Current Biology of Stem Cell Homing and Mobilization: Dynamic Interactions Between Hematopoietic Stem and Progenitor Cells and Their Surrounding Bone Marrow Microenvironment

Introduction

Hallmarks of hematopoietic stem and progenitor cell (HSPC) function include their dynamic metabolism, active bi-directional migration (bone marrow [BM] homing, egress, recruitment, and mobilization to the blood), durable multilineage BM and blood repopulation potential, self-renewal and chemotherapy resistance. Hematopoietic stem cells (HSCs) mainly reside in the BM, and their chemotherapy resistance requires their quiescence, adhesion, and metabolic interactions with bone-forming stromal cells. However, HSPCs are also actively released daily to the circulation at low levels as part of steady-state homeostasis and blood replenishment. This minute egress is profoundly accelerated via metabolic elevations of reactive oxygen species (ROS) levels leading to enhanced HSPC development, mobilization, and recruitment to the circulation during stress-induced hematopoiesis (e.g., blood loss and infections) as part of host defense and repair mechanisms. BM reconstitution requiring HSPC migration in the opposite direction, from the circulation across the blood-BM endothelial barrier into the BM and their lodgment in their stromal niches, also requires metabolically active directional navigation, a process termed homing. The ability of intravenously transplanted HSPCs to actively home to the BM is the first and essential step for successful engraftment and durable repopulation, following clinical BM transplantation. In recent years, several key players and mechanisms controlling HSPC function and dynamic regulation of the BM microenvironment, including the central role of the nervous, immune, coagulation, purinergic, and complement systems, have been revealed. In functional preclinical experimental mice models, during steady-state daily-light onset induces transient elevations of BM norepinephrine (NE) and tumor necrosis factor (TNF), which further lead to a transient rise in BM HSPC ROS levels. These dynamic changes metabolically program BM HSPC proliferation, differentiation, migration, and egress to replenish the blood with new mature cells, including the active daily release of immature HSPCs. Darkness onset induces lower transient elevations of BM NE and TNF, leading to a lower burst of BM HSPC ROS levels. These transient elevations at night activate local BM production and secretion of the darkness hormone melatonin, which together with other factors metabolically reprogram BM HSPC self-renewal and retention accompanied by increased short and long-term repopulation potential.

Stress signals caused by bleeding, injury, as well as by viral and bacterial infections induce massive proliferation and differentiation of BM HSPCs, accompanied by accelerated progenitor and mature cell recruitment to the circulation as part of host defense and repair. This process is mimicked by clinical mobilization protocols, which transiently expand the circulating HSPC pool, allowing the efficient harvest of stem and progenitor cells for clinical BM transplantation. Importantly, clinical mobilization of patients for autologous transplantation is often associated with poor yields of circulating HSPC due to the advanced disease state, heavy toxicity of clinical pretreatments, and old patient age. Both HSPC homing and mobilization involve metabolically active multi-step interactions with the BM microenvironment, including the adhesion machinery, proteolytic enzymes, cytokines, and chemokines. A dominant mechanism involves the interactions between the key BM stromal chemokine CXCL12 (also termed SDF-1) and its major receptor CXCR4, functionally expressed also by HSPCs. These interactions regulate HSPC motility (both homing and mobilization), proliferation, adhesion, BM retention, quiescence, and chemotherapy resistance. In addition to CXCL12, BM stromal and endothelial cells also functionally express CXCR4, and many mobilizing agents induce BM CXCL12 secretion to the blood, creating a gradient leading to HSPC mobilization. For clinical requirements, HSPC mobilization is induced by a variety of agents. Among them are repeated stimulation with the myeloid cytokine granulocyte colony-stimulating factor (G-CSF), the CXCR4 antagonist AMD3100 (also termed Plerixafor), and DNA damaging chemotherapy drugs such as cyclophosphamide and total-body radiation, which all induce BM CXCL12 secretion. HSPC mobilization involves in addition to the CXCL12/CXCR4 axis among others, also the stem cell factor (SCF)/c-Kit axis, the pro-inflammatory factors NE, TNF, sphingosine 1 phosphate (S1P), hepatocyte growth factor (HGF), and their receptors, complement, thrombin, and proteinase activated receptor one (PAR1).

This chapter discusses recent findings concerning the biology of HSPC homing and release during steady-state and their mobilization and recruitment during stress-induced conditions. This chapter emphasizes major signals controlling HSPC homing, BM retention, chemotherapy resistance, and clinical mobilization with a major focus on the CXCL12/CXCR4 axis.

Lessons from Steady-State Homeostatic Regulation of Hematopoietic Stem and Progenitor Cells

Daily Light- and Darkness-Onset Induced Signals Maintain Blood Replenishment Together With Self-Renewal of Long-Term Repopulating Hematopoietic Stem Cells

BM retained HSPCs daily give rise to the entire yet diverse repertoire of mature blood and immune cells while maintaining the BM reservoir of undifferentiated progenitor cell pool including long-term repopulating stem cells (LT-HSCs) throughout adult life. How these contrasting tasks, requiring induction of proliferation, differentiation, and migration versus quiescence, self-renewal, and BM retention are regulated and controlled is not fully understood. Recent studies revealed that daily light and darkness onset and circadian rhythms metabolically regulate mouse HSPC migration and development as well as their BM retention and self-renewal. One hour post light onset a transient increase in the level of ROS within BM retained HSPCs was observed. This increase is driven by and requires a transient increase in BM NE and TNF levels, induced by the onset of light. These modulations in the surrounding BM microenvironment including reduced levels of CXCL12 and higher surface expression of CXCR4 by HSPCs metabolically regulate HSPC differentiation and migration, accompanied by increased permeability of BM-blood vessels. Consequently, immature and maturing leukocytes are released from the BM to the blood, peaking five hours post light initiation, to replenish the blood. In contrast, one hour following the onset of darkness, similar triggering processes are observed, although weaker signals are generated: lower transient increases in ROS levels within BM retained HSPCs are following lower increases in BM NE and TNF. These darkness-onset mediated transient increases, metabolically inducing local synthesis and secretion of the dark hormone melatonin in the mouse BM. Melatonin together with other cytokines such as PGE2, directly and indirectly, promotes retention of HSPCs in the BM. Directly, ROS levels within HSPCs and primitive LT-HSCs are further reduced following melatonin and PGE2 stimulation. Indirectly, melatonin and PGE2 also reduce the permeability of mouse BM endothelial vessels via nocturnal S1P augmentation-mediated COX2 elevation. Acting together, retention of BM HSPCs is upregulated, and these primitive cells, when harvested for transplantation at darkness during the night, are endowed now with higher homing and LT-HSC repopulation potential. Taken together, these results reveal how light onset metabolically regulates HSPC differentiation and migration to replenish the blood with new mature blood and immune cells with a finite life span. Furthermore, the onset of darkness metabolically reprograms HSPCs, facilitating their BM retention, maintenance, and self-renewal via daily BM melatonin and PGE2 bursts.

Mechanisms of Long-term Repopulating Stem Cell Chemotherapy Resistance via Anchorage to Their Supportive Bone Marrow Niches: The Dual Roles of the Coagulation-Related, Proteinase-Activated Receptor One Axis

The major coagulation receptor, PAR1, is well known in the context of coagulation and inflammation. PAR1 has two different signaling cascades leading to opposite outcomes. One cascade leading to pro-inflammatory and pro-coagulant activity is induced by thrombin-mediated PAR1 signaling. The other cascade, leading to anti-inflammatory and anti-coagulation, is initiated by interactions of activated protein C (aPC) with endothelial protein C receptor (EPCR) and PAR1. Recent studies reported that PAR1 is functionally expressed not only in the blood involving coagulation processes but also within the BM, a site with no coagulation. Hence, PAR1 is expressed by BM stromal, endothelial, and hematopoietic cells. EPCR as well is functionally expressed by BM endothelial cells and also by some other hematopoietic cells, including the most primitive human and mouse LT-HSCs that are endowed with the highest multilineage long-term repopulating potential in transplanted mice. Recent results revealed that PAR1 signaling in HSPCs determines their location, either targeting their retention in the BM (including LT-HSCs) or their egress and mobilization, independently of coagulation. In mice, thrombin-induced PAR1 mediated secretion of CXCL12 by BM stromal cells and increased the permeability of BM endothelial vessels. In parallel, thrombin/PAR1 signaling elevated generation of the signaling free radical nitric oxide (NO) in HSPCs, including in primitive EPCR ⁺ LT-HSCs. Other effects promoted by the NO elevation in HSPCs were: shedding of EPCR, increased expression of surface PAR1 and CXCR4, higher activity of the cell-cycle and motility regulator Cdc42, enhanced CXCL12/CXCR4-directed migration, which altogether led to rapid HSPC mobilization. Interestingly, despite the shedding of EPCR, primitive LT-HSCs still contain intracellular storage of EPCR that, upon their transplantation and homing to the BM, is re-expressed on the surface and is required for long-term BM repopulation. In contrast, aPC/EPCR/PAR1 signaling in primitive BM retained EPCR ⁺ LT-HSCs exert the opposite effects, namely, inhibition of NO generation, reduction of Cdc42 activity, and increase of VLA4-mediated adhesion, which all together boost BM retention. The dual activity of PAR1 is clinically relevant. Activation of this pathway in mouse BM retained EPCR ⁺ LT-HSCs is essential for acquiring chemotherapy resistance. Furthermore, the manipulation of NO levels can be applied to increase HSPC homing or mobilization as well as for increasing their BM retention and chemotherapy resistance.

Finally, expression levels of PAR1 by circulating human HSPCs and mature leukocytes is highly important for the outcome of clinical stem cell transplantation. PAR1 signaling is essential for CXCR4 mediated directional migration of both human and mouse HSPCs toward a gradient of CXCL12 in vitro. Importantly, in clinical settings, the levels of PAR1 expression by mature mononuclear cells in the blood of healthy donors during steady-state correlate with their CXCR4-dependent in vitro migration toward CXCL12. Moreover, upon G-CSF treatment in these healthy donors, the yield of their mobilized CD34 ⁺ HSPCs also correlated with the levels of PAR1 expression by circulating HSPC and mature leukocytes before treatment. Furthermore, higher PAR1 basal expression correlated with faster kinetics of hematological recovery in allogeneic matched transplanted patients.

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