ESAM Is a Novel Human Hematopoietic Stem Cell Marker Associated with a Subset of Human Leukemias
Authors: Ishibashi Tomohiko , Yokota Takafumi , Tanaka Hirokazu , Ichii Michiko , Sudo Takao , Satoh Yusuke , Doi Yukiko , Tanimura Akira , Hamanaka Yuri , Ezoe Sachiko , Shibayama Hirohiko , Oritani Kenji , Kanakura Yuzuru 
Published: October 2019

Abstract
Murine hematopoietic stem cells (HSCs) can be isolated with high efficiency as Lineage- Sca-1+ c-kitHigh (LSK) CD34-/Low CD150+ CD48- cells. In humans, however, the same method is not useful because of critical differences between murine and human HSC phenotypes. Such discrepancy has hampered the translation of findings in mice into a human preclinical or clinical context. Therefore, the identification of common HSC antigens between the two species would be a significant advance with respect to translational studies of HSC biology. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel maker for HSCs in mice (Blood, 2009). We also found that ESAM is functionally important for murine HSCs to reconstitute hematopoiesis after 5-FU treatment (J Immunol, 2012). In the present study, we have extended our research of ESAM to human HSCs and leukemia.
We first examined whether ESAM expression showed potential as a marker of human HSCs. In addition to adult BM, the majority of CD34+ CD38- cells in cord blood (CB) and G-CSF mobilized peripheral blood expressed ESAM. The addition of anti-CD90 and CD45RA antibodies divides the adult BM CD34+ CD38- fraction into three subpopulations, namely HSCs, multipotent progenitors (MPPs), and multi-lymphoid progenitors (MLPs). We found that HSCs expressed high levels of ESAM whereas MPPs expressed lower levels and many MLPs lost ESAM expression. Functional assessment for ESAM-/Low and ESAMHigh cells in the CD34+ CD38- fractionconfirmed that high ESAM expression distinguishes progenitors that are more primitive and multipotent.
We also identified a subset of CD34+ CD38- cells in adult BM and CB that expressed extremely high levels of ESAM, namely ESAMBright cells. Gene expression profiles of the CD34+ CD38- ESAMHigh and CD34+ CD38- ESAMBright populations showed that the former cells expressed HSC-related genes whereas the latter showed more endothelial-related profiles. Indeed, the CD34+ CD38- ESAMBright cells produced CD31+ endothelial cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells. These results suggest that the CD34+ CD38- fraction, which is conventionally considered the human HSC fraction, also contains a substantial number of non-hematopoietic progenitors. Thus, the inclusion of ESAM provides a more accurate estimation of HSC numbers.
Since some of HSC-related antigens are useful for determining leukemia lineage and have utility as prognostic indicators, we determined whether ESAM might also be a valuable addition to this antigen panel. First, we examined human leukemia cell lines. Tested myeloid leukemia lines including KG-1a, HL60, THP1, U937 and Kasumi were uniformly negative for ESAM expression. Jurkat and MOLT4, lymphoid lineage lines were also negative. On the other hand, HEL, an erythroid leukemia cell line, and CMK, a megakaryocytic leukemia cell line, exhibited high expression of ESAM. Additionally, K562 cells, which originated from CML that subsequently transformed into acute erythro-leukemia, also express ESAM. We then evaluated ESAM expression on primary acute leukemia cells, which were isolated from patients upon diagnosis. Interestingly, while all of ALL cases were virtually negative for ESAM, more than half of AML cases were ESAM-positive. Notably, the ESAM expression pattern on AML cases substantially differs even in the same FAB classification.
We inferred that AML cells might change their ESAM expression levels according to cell intrinsic features and/or the surrounding environment in vivo. Therefore, we inoculated ESAM- KG-1a cells into NOD/SCID mice and harvested reconstituted KG-1a (rKG-1a) cells after the inoculation. They were then cultured in vitro and inoculated again into NOD/SCID mice. FACS analyses revealed that, although parental KG-1a cells were ESAM-negative, rKG-1a cells expressed a substantial amount of ESAM. Notably, rKG-1a cells were more aggressive and killed the recipient mice in a shorter period. This observation indicates that leukemia cells change their surface phenotype according to the environment, and that ESAM expression may be related to the acquisition of a more aggressive phenotype.
In conclusion, we demonstrate that ESAM is a reliable marker of HSCs in humans as well as in mice. Additionally, ESAM is expressed on some of human acute leukemia cells and might be useful for lineage determination and as prognostic indicator.

Disclosures
Yokota: SHIONOGI & CO., LTD.: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.