Hematopoietic Stem/Progenitor Cell Retention in the Bone Marrow Depends On Tissue Specific Heparan Sulfate Proteoglycans
Authors: Saez Borja , Ferraro Francesca , Yusuf Rushdia Z. , Cook Colleen M , Sykes Stephen M. , Pardo Ana , Yu Vionnie W.C. , Lotinun Sutada , Palchaudhuri Rahul , Lee Dongjun , Lymperi Stefania , Day Robyn , Vasic Radovan , Acharya Sanket S. , Baron Roland , Wagers Amy J. , Scadden David T. 
Published: October 2019

Abstract
Abstract 637
The hematopoietic stem cell niche is known to govern the function of hematopoietic and leukemic stem cells. One constituent of the niche is the extracellular matrix comprised of several elements including a group of molecules called heparan sulfate proteoglycans (HSPGs). These molecules are present in the microenvironment of all tissue stem cells and are critical in the dynamic interactions between stem cells and niche elements.
Heparan sulfate proteoglycan synthesis depends upon the glycosyltransferase, Ext1, a known tumor suppressor with a skeletal phenotype (exostoses) when mutated in affected humans. In order to investigate the role of HSPGs in the hematopoietic stem cell niche we conditionally deleted the Ext1 gene in the recently described Mx1+ osteolineage stem/progenitor cell population (Park et al., 2012). Our studies show that Ext1 and its biological product, HSPGs, play a major role in the maintenance of hematopoietic stem cells (HSCs) by modulating the bone marrow microenvironment.
Ext1 deletion in osteolineage cells results in a marked loss of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow and an increase in circulating colony forming units (CFU-Cs) and HSCs (designated as lineage -, c-Kit+, Sca1+, CD 150 + and CD48-) both in the spleen and peripheral blood, suggesting a key role of heparan sulfate proteoglycans in the retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. Importantly, histomorphometry analysis showed no defect in bone remodeling upon Ext1 deletion.
In order to investigate whether the above phenotype could be therapeutically exploited in the context of bone marrow stem/progenitor cell mobilization, we administered the heparan sulfate proteoglycan inhibitors, heparin and protamine sulfate to mice in combination with conventional mobilization regimes such as G-CSF. Pharmacological inhibition of HSPG induced a significant improvement of hematopoietic stem/progenitor cell mobilization over G-CSF treatment alone, an effect that was completely abrogated in the absence of endogenous HSPGs. Furthermore, by combining HSPG inhibition with G-CSF, we could mobilize a population of HSPCs with superior in-vivo self-renewing ability.
Mechanistically, we observed that Ext1 deletion in stromal cells leads to a constitutive activation of the AKT pathway through the PDK1-mediated phosphorylation of AKT-Thr-308, which in turn phosphorylates and inactivate FOXO1 transcription factor. Expression profiling of genes transcriptionally regulated by FOXO1 revealed that VCAM1, a key molecule driving HSCs adhesion to their niche, was highly down regulated in Ext1 deficient cells. VCAM1 protein levels were also decreased accompanied by a profound reduction in HSC adhesion to Ext1 deficient cells in vitro.
Taken together our results suggest that Ext1/HSPGs control hematopoietic stem/progenitor cells retention in the bone marrow through an AKT/FOXO1/VCAM1 dependent pathway and that targeting heparan sulfate proteoglycans is a strategy for mobilizing stem cells with superior self renewing capability to the blood.
We are grateful to Lexicon Genetics and the MMRRC for kindly providing the Ext1 mouse model.

Disclosures:
Wagers: BD Biosciences: Consultancy; iPierian, Inc.: Consultancy; MPM Capital: Consultancy; Novartis: Honoraria. Scadden:Fate Therapeutics: Consultancy, Equity Ownership; Genzyme: Consultancy.