EPCR Guides Hematopoietic Stem Cells Homing to the Bone Marrow Independently of Niche Clearance
Authors: Gur-Cohen Shiri , Avemaria Francesca , Kollet Orit , Avci Seymen , Itkin Tomer , Ramasamy Saravana K. , Kusumbe Anjali P. , Golan Karin , Khatib-Massalha Eman , Kumari Anju , Carolina Mayela M. , Lin Wei-Ling , Yu Qing-Cissy , Wu Hua-Lin , Zeng Yi Arial , Adams Ralf H. , Edward Conway M. , Shpall Elizabeth J. , Erez Ayelet , Sagi Irit , Esmon Charles T. , Ruf Wolfram , Lapidot Tsvee 
Published: October 2019

Abstract
Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) are active and essential first steps during embryonic development and in clinical stem cell transplantation. Rare, BM LT-HSCs endowed with the highest self-renewal and durable repopulation potential, functionally express the anticoagulant endothelial protein C receptor (EPCR) and PAR1. In addition to coagulation and inflammation, EPCR-PAR1 signaling independently controls a BM LT-HSC retention-release switch via regulation of nitric oxide (NO) production within LT-HSCs. EPCR+ LT-HSCs are maintained in thrombomodulin+ (TM) periarterial BM microenvironments via production of activated protein C (aPC), the major ligand for EPCR. Restriction of NO production by aPC-EPCR-PAR1 signaling, activates VLA4-mediated adhesion, anchoring EPCR+ LT-HSCs to the BM and protecting them from chemotherapy insult, sparing hematological failure and premature death (Gur-Cohen S. et al, Nat Med 2015).
We report that transplanted EPCR+ LT-HSCs preferentially homed ‎to and were retained in the BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing treatment that block aPC binding and attenuate EPCR+ LT-HSC BM homing. Furthermore, short term aPC in vitro pretreatment dramatically augmented EPCR+ LT-HSC BM homing, lodgment and long-term repopulation. PAR1 deficient stem cells were irresponsive to treatment with aPC and displayed reduced BM homing efficiency, all pointing to the aPC-EPCR-PAR1 axis as a crucial mediator of BM LT-HSC homing. Additionally, aPC pretreated EPCR+ LT-HSCs had a striking advantage to competitively home to the BM. Consistently, BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with wild type stem cells in competitive repopulation assays. Importantly, the competitive homing results strongly imply that the BM available niches for newly arrived EPCR+ LT-HSCs are limited. Indeed, aPC pretreated EPCR+ LT-HSCs BM homing reached a plateau, as increasing the transplanted cell dose above 5x106 BM mononuclear cells, did not yield higher donor EPCR+ LT-HSC homing. These results reveal that there is a limited BM space for newly arrived transplanted EPCR+ stem cells to non-irradiated hosts. Importantly, we found that EPCR+ LT-HSCs can engraft the BM of non-conditioned mice with high efficiency, while remaining in a dormant, non-cycling state. Furthermore, the dormant homed EPCR+ LT-HSCs were later awakened and activated solely by treating the engrafted hosts with a low dose 5-FU chemotherapy, or with NO donor SNAP, revealing that preconditioning and clearance of occupied BM HSC niches are not required.
To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by unique BM arterioles, and not in the spleen. BM homed EPCR+ LT-HSCs were found adjacent to TM+ arterioles, imposing their retention. Homed BM EPCR+ LT-HSCs highly express full-length TM with intact lectin-like domain, and the BM TM+ endothelium was found to be enriched with a Glycocalyx layer, in particular with Heparan Sulfate Proteoglycan-2 (HSPG-2). HSGP-2 might specifically interact with the lectin-like domain of TM-expressingLT-HSCs, providing BM specific recognition and accelerated homing. Intriguingly, stabilizing TM function by in vitro pretreatment with platelet factor-4 (PF4) bypassed BM-derived cues and increased EPCR+/TM+ LT-HSC homing also to the spleen, suggesting a supportive role for PF4, highly secreted by BM megakaryocytes, in guiding EPCR+/TM+ LT-HSCs to the BM.
Herein we define EPCR as a guidance molecule, navigating LT-HSC specifically to BM TM+ aPC-secreting blood vessels, allowing stem cell retention and protection from DNA damaging agents. The BM harbors a limited number of available stem cell niches for newly arrived transplanted EPCR+/TM+ LT-HSCs, and in vitro aPC pretreatment dramatically augments EPCR+/TM+ LT-HSC BM homing. Our findings provide new mechanistic insights and identify key players concerning LT-HSC homing specifically to the BM, leading to better repopulation following transplantation. This up-to-date approach and new knowledge may potentially lead to improved BM transplantation protocols and to prevent chemotherapy resistance of EPCR-expressing cancer stem cell mediated relapse.

Disclosures
Ruf: Iconic Therapeutics: Consultancy.