Mesenchymal Stem Cells, Regulated by the Sympathetic Nervous System, Form the Hematopoietic Stem Cell Niche
Authors: Mendez-Ferrer Simon , Enikolopov Grigori N. , Lira Sergio , Frenette Paul S. 
Published: April 2020

Abstract
The identity of mesenchymal stem cells (MSCs) and their relationship to hematopoietic stem cells (HSCs) remain poorly defined. In addition, there are discrepancies regarding the cellular constituents of the HSC niche, with studies suggesting a role for bone-lining osteoblasts, and other data implicating sinusoidal endothelial and adventitial reticular cells. Previous work from our group has demonstrated that the sympathetic nervous system (SNS) is critical for both physiological and enforced egress of HSCs from the bone marrow (BM). HSC mobilization induced by G-CSF requires signals from the SNS (Katayama et al. 2006; Cell124:407–21). Physiological release of HSCs into the bloodstream follows circadian oscillations governed by the molecular clock and triggered by cyclical norepinephrine secretion by the SNS in the BM, activation of the β3-adrenergic receptor (encoded in Adrb3), degradation of Sp1 transcription factor and downregulation of Cxcl12 (Mendez-Ferrer et al. 2008; Nature452:442–7). Here, we have identified the cell targeted by the SNS in the BM as a perivascular stromal cell expressing Nestin, an intermediate filament protein characteristic of neuroectoderm-derived stem cells. Using transgenic mice expressing GFP under the regulatory elements of the Nestin promoter, we show that virtually all catecholaminergic fibers in the BM are associated with Nestin+ cells, which represent 4.0 ± 0.6% of BM CD45− cells and 0.08 ± 0.01% of total BM nucleated cells, as determined by FACS analyses. Quantitative real-time PCR (QPCR) analyses have revealed a ~30-fold higher expression of the gene encoding the chemokine CXCL12 in Nestin+ cells than in the rest of BM CD45− cells, whereas Adrb3 was exclusively expressed in Nestin+ cells and not detectable in Nestin− CD45− cells. Detailed immunofluorescence analyses of the spatial distribution of HSCs in longitudinal BM sections revealed that 60% of CD150+ CD48−/Lineage− cells were directly attached to Nestin+ cells, and 90% of HSCs were located within 5 cell diameters from Nestin+ cells in the endosteal or sinusoidal regions of the BM (n=30). In long-term BM cultures, Nestin+ cells were rare, but located near HSCs/progenitors-enriched cobblestone-forming areas. BM Nestin+ cells were associated with HSCs not only physically but also functionally, because core HSC retention signals (Cxcl12, Kitl, Vcam1, Angpt1, Il7) were highly expressed by Nestin+ cells and significantly downregulated during G-CSF-induced mobilization, whereas the expression of the same genes was significantly lower and was not downregulated by G-CSF in Nestin− CD45− cells, as measured by QPCR. A non-selective β- or a selective β3-adrenergic receptor agonist also downregulated these core HSC retention genes, underscoring the role of the SNS in regulating HSC adhesion in the BM niche. Cell sorting of Nestin+ CD45− and Nestin− CD45− cells revealed that all the mesenchymal progenitor activity of the bone marrow (CFU-F) was contained in the Nestin+ cell fraction. Further, Nestin+ cells could robustly differentiate into osteoblasts and adipocytes. Lineage-tracing studies using a Nestin-CRE transgenic line bred to R26R reporter mice have confirmed the contribution of Nestin+ cells to osteoblasts and chondrocytes during development. G-CSF, which induces proliferation of hematopoietic cells in the BM at the expense of non-hematopoietic lineages, significantly downregulated markers of osteoblastic and adipogenic differentiation in BM Nestin+ CD45− cells but not in Nestin− CD45− cells. By contrast, daily administration of parathyroid hormone over five weeks, a treatment previously shown to expand both the osteoblastic and HSC pools, induced proliferation of Nestin+ cells and favored their differentiation into Col1a1-LacZ+ osteoblasts. Finally, we have found that Nestin+ CD45− cells, but not Nestin− CD45− cells, can form self-renewing spheres in clonal density culture, with a frequency similar to other neural crest-derived stem cells. After two weeks in culture, clonal spheres showed spontaneous multilineage differentiation into adipocytes and Col1a1-LacZ+ osteoblasts. Altogether, these results suggest that the HSC niche is composed of a heterotypic MSC-HSC pairing that is tightly regulated by the SNS. This association may reconcile divergent views regarding the vascular and osteoblastic locations of the HSC niche, and its regulation by the SNS might explain the crosstalk between hematopoietic and mesenchymal lineages in the BM during health and disease.