Background. Discovery of niche-derived HSPC regulators is critical for further development of novel therapeutic approaches to promote HSPC regeneration. We have previously reported a proximity-based approach to the study of the bone marrow niche, which is based on the transcriptional comparison of osteolineage cells (OLCs) located close (proximal OLCs) or further away from transplanted HSPC (Blood 2014;124: 773), which lead to identification of IL18 as a quiescence regulator of early progenitors. We now report the results of functional validation for another molecule - Embigin - identified through this strategy as a hematopoietic regulator.

Results. Embigin is a cell adhesion molecule with poorly characterized function. Our analysis showed that Embigin expression in proximal OLCs was significantly higher. Since the genetic tools for this molecule do not exist, we used a neutralizing antibody against Embigin to investigate the effect of Embigin blockade on primitive hematopoietic cells. We found that injection of anti-Embigin resulted in mobilization of myeloid progenitors and colony-forming cells (CFC) into the blood. In contrast, Embigin blockade was associated with a homing defect when LKS cells [known to express Embigin] were either pre-incubated with anti-Embigin antibody or injected into anti-Embigin pretreated animals, overall suggesting that Embigin regulates retention and localization of primitive hematopoietic cells in the bone marrow. Moreover, animals treated with anti-Embigin antibody had a higher frequency and proliferative activity of primitive hematopoietic cells, as demonstrated by cell cycle and BrdU incorporation studies and an increased CFC, consistent with Embigin-mediated localization also affecting HSPC cell cycling. Bone marrow from anti-Embigin treated mice reconstituted poorly when competitively transplanted with untreated animal marrow into irradiated recipients, likely due to the impaired homing and increased cell cycling. Finally, pre-treating irradiated recipients with anti-Embigin resulted in increased proliferation of transplanted WT LKS cells. Collectively, these data are consistent with Embigin serving as a regulator of HSPC localization and quiescence.

Given a functional role of Embigin in the bone marrow niche and an overlapping pattern of expression with VCAM1, a known niche-derived HSPC regulator, in proximal OLCs we explored the use of Embigin in conjunction with VCAM1 as cell surface markers for prospective isolation of niche factor-enriched OLC subset by flow cytometry. Using this strategy, we were able to purify a rare OLC subset of CD45- Ter119-V CAM-1+E mbigin+ cells (termed VE cells), which were enriched for niche factor expression as compared to their non-VE counterparts. VE cells were transcriptionally distinct from other, previously defined niche subsets such as nestin-GFPdim mesenchymal stem cells, nestin-GFPbright pericytes and N-cadherin-positive osteoblastic cells. In particular, VE cells expressed higher levels of most niche factors than nestin-GFPbright cells - a cell population recently characterized as regulating HSPC quiescence. Interestingly, the expression profile of VE cells from animals transplanted with LT-HSCs and those which were irradiated but injected with saline alone demonstrated upregulation of cell adhesion molecules in the LT-HSC-injected group, suggesting that VE cells are involved in bidirectional communication within the niche.

To investigate a functional role of VE cells, we performed co-culture experiments in which we compared the effect of VE and non-VE cells on HSPC proliferation and engraftment. We found that in the presence of VE cells, HSPCs proliferated at slower rate and generated a lower hematopoietic colony number, consistent with quiescence-inducing effect. Upon transplantation, VE-cultured HSPCs generated a higher level of chimerism compared to those cultured on non-VE layer, indicating a superior ability of VE cells to support engraftment and reconstitution properties of HSPC during the in vitro culture.

Conclusion. Our work defines Embgin as a previously unrecognized hematopoietic regulator and a cell surface marker for a niche factor-enriched subset of the osteolineage cells which regulates HSPC quiescence. Pharmacological blockade of Embigin signaling may serve as a potential therapeutic tool to enhance hematopoietic regeneration.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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