerant to

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5-FU treatment, and the recovery of their HSC fraction was significantly delayed compared

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with wild-type mice [12]. Furthermore, we found that ESAM is vital for the early stages of

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erythrocyte recovery after BM injury (T. Sudo, manuscript in preparation). To date, few

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functional markers that are commonly expressed on both human and murine HSC have been

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reported. Thus, ESAM is important not only as a marker but also for determining the identity

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of HSCs across species.

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Recently CD166, a member of the immunoglobulin superfamily, was reported as a universal marker for murine and human HSCs [23]. This molecule was also expressed on

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endosteal niche components and immature osteoblasts, which exhibit high HSC-supporting

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activity [24, 25]. Reciprocal transplantation of CD166-deficient HSCs into wild-type mice

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and wild-type HSCs into CD166-deficient mice indicated that hemophilic CD166-CD166

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interaction likely mediates the HSC-niche association and affects HSC maintenance. Given

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that ESAM is also a member of the immunoglobulin superfamily with hemophilic interacting

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features, it may also be involved in such HSC-niche interactions. However, because ESAM is

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an endothelial lineage-related antigen, this molecule may mediate the interaction between

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HSCs and endothelial cells. Indeed, irradiation or 5-FU treatment up-regulates ESAM

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expression on murine HSCs, resulting in an intimate association between ESAMHigh HSCs

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and the vasculature of BM [12]. In this context, both irradiation and 5-FU decreased CD166

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expression on HSCs [23]. Therefore, we propose that these two markers are important for

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HSC maintenance but they do not necessarily have overlapping functions.

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Although it is widely accepted that human HSCs reside in the CD34+ CD38–

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ACCEPTED MANUSCRIPT fraction, recent studies reported that a CD34+ CD38Low fraction also contains human

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hematopoietic repopulating cells [26, 27]. Our flow cytometry analysis revealed high ESAM

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expression on CD34+ CD38Low cells (Figure 1A, right). Thus, high ESAM expression may

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enrich HSCs regardless of CD38 expression. Furthermore, long-term reconstitution activity

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has also been observed in the CD34– population [28-31]. Previous studies by Sonoda et al.

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have verified that cells negative for 18 lineage-related antigens and positive for CD133

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effectively enrich SCID-repopulating HSC activity in human CB CD34– populations [32-34].

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Since ESAM expression covers a broader range of CB cells that are negative for

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lineage-related antigens compared to CD34 expression (T. Yokota, unpublished observation),

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ESAM as well as CD133 may be useful as positive markers for the CD34– HSCs. Future

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studies will provide a more accurate marker set for estimating HSC numbers in human CB.

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Human CB contains various types of progenitor cells that are useful for tissue regenerative therapy [35]. For example, the CB CD34+ fraction can contribute to vasculature

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development in zebrafish [36]. Furthermore, the administration of human CB CD34+ cells

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ameliorates neurological damage after brain injury in rodents, presumably by stimulating

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angiogenesis [37]. Thus, CB CD34+ cells likely contain a substantial amount of angiopoietic

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progenitors. In the present study, we identified a new subpopulation of CB CD34+ CD38–

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cells that express high levels of ESAM; these cells are apparently related to the endothelial

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lineage. The ‘ESAMBright’ phenotype may be useful for enriching angiopoietic progenitors.

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However, thorough examination of their differentiation potential in vitro and in vivo is

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required to evaluate this possibility.

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Since high ESAM expression was related to the totipotent nature and robust growth

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potential of normal HSCs, we expected that ESAM would be expressed in many leukemia

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cell lines. However, all tested myeloid and lymphoid cell lines were ESAM-negative. In

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contrast, erythroid and megakaryocyte cell lines exhibited robust ESAM expression. Given

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that ESAM is indispensable for normal erythropoiesis and thrombocyte function [12, 38], the

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molecule may be intrinsically involved in the erythrocyte and megakaryocyte lineages. We observed that the human AML cell line KG-1a, which is normally ESAM–,

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became ESAM+ concomitant with engraftment in immunodeficient mice. Although we

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cannot exclude the possibility that serial xenogeneic transplantation induced some

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transformation in KG-1a cells, there are other biological explanations for this phenotypic

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change. For example, ESAM may be expressed in a rare subpopulation of KG-1a cells that

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comprise the leukemic stem cell (LSC) compartment. In this context, a recent approach for

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identifying LSC markers revealed ESAM in the plasma membrane proteome of

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LSC-enriched fractions obtained from myeloid leukemia patients with poor prognosis [39].

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Furthermore, such rare ESAM+ KG-1a cells may acquire higher proliferative activity in vivo

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by taking advantage of the interaction with the leukemia-supporting microenvironment in the

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BM [40]. Indeed, ESAM+ rKG-1a cells showed higher engraftment capacity compared with

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ESAM– rKG-1a cells. In this case, targeting the leukemia-microenvironment interaction via

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ESAM may be a therapeutic strategy, as ESAM may confer a more aggressive clinical course

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in some types of leukemia. As shown in Figure 6, the pattern of ESAM expression varied

344

significantly among primary AML cases. Although we observed no correlation between

345

ESAM expression and clinical features at this stage, further studies and longer observation

346

periods may reveal the true clinical value of ESAM in the diagnosis and treatment of acute

347

leukemia.

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In summary, we demonstrated that ESAM is a robust functional marker of HSCs in

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humans as well as in mice. Additionally, ESAM is expressed on some types of human

350

leukemia cells and may be useful for lineage determination and as a prognostic indicator. The

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cross-species properties of ESAM expression will accelerate the translation of findings in

352

animal models to studies in humans; this will ultimately increase our understanding of the

353

identity and biology of human HSCs and leukemia cells.

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354 Acknowledgments

356

We thank Dr. Satoko Fujita (Obstetrics and Gynecology, Osaka University) and Dr. Takashi

357

Sakai (Orthopedic Surgery, Osaka University) for providing human samples. This work was

358

supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers

359

24591423 and 26461446).

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360 Author contributions

362

T.I. and T.Y. designed and performed the research, analyzed the data, and wrote the

363

manuscript; Y.K. designed the research, analyzed the data, and wrote the manuscript; H.T.

364

established rKG-1a cells; M.I., T.S., and Y.S. designed and performed the microarray

365

analyses, and analyzed the data. Y.D., T.U., A.T., Y.H., S.E., H.S., and K.O. designed the

366

research and analyzed the data.

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Conflicts of interest disclosure

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The authors declare no conflict of interest.

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Supplementary methods

373

Mononuclear cell (MNC) preparation

374

Cord blood was diluted 1:1 with Dulbecco’s phosphate-buffered saline without

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ACCEPTED MANUSCRIPT calcium and magnesium (PBS(–); Nacalai Tesque, Kyoto, Japan) and was overlaid onto an

376

equivalent volume of Ficoll-Paque PLUS (GE Healthcare Life Sciences, Little Chalfont, UK).

377

Cells were centrifuged for 25 min at 20°C at 400 ×g. MNCs were isolated and washed twice

378

with RPMI 1640 medium (Nacalai Tesque).

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Flow cytometry analysis and cell sorting

Allophycocyanin (APC)-conjugated anti-CD34 (HPCA-2), fluorescein

382

isothiocyanate (FITC)-conjugated anti-CD38 (HIT2) and CD45 (HI30), and Phycoerythrin

383

(PE)-conjugated anti-CD4 (SK3), CD8 (SK1), CD13 (WM15), and CD33 (P67-6)

384

monoclonal antibodies (mAbs) were purchased from BD Biosciences (San Diego, CA, USA).

385

PE-conjugated anti-CD45 (HI30), CD90/Thy1 (5E10), CD133 (AC133), Flk1/VEGFR-2

386

(7D4-6), VE-cadherin (BV9), and CD19 (HIB19) were purchased from BioLegend (San

387

Diego, CA, USA). ESAM mAb (Clone 408519) was purchased from R&D Systems

388

(Minneapolis, MN, USA) and biotinylated using EZ-Link Sulfo-NHS-Biotin and

389

biotinylation kits (Thermo Scientific, Waltham, MA, USA). Streptavidin-conjugated with

390

PE-Texas Red (Life Technologies Corp., Carlsbad, CA, USA), PE, and APC (BD

391

Biosciences) were used for visualization of biotinylated antibodies. Dead cells were excluded

392

by staining with 7-amino actinomycin D (7-AAD; Calbiochem, San Diego, CA, USA). Cells

393

were washed and then resuspended in PBS(–) containing 3% fetal bovine serum (MP

394

Biomedicals, Irvine, CA, USA). Cells were analyzed using FACSAria or FACSCanto II (BD

395

Biosciences, Franklin Lakes, NJ, USA). In some experiments, CD34+ CD38– ESAM–/Low,

396

CD34+ CD38– ESAMHigh, and CD34+ CD38– ESAMBright cells were sorted using a FACSAria

397

and used in subsequent experiments. The FACS data was analyzed using FlowJo software

398

(Tree Star, San Carlos, CA, USA).

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Colony-forming cell assays

401

For myeloid colony formation, cells were cultured for 14 days in MethoCult H4434 (StemCell Technologies, Vancouver, Canada). Subsequently, colonies were enumerated and

403

classified as colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming

404

unit-erythroid (BFU-E), or CFU-Mix according to their shape and color, as observed under

405

an inverted microscope. All cultures were incubated at 37°C in a humidified chamber with

406

5% CO2.

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408 409

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407 Limiting dilution assays

Cord blood CD34+ CD38– ESAMHigh or CD34+ CD38– ESAMBright cells were plated at various concentrations from 1 to 100 cells/well. Each well contained 200 µL MSC growth

411

medium with 10 ng/mL stem cell factor (SCF) and 5 ng/mL Flt-3 ligand. Half of the culture

412

medium was replaced with fresh medium containing the same cytokines twice per week.

413

After 28 days of culture, wells containing cells that had expanded were scored.

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SCID-repopulation assays

NOD/Shi-scid, IL-2γnull (NOG) mice were purchased from the Central Institute for

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Experimental Animals (Kawasaki, Kanagawa, Japan). Sorted CB cells (CD34+ CD38–

418

ESAM–/Low, ESAMHigh, or ESAMBright) were injected via the tail vein after whole-body

419

irradiation at a dose of 1.8 Gy. After 3 months, BM cells were obtained and analyzed by flow

420

cytometry. Whole BM MNCs (2 × 106 cells/mouse) from primary recipients were injected

421

into secondary recipients via the tail vein after whole-body irradiation. After 4 months, BM

422

cells were obtained and analyzed by flow cytometry. Animal studies were performed with the

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ACCEPTED MANUSCRIPT 423

approval of the Institutional Review Board of Osaka University.

424

426

Microarray Total RNAs were extracted from CD34+ CD38– ESAMHigh or ESAMBright cells of

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collagenase-treated BM using an RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA

428

samples were subjected to gene expression analysis using a SurePrint G3 Human GE 8 × 60K

429

v2 Microarray (Agilent Technologies, Santa Clara, CA, USA).

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430

432

KG-1a mouse leukemia model

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KG-1a cells derived from a human acute myeloid leukemia (AML) sample were transplanted into the tail vein of 2 Gy-irradiated NOD/SCID mice. Ten weeks after

434

transplantation, reconstituted KG-1a (rKG-1a) cells were obtained from the BM of recipients.

435

The rKG-1a cells were cultured in vitro for 3 weeks, and then transplanted into the tail vein

436

of 2 Gy-irradiated NOD/SCID mice.

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risk classification in AML independent of established biomarkers: ECOG phase 3 trial,

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pluripotent stem cells. Int J Hematol. 2012;95:617-623. doi:10.1007/s12185-012-1094-x

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advances and challenges toward de novo generation of hematopoietic stem cells. Blood.

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osteoblasts reveals the significant role of CD166 in hematopoietic stem cell maintenance and

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function. Bone. 2013;54:58-67. doi:10.1016/j.bone.2013.01.038

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highly variable proliferation and self-renewal properties comprise the human hematopoietic

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stem cell compartment. Nat Immunol. 2006;7:1225-1233. doi:10.1038/ni1393

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hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in

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multiple species. Nat Med. 1997;3:1337-1345. doi:10.1038/nm1297-1337

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human hematopoietic cells with SCID-repopulating activity. Nat Med. 1998;4:1038-1045.

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characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hematopoietic stem cells using

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cell surface markers AC133 and CD7. Blood. 2000;95:2813-2820.

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blood-derived CD34- cells assured by intra-bone marrow injection. Blood.

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Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly discovered class of

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method for precise functional characterization of primitive human cord blood-derived

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CD34-negative SCID-repopulating cells. Exp Hematol. 2011;39:203-213 e201.

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distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells.

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Leukemia. 2014;28:1308-1315. doi:10.1038/leu.2013.326

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mesenchymal stem cells are enriched at different gestational ages in human umbilical cord

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blood. Pediatr Res. 2008;64:68-73. doi:10.1203/PDR.0b013e31817445e9

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human CD34+ progenitor cells in zebrafish. Arterioscler Thromb Vasc Biol.

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2011;31:1589-1597. doi:10.1161/ATVBAHA.111.226969

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umbilical cord blood CD34(+) cells in a mouse model of neonatal stroke. Neuroscience.

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(ESAM) localizes to platelet-platelet contacts and regulates thrombus formation in vivo. J

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Thromb Haemost. 2009;7:1886-1896. doi:10.1111/j.1538-7836.2009.03606.x

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proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers. Mol

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Cell Proteomics. 2013;12:626-637. doi:10.1074/mcp.M112.021931

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Javed MJ, Mead LE, Prater D, et al. Endothelial colony forming cells and

Pozzoli O, Vella P, Iaffaldano G, et al. Endothelial fate and angiogenic properties of

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Tsuji M, Taguchi A, Ohshima M, et al. Effects of intravenous administration of

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Stalker TJ, Wu J, Morgans A, et al. Endothelial cell specific adhesion molecule

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Tabe Y, Konopleva M. Advances in understanding the leukaemia microenvironment.

Ciraci E, Della Bella S, Salvucci O, et al. Adult human circulating

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CD34(-)Lin(-)CD45(-)CD133(-) cells can differentiate into hematopoietic and endothelial

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cells. Blood. 2011;118:2105-2115. doi:10.1182/blood-2010-10-316596

560 Figure legends

562

Figure 1. Expression of ESAM on human BM and GMPB.

563

Mononuclear cells (MNCs) from human BM were stained with anti-CD34, anti-CD38, and

564

anti-ESAM antibodies and analyzed by flow cytometry (A). The ESAM expression patterns

565

of the CD34+ CD38– fraction of BM (B) and GMPB (C) are shown. The solid black lines and

566

dashed lines show ESAM and background levels, respectively. The CD34+ CD38– fraction

567

was subdivided into two populations: ESAM–/Low and ESAMHigh, and the percentages of cells

568

in each gate are shown in each panel. (D) CD34+ CD38– cells in ABM were stained with

569

CD45RA and CD90 (Thy-1) and subdivided into 3 fractions, including CD45RA– CD90+

570

hematopoietic stem cells (HSC); CD45RA– CD90– multipotent progenitors; and CD45RA+

571

CD90– multi-lymphoid progenitors (MLP) (left panel). The percentage of cells in each

572

fraction is shown in the upper right section of the panel. In the middle panel, ESAM

573

expression levels in HSCs (solid line), multipotent progenitors (dashed line), and MLPs

574

(dotted line) are shown. Grey line shows background level of CD34+ CD38– cells.

575

Differences between background and mean ESAM intensities in each fraction are shown

576

(right panel).

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578

Figure 2. High ESAM expression marks primitive hematopoietic progenitors.

579

(A) ESAM–/Low and ESAMHigh cells of the CD34+ CD38– fraction of BM were sorted and

580

subjected to a methylcellulose colony formation assay. The numbers of colony-forming units

26

ACCEPTED MANUSCRIPT 581

(CFUs) are shown. CFU-G/GM/M, CFU- granulocyte/granulocyte-macrophage/macrophage;

582

BFU-E, burst-forming unit erythrocyte; CFU-Mix, mixed colony-forming unit. (B) ESAM–

583

/Low

584

with MS5 in the presence of stem cell factor, Flt3 ligand, and interleukin-7. Cell numbers of

585

each fraction after 1–4 weeks are shown. *p < 0.05; **p < 0.005 by Student’s t-test. (C)

586

Numbers of CD19+ CD13– B-lineage cells after 4-week-co-culture with MS5 are shown.

587

****p < 0.0001 by Student’s t-test.

588

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and ESAMHigh cells of the CD34+ CD38– fraction of BM were sorted and co-cultured

Figure 3. Expression of ESAM on human CB cells.

590

(A) CB MNCs were stained with anti-CD34 and CD38 antibodies and analyzed by flow

591

cytometry (left panel). The percentages of cells in each subpopulation are shown in the upper

592

right section of the panel. ESAM expression on CD34– CD38+, CD34+ CD38+, CD34– CD38–,

593

and CD34+ CD38– fractions were analyzed by flow cytometry (right panel). According to

594

ESAM expression levels, the CD34+ CD38– fraction was subdivided into three populations:

595

ESAM–/Low, ESAMHigh, and ESAMBright (lower right). The same gates were applied to other

596

fractions, and the percentages of cells in each gate are shown in each panel. The solid black

597

lines and dashed lines show ESAM and background levels, respectively. (B) Ten CB samples

598

were analyzed in the same manner, and the percentages of ESAM–/Low, ESAMHigh, and

599

ESAMBright cells are shown. The percentage of each population of the same CB sample is

600

represented by the same symbols.

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Figure 4. High ESAM expression marks authentic HSCs in CB.

603

(A) ESAMHigh and ESAMBright cells in the CB CD34+ CD38– fraction were sorted and

604

subjected to methylcellulose colony formation assay. Numbers of colony-forming units

27

ACCEPTED MANUSCRIPT (CFUs) are shown. BFU-E, burst-forming unit erythrocyte; CFU-GM/M, colony-forming unit

606

granulocyte-macrophage/macrophage; CFU-Mix, mixed colony-forming unit. (B) ESAMHigh

607

and ESAMBright cells of CB CD34+ CD38– fraction were subjected to limiting dilution

608

analyses in the MS5 co-culture system in the presence of stem cell factor and

609

granulocyte-colony-stimulating factor (G-CSF). The input cell numbers corresponding to

610

37% negative value are shown in rectangles. (C) ESAM–/Low, ESAMHigh, and ESAMBright cells

611

in the CB CD34+ CD38– fraction were purified and transplanted into sub-lethally irradiated

612

NOG mice (ESAM–/Low 5 × 103 cells/mouse, ESAMHigh 2 × 104 cells/mouse, ESAMBright 1 ×

613

103 cells/mouse, n = 6 for each group). Three months after transplantation, all recipients were

614

sacrificed and flow cytometry analyses of BM were performed. BM MNCs were stained with

615

mouse CD45 and human CD45, and the percentages of human CD45+ cells are shown. (D)

616

The representative results of the recipient transplanted ESAMHigh cells are shown. Human

617

CD45+ cells were gated and the percentages of each lineage marker-positive cells are shown.

618

(E) Whole BM MNCs were obtained from primary recipients of ESAMHigh cells and were

619

transplanted into sub-lethally irradiated NOG mice (2 × 106 cells/mouse, n = 5). Four months

620

after transplantation, all recipients were sacrificed and flow cytometry analyses of BM were

621

performed. The percentages of human CD45+ cells are shown. (F) The figures shows

622

representative results for the secondary recipient.

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Figure 5. Morphological, immunophenotypic, and gene expression profiling differences

625

between ESAMHigh and ESAMBright cells of the CD34+ CD38– fraction.

626

(A) ESAM–/Low, ESAMHigh and ESAMBright cells of CB CD34+ CD38– fraction were sorted,

627

and cytospin slides were stained by May-Grünwald/Giemsa staining. Horizontal bars

628

represent 20 µm. (B) CB CD34+ CD38– cells were stained with ESAM and CD45,

28

ACCEPTED MANUSCRIPT hematopoietic cell marker (left panel), and CD133, HSC marker (right panel). (C) ESAMHigh

630

and ESAMBright cells of the CB CD34+ CD38– fraction were back-plotted onto the CD34 and

631

CD38 profile. ESAMHigh cells were found as CD34Low, whereas ESAMBright cells were

632

CD34High. (D) CB CD34+ CD38– cells were stained with ESAM and the endothelial markers,

633

VEGFR-2 (left panel) and VE-cadherin (right panel). (E) ESAM expression of the CD34+

634

CD38– fraction from collagenase-treated bone was determined by flow cytometry. ESAM–/Low,

635

ESAMHigh, and ESAMBright gates and the percentages of cells in each gate are shown. The

636

solid black line and dashed line show ESAM and background levels, respectively. (F) The

637

gene expression profiles of CD34+ CD38– ESAMHigh and ESAMBright cells from

638

collagenase-treated bone were determined by microarray. Heat maps of selected genes related

639

to endothelial and hematopoietic cells are shown. (G) The CD34+ CD38– ESAMBright cells

640

from collagenase-treated bone were cultured with MS5 stromal cells in the presence of VEGF,

641

stromal cell-derived factor (SDF) and IL-16. This co-culture condition supports both

642

endothelial and hematopoietic cell growth simultaneously [41]. After 5 weeks, the cells were

643

harvested and analyzed by flow cytometry. Recovered cells were stained with anti-CD34 and

644

CD45 antibodies (left panel). The expression of endothelial marker CD31 on the CD34+

645

CD45– cells is shown in the right panel.

646

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647

Figure 6. ESAM expression on various human leukemia cell lines and primary leukemia

648

cells.

649

(A) The ESAM along with CD34 and CD38 expression patterns on various human leukemia

650

cell lines were analyzed by flow cytometry. (B) Primary leukemia cells were analyzed by

651

flow cytometry. Leukemia cells were gated in the CD34 and CD38 profiles, and the ESAM

652

expression patterns of leukemia cells are shown. The solid black lines and dashed lines show

29

ACCEPTED MANUSCRIPT 653

ESAM and background levels, respectively.

654 Figure 7. KG-1a leukemic mouse model.

656

(A) Scheme of the transplantation protocols. KG-1a, a human AML cell line, was

657

transplanted into sub-lethally irradiated NOD/SCID mice. Ten weeks after transplantation,

658

the mice were killed and leukemic cells in BM were collected. The reconstituted KG-1a

659

(rKG-1a) was cultured in vitro for 3 weeks and re-transplanted into sub-lethally irradiated

660

NOD/SCID mice. (B) Kaplan-Meier survival curves of transplanted mice are shown. (C)

661

ESAM expression pattern of KG-1a and rKG-1a were analyzed by flow cytometry by using a

662

monoclonal antibody specific for human ESAM. The solid black line and dashed line show

663

ESAM expression level in rKG-1a and KG-1a, respectively. (D) ESAM– and ESAM+ rKG-1a

664

cells were sorted and transplanted into sub-lethally irradiated NOD/SCID mice (5 × 104

665

cells/mouse, n = 4 for each group). Three months after transplantation, the mice were

666

sacrificed and BM cells were analyzed by flow cytometry. The percentages of human CD45+

667

cells are shown. *p < 0.05 by Student’s t-test. (E) Representative results of the mice

668

transplanted with ESAM– and ESAM+ rKG-1a cells are shown. The percentages of human

669

CD45+ cells (mean ± SD) are shown in the rectangles.

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EP

Figure 1. CD34+ cells

BM MNCs

A 10 5

10 5

21

10 3

2

0

3

10

2

0

10 2

0

10 3

10 4

10 5

CD34

B

C

80

% of Max

80

60

40

40

16.7

83.3

20

0 0

10

2

10

3

10

4

10

5

0

ESAM

10

2

10

3

10

4

10

5

ESAM 300

100

105

HSC

24

MPP

80 104

60

13 % of Max

HSC 103

102

MLP 60

40

200

100

20

MLP 103

CD45RA

104

105

0 0 102

103

ESAM

104

105

0

MLP

0

MPP

MPP

HSC

CD90

10 5

60

89.6

0

0

10 4

mobilized PB 100

20

D

10 3

CD38

100

10.4

10 2

0

adult BM

% of Max

10

⊿ESAM Mean

10

AC C

10 4

ESAM

ESAM

10 4

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ESAMHigh

30

20

6

4

10

2

0

0

-E

H ig h

w

ES

AM

Lo –/

AM

1

2

3 Week

4

1000 0 H ig h

* 0

0

2000

w

1 x105

3000

AM

**

****

4000

Lo

2 x105

1x104

5000

–/

**

C

ES

ESAM –/Low ESAMHigh

*

AM

4 x105

*

3 x105

ES

Cell Number

2x104

ES

B

CD19+ CD13– Cell Number

C

FU

-G

/G

BF

U

M

Colonies / 200 input cells

8

ESAM–/Low

/M

Colonies / 200 input cells

40

M ix

A

AC C

Figure 2.

AC C

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Figure 3.

CD34–CD38+

A

10

4

34 0.1 10

3

10

2

80

80

60

CD38

60

69

40 10

10

1

0

10

0

1

10

10

2

10

3

10

CD34+CD38+

100

100

66 0.2

4

20

20

0

0 0

4

10

10

1

10

2

10

3

10

4

% of Max

100

100

80

80

60

10

1

10

2

10

23

10

3

10

4

60

91

40

9

40

8

0.5

33

59

20

0

0 0

10

1

10

2

10

3

ESAM 100

% of CD34+ CD38– cells

0

68

CD34+CD38–

20

80

60

40

20

0

ESAM –/Low

10

CD34–CD38–

CD34

B

7

40

27

ESAM High

ESAM Bright

4

10

0

10

10

1

10

2

10

3

10

4

Figure 4. CFU-Mix BFU-E CFU-GM/M

120

12

0

20

100

37

100

10

80

% negative

Colony counts / 1000 cells

Input cell number

B

140

60

40

40

60

80

100

ESAM Bright

1

20

63

ESAM High

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ESAM High ESAM Bright

D

BM MNC

10

Mouse CD45

40

20

0

10

4

10

3

43

B lymphoid

300

200

200

15

72

15

50 100

100

10 2

10 3

10 4

10 5

0

0 0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

0

10

2

10

3

10

4

10

5

rig ht

ig h

300

0

ES AM B

/L

ES AM H

400

100

0

ES AM –

T lymphoid

400 150

10 2

0

E

Human CD45

F

Human CD13 and CD33

Human CD19

Human CD4 and CD8

BM MNC

15

Myeloid

10 5

B lymphoid

T lymphoid

12 12

10 4

5

0

15

1.1

9 9

# Cells

Mouse CD45

10

10 3

10

16

6 6

ig h

12

77

5

10 2

3

3

0 0

ES AM H

human CD45 % of BM MNCs

Myeloid

5

# Cells

60

ow

human CD45 % of BM MNCs

C

AC C

0.1

0

0

10 2

10 3

Human CD45

10 4

10 5

0 0

10

2

10

3

10

4

10

5

Human CD13 and CD33

0 0

10 2

10 3

Human CD19

10 4

10 5

0

10 2

10 3

10 4

Human CD4 and CD8

10 5

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E

ESAMBright cells

100 80 60 40

4

CD133

2

10

2

3

0 10

4

10

10

5

10

10

2

3

0 10

4

10

ESAMHigh 10

4

10

4

10

3

3

10

2

0 2

0 10

3

10

4

10

2

10

0

5

2

10

3

0 10

CD34

10

4

10

5

10

4

10

VEGFR2

3

10

2

10

0 2

0 10

3

10

ESAM

4

10

5

10

ht rig

MH

MB A

ES A

HSC self-renewal and expansion

Hematopoietic

5

2

G

5

10

HSC development

10

CD34

10

KDR HHEX FLI1 TEK PECAM1 ESAM FLT1 EMCN TIE1 LMO2 TAL1 CBFB RUNX1 PBX1 EZH2 TCEA1 PTEN MEIS1 SOX17 CITED2 GATA2 MYB GFI1 BMI1 CBFA2T3 HOXB4 HBE1 HBG1 GYPA ITGA2B NFE2 VAV1 GATA1 MPO

Angiohematopoietic

3

4

5

100

5

10

4

10

3

10

80

4

% of Max

CD38

10 10

105

ig h

ht

Typical endothelial genes

5

104

rig

10

ESAMBright

5

103

A MB

5

10

ESAM

10

CD38

h

0

ESAM

VE-cadherin

ig

2

10

3

10

CD45

CD45

10

F11R JAM3 TJP1 TJP2 CDH5 CDH2 CLDN5 HIF1A AAMP F2R EDF1 PROCR SCARF1 NOS3 CAV1 S1PR1 CTGF APOLD1 PPAP2B AMOTL1 AMOTL2 AMOT TNFAIP1 COL18A1 MMRN1 SERPINE1 MALL ANGPT1 ANGPTL2 ROBO4 EPAS1 PGF ENG

Endothelial junctions

3

0

D

A MH

4

10

3

0 102

ESAM

ES

10

10

C

F

5

10

0

ES

5

AC C

B

60

20

20

20

ES

ESAMHigh cells

EP

ESAM–/Lowcells

% of Max

A

TE D

Figure 5.

2

10

0

60 40

2

10

20

0 2

0 10

3

10

ESAM

4

10

5

10

2

0 10

3

10

CD34

4

10

5

10

0

2

0 10

3

10

CD31

4

10

5

10

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Figure 6. A

Myeloid 10 4

10 3

10

10 2

10 2

0

10 4

10 5

10 5

10 5

10 4

10 4

10 3

10 3

10 2

10 2

10 3

10 4

10 5

10 4

10 4

3

10

10 2

2

10

3

10

4

10

5

10

10 2

10 3

10 4

10 5

0

10 2

10 3

10 4

10 5

10 5

10 5

10 4

10 4

10 3

10 3

10 2

10 5

10

2

10

0

3

10

4

10

5

2

10

3

10

4

10

5

Erythroid / Megakaryocyte

K562

3

0

10

10

2

10

3

10

4

10

5

2

0

0

10 2

10 3

10 4

10 5

10 2

0

10 5

10 5

10 4

10 4

10

3

10 3

10

2

10

0

2

0

0

10 2

10 3

10 4

10 5

10 5

10 5

10 4

10 4

10 3

10 3

10 4

10 5

10 3

10 2

10 2

0 10 5

0

10 2

10 3

10 4

10 5

0

10 2

10 3

10 4

10 5

0

10 2

10 3

10 4

10 5

0

10 5 0

4

10

CMK 3

10

10 2

0

0

10

2

10

3

10

4

10

5

10 3

10 4

10 5

10 5

10 5

10 4

10

4

10 3

10

3

2

10

2

3

10 2

0

10 2

4

0

10

2

10

3

10

4

10

ESAM

ESAM

10

10 4

10 3

10 2 0

5

10

0

0

CD34

CD38 0

10

2

10

3

10

4

10

5

CD34

B

0

10 3

10 2

0

HEL

10 3

10

10 2

10 4

10 4

10

0

10 5

10 5

Kasumi

3

10 2

0

10 2

0

10

10 4

3

0

10 2

0

ESAM

10 5

MOLT4

0

10 5

0

KG1a

10 4

AC C

ESAM

10 2

10 5

10

10 3

0

0

10

0

10 2

0

ESAM

10 3

0

THP1

3

EP

ESAM

U937

10 2

10 5

10 4

0

0

10 5

TE D

10 4

Lymphoid Jurkat ESAM

10 5

ESAM

10 5

ESAM

ESAM

HL60

AML (M2)

AML (M2)

APL

CD38

APL

ALL

% of Max

100 80 60 40 20 0 0 10

2

10

3

ESAM

10

4

10

5

0 10

2

10

3

ESAM

10

4

10

5

0 10

2

10

3

ESAM

10

4

10

5

0 10

2

10

3

ESAM

10

4

10

5

0 10

2

10

3

ESAM

10

4

10

5

KG-1a

1.0×106

80

cells

i.v. 10 weeks

60 40 20

0 0 10

bone marrow from leukemic mouse

10

3

10

4

10

5

ESAM

E in vitro culture for 3 weeks

Mouse CD45

reconstituted KG-1a (rKG-1a) 5.0×105 cells i.v.

B

ESAM– rKG-1a 10 5

10 4

10 4

10

3

10

KG-1a (n=5) rKG-1a cl 1 (n=5)

60

rKG-1a cl 2 (n=5)

40 20 0 0

2

4

6

8

Weeks after transplantation

10

0.1

0.0

10 2

3

10 2

0.0056 ± 0.00324

0

10

2

10

3

10

4

10

5

0.1634 ± 0.0586

0

0

10

Human CD45

80

0.2

ESAM+ rKG-1a

10 5

0

100

% Survival

2

*

0.3

ES AM –

NOD/SCID

D

100

ES AM +

C

2.4 Gy

% of Max

A

Human CD45 % of BM MNCs

Figure 7.

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2

10

3

10

4

10

5

ACCEPTED MANUSCRIPT

ESAM marks hematopoietic stem cells in humans as well as in mice.

RI PT

ESAM enriches human hematopoietic stem cells in all clinically available sources.

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ESAM marks a subset of human leukemia cells and reflects some of their features.