Hyaluronan Associated with the Bone Marrow Hematopoietic Microenvironment Is Required for the Recruitment and Retention of Hematopoietic Stem and Progenitor Cells In the Bone Marrow.

Abstract 2593

The bone marrow microenvironment regulates a variety of hematopoietic stem cell (HSC) functions, including their recruitment into the marrow following transplantation. However, an insufficient understanding of the biology of the hematopoietic microenvironment reflects gaps in our knowledge of fundamental stem cell biology and remains an obstacle to an optimal therapeutic approach. While the involvement of hyaluronan (HA), one of the major components of the bone marrow ECM, in normal cell and tumor biology is generally appreciated, little is known of how HA contributes to the regulation of the hematopoietic microenvironment.

In this study we investigated whether HA contributes to the recruitment of circulating HSCs into marrow. Stimulation of bone marrow cells with HA induced production of chemokines including SDF-1, MIP-1a, MIP-1b, IL-8, eotaxin, CXCL16, RANTES, LIX and MCP-1. HA-induced conditioned media containing these chemokines enhanced SDF-1-mediated HSC transmigration in vitro, whereas HA alone had no effect, suggesting that endogenous HA participates in the recruitment of HSC indirectly, via regulation of the production of chemokines. We next tested the relevance of this finding in vivo. Since HA synthase 2 knockout mice are embryonically lethal, we developed an alternative in vivo model that allows deprivation of HA. Mice were lethally irradiated to eliminate HSCs and to degrade endogenous HA, and injected with 4-MU, an HA synthase inhibitor, to prevent de novo synthesis of HA. Pre-treated mice were transplanted with HSCs, and after 24 hours the bone marrow was assayed by competitive reconstitution. This assay demonstrated that the number of HSCs which migrated into the marrow of mice pretreated with 4MU was significantly lower as compared to control.

To further understand the role of endogenous HA in bone marrow microenvironment we used compound mutant mice in which Has1 and Has3, two genes encoding hyaluronan synthase, were knocked out (Has1^-/-;Has3^-/-). The number of hematopoietic progenitors (HPs) circulating in peripheral blood was significantly increased in the double knockout (dKO) mice as compared to the wild type (WT) mice. This increase in the number of circulating HP was further enhanced in triple Has knockout mice (Prx1-Cre;Has2^flox/flox;Has1^-/-;Has3^-/-). This correlated with the higher number of hematopoietic progenitors in spleens of Has1^-/-;Has3^-/- double KO mice and Prx1-Cre;Has2^flox/flox;Has1^-/-;Has3^-/- triple KO mice as compared to the WT mice.

In summary, our data indicate that low levels of HA correlate with re-distribution of HSCs and HPs from bone marrow into peripheral blood and spleen. This suggests that HA associated with the hematopoietic microenvironment is important for recruitment and retention of HSCs and HPs, confirming that HA plays a critical regulatory role in the hematopoietic microenvironment.