Replenishment of Damaged Bone Marrow Niches By Circulating Hematopoietic Stem Cells

Hematopoietic Stem and Progenitor Cells (HSPC) support life-long production of blood cells through multipotent differentiation and self-renewal. Adult HSPC reside predominantly in the bone marrow (BM) but low levels of HSPC can be found in the bloodstream (Wright et al, Science 2001). The spontaneous egress of HSPC from the BM into the circulation follow circadian cycles which are in anti-phase with the chemokine Cxcl12 produced by the niche microenvironment (Mendez-Ferrer et al, Nature 2008). This phenomenon is regulated exogenously by the molecular clock, which orchestrates the circadian release of noradrenaline by the sympathetic nervous system. Medullary HSPC are crucial for haematopoiesis under normal and stress conditions; in contrast, the physiological function of circulating HSPC remains largely unknown. Here we aimed at exploring the role of circulating HSPC.

To study the role of circulating HSC under stress conditions we used a mouse model in which genotoxic damage was specifically applied to the lower limbs (femur and tibiae) without affecting other parts of the body. Affected bones were collected at different times after irradiation and the number of LSK (Lin^NEG Sca1⁺ c-Kit⁺) and myeloid progenitors (Lin^NEG Sca1^NEG c-Kit⁺) were determined by flow cytometry. We found that LSK numbers were restored in the irradiated marrows from two weeks after irradiation, indicating that endogenous circulating HSPC can home into and re-populate genotoxic-damaged niches. To further confirm this observation we performed parabiotic assays in which we surgically conjoined unperturbed or sub-lethally irradiated mice together with non-irradiated wild type (WT) GFP reporter mice. Parabionts were surgically separated after 3 weeks of shared circulation and irradiated mice were analysed for partner-derived HSC repopulating activity. Our results indicate that circulating HSPC efficiently travel through the circulation of the parabiotic partner, home to the BM and repopulate damaged niches.

We next examined the contribution of circulating HSC in the absence of exogenous stress. This, however, represented a challenge as no models have been reported to display basal deficiency of circulating HSPC in blood. While analysing mice with genetic deficiency in CXCR2, a chemokine receptor needed for the egress of neutrophils from the BM, we noticed that mice haploinsuficient for CXCR2 (CXCR2^HET) displayed a 6-fold reduction in the number of circulating HSPC compared with WT mice. Additional experiments demonstrated that this chemokine receptor was expressed at low levels on HSPC as determined by flow cytometry and quantitative PCR, and was functional as determined by in vitro migration assays. CXCR2 was also expressed in wild-type HSPC with long-term reconstituting capacity (LT-HSC), as BM cells that migrated to the CXCR2 ligand CXCL1 had LT reconstituting capacity following BM transplantation. Remarkably, CXCR2^HET resulted in undetectable levels of the receptor on HSPC and impaired in vitro and in vivo migration, which suggested that HSC with LT-repopulating activity need two copies of CXCR2 for homeostatic egress from the BM. These observations also provided a model to study the functional contribution of blood HSC during homeostasis. We therefore conjoined WT or CXCR2^HET with S cid mice by parabiosis. Scid mice display an intrinsic HSPC defect that becomes evident in the presence of normal, competing HSC (Qing et al, Blood 2012). After 1 month of shared circulation, the parabionts were surgically separated and partner-derived LT repopulating activity was measured in the S cid partners. While exogenously injected marrow cells from WT or CXCR2^HET mice were able to home into and repopulate the BM of the Scid mice, only WT partners were capable to efficiently outcompete Scid HSPC.

Our results thus indicate that an important function of endogenously circulating HSPC is to repopulate niches that either become vacant after a stress or are populated by intrinsically defective HSPC, and that this process relies on CXCR2-mediated homeostatic egress. These studies pave the way to understanding the biology of circulating HSC and to extend their application in haematological deficiencies, leukaemia or bone marrow regeneration.