The Endothelial Antigen ESAM Monitors Reversible Conversion of Hematopoietic Stem Cells Between Dormancy and Self-Renewal

Abstract 2337

Although hematopoietic stem cells (HSC) are characterized with self-renewal and pluri-potential, their cell-cycle status and differentiating behavior do fluctuate according to the physiological requirement. In the homeostatic state of adult bone marrow (BM), HSC are likely to be quiescent so that they can evade exhaustion or mutation. However, when BM is injured by irradiation and/or anti-cancer drugs, HSC need to proliferate to restore normal hematopoiesis. Then, after re-establishment of homeostasis, activated HSC return to be quiescent. Molecular crosstalk between HSC and BM microenvironment is thought to elaborately control the status of HSC, but precise mechanisms remain unknown. If the conversion of HSC between dormancy and self-renewal could be accurately monitored, the method should be useful to understand how the HSC status is regulated.

Our previous study demonstrated that endothelial cell-selective adhesion molecule (ESAM) is a useful marker for murine HSC throughout life. In the present study, we examined if the ESAM level reflects the HSC status between dormancy and activation. Firstly we monitored ESAM levels of the Lin⁻ Sca1⁺ c-kit⁺ (LSK) HSC-enriched fraction in BM after a single 5-FU injection (150 mg/kg) by flow cytometry. From 2 to 9 days after the 5-FU injection, ESAM levels on the LSK fraction remarkably increased. Indeed, the mean fluorescence intensity of ESAM expression on HSC increased by 9.6-fold in 5 days after 5-FU injection. The increase of ESAM expression was more drastic than that of other endothelial-related markers such as CD34 (1.6-fold). After reaching to the maximum peak around day 5–6, ESAM level gradually decreased and returned to the homeostatic level by 12 days after 5-FU. Interestingly, the ESAM up-regulation on HSC was abrogated when inhibitory drugs for NF-kB and topoisomerase-II were given after 5-FU injection. Furthermore, short-term BrdU exposure proved that the ESAM^hi cells after 5-FU treatment are actually active in the cell cycle status. Then, the immuno-histochemical analyses were performed to locate the activated HSC in 5-FU treated BM. Since more than 80% of the Lin⁻ ESAM^hi Sca1⁺ cells were found within 20 μm from vascular endothelium, the activated HSC seemed to be intimate with endothelial cells and/or vascular-related cells.

Next, we performed functional assessments of the ESAM^low LSK and ESAM^hi LSK fractions sorted from 5-FU-treated BM. In methylcellulose cultures, while both fractions contained a number of hematopoietic progenitors, CFU-Mix, primitive multipotent progenitors, were significantly enriched in the ESAM^hi fraction (10±0 vs. 48.5±2.1 per 200 ESAM^low or ESAM^hi LSK cells, respectively). In the in vivo long-term reconstitution assays, we transplanted 2,000 CD45.1⁺ ESAM^low or ESAM^hi LSK cells with 2 × 10⁵ CD45.2⁺ competitor BM cells into lethally irradiated CD45.2⁺ mice. Sixteen weeks after transplantation, the mice transplanted with ESAM^hi LSK cells showed significantly higher chimerisms of CD45.1⁺ cells than those transplanted with ESAM^low LSK, suggesting that long-term HSC are enriched in the ESAM^hi fraction. It is noteworthy that the ESAM^hi CD45.1⁺ LSK fraction re-constituted a CD45.1⁺ LSK population in the CD45.2⁺ recipient BM, whose ESAM expression levels lowered to the homeostatic level.

The results above suggested that ESAM expression level mirrors the activation status of HSC after BM injury. However, it remains unclear if ESAM plays an important role in the hematopoietic recovery. Although we did not observe significant phenotypes except slight anemia in homeostatic ESAM KO mice, we presumed that substantial BM stress might reveal physiological importance of the ESAM expression. At day 5 after injecting 200mg/kg 5-FU, we found that leukocytes and platelet were remarkably decreased in KO mice. Furthermore, the KO mice showed severe anemia (Hb; WT 10.4±1.1 g/dl vs. KO 6.0±1.7 g/dl at day 10), and two of five mice died at day 12. In addition, we observed LSK Flt3⁻ HSC as well as total mononuclear cells more significantly decreased in the KO mice.

In summary, our data have shown that ESAM serves as a strong tool to monitor the conversion between dormancy and proliferation of adult BM HSC. In addition, the data from ESAM KO mice have suggested that ESAM is indispensable for normal hematopoietic recovery after BM injury. Further studies should address physiological meanings of the high ESAM level on active HSC.