Spatiotemporal Regulation of Hematopoietic Stem Cell Niches By Dual Cholinergic Signaling

Hematopoietic stem cells (HSCs) are known to be heterogeneous, but it is unclear whether, or how, different bone marrow (BM) microenvironments can imprint distinct HSC states. In the BM, quiescent HSCs have been found enriched in endosteal niches, whereas activated HSCs traffic in and out the BM through sinusoids localized further from bone. However, how these separate niches are integrally regulated to maintain BM homeostasis remains largely unknown. We have shown previously that sympathetic adrenergic nerve terminals innervating nestin+ mesenchymal stem cells (MSCs) regulate HSC egress from BM to circulation (Méndez-Ferrer S et al, Nature 2008 and 2010). This regulatory network is required to control HSC proliferation and migration, since its damage caused by mutated HSCs can lead to the manifestation of diseases such as myeloproliferative neoplasms and its protection can block the progression of these disorders (Arranz L et al, Nature 2014). Here we aimed to study the possible cooperation of both branches of the autonomic nervous system, the parasympathetic and the sympathetic nervous systems, which have antagonistic actions in other systems, in HSC regulation in endosteal and sinusoidal niches. We describe for the first time different cholinergic neural signals derived from both autonomic branches that regulate spatially and temporally distinct BM HSC niches. We used mice deficient in cholinergic nerve fibers (secreting acethylcholine, the main postsynaptic neurotransmitter of the parasympathetic nervous system), due to the lack of the neurturin receptor Gfrα2, a member of the glial-cell-derived family of neurotrophic factors (Rossi J et al. Neuron 1999). We found that Gfrα2^-/- mice exhibited a circadian-specific defect in HSC traffic. Parasympathetic deficiency caused exacerbated sympathetic tone, manifested by ~2-fold increased BM sympathetic adrenergic fibers and nocturnal urine norepinephrine. Circulating HSCs, measured by long-term competitive repopulating assays, were 3-fold higher in Gfrα2^-/- mice only during the resting period, and this was rescued by deletion of the β₃-adrenergic-receptor. Therefore, systemic parasympathetic cholinergic signals antagonize BM sympathetic adrenergic activity during the resting phase, contributing to β₃-adrenergic-receptor-orchestrated circadian HSC traffic through sinusoidal niches. On the other hand, sympathetic cholinergic nerve fibers, described here for the first time in the BM and running along Haversian canals of bone, regulate HSC maintenance and quiescence in endosteal niches. In neonatal mice, we found that some endosteal sympathetic nerve fibers, sensitive to chemical sympathectomy by 6-hydroxydopamine, switch from catecholaminergic to cholinergic fate and help direct developmental HSC migration to BM. This migration, dependent in perinatal life on Cxcl12 produced by BM nestin+ MSCs (Isern et al, eLife 2014), was impaired in Gfrα2^-/- mice, which exhibited one week after birth a reversible ~40% reduction in BM HSCs, associated with the lack of BM sympathetic cholinergic fibers. In adult mice, these sympathetic cholinergic fibers activate nicotinic receptors and induce Cxcl12 expression in bone-associated nestin+ MSCs, an effect reproduced in vitro with the MS-5 stromal cell line. Cxcl12 expression was 2.5-fold higher in Nes-GFP⁺ cells associated with the bone, compared with those that were not, and was 4-fold lower in Gfrα2^-/- Nes-GFP⁺ cells localized only in the endosteal BM. Concomitantly, Gfrα2^-/- mice exhibited reduced allocation of quiescent HSPCs in the endosteal BM, compared with control mice, in which the HSPC fraction in the G0 cell cycle phase was 2-fold higher in the endosteal than in non-endosteal BM. Moreover, HSCs from Gfrα2^-/- mice showed increased proliferation, even 6 months after transplantation into secondary wild-type recipients. As a result, Gfrα2^-/- mice exhibit accelerated hematopoietic recovery after myeloablation, a phenotype mimicked by mice deficient in the α7-nicotinic receptor. Increased HSC proliferation was associated with loss of self-renewal, since bone-associated HSCs from Gfrα2^-/- mice exhibited 50% reduced reconstituting capacity and myeloid potential 24 weeks after competitive transplantation into wild-type recipients. Thus, both branches of the autonomic nervous system regulate HSC maintenance and function in temporally and spatially separate niches.