1. Volume 20, Issue 5, Pages 648-658.e4 (May 2017)
Cholinergic Signals from the CNS Regulate G-CSF-Mediated HSC Mobilization from Bone Marrow via a Glucocorticoid Signaling Relay 
Halley Pierce, Dachuan Zhang, Claire Magnon, Daniel Lucas, John R. Christin, Matthew Huggins, Gary J. Schwartz, Paul S. Frenette 
Cell Stem Cell 
Volume 20, Issue 5, Pages e4 (May 2017)
DOI: /j.stem
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2. Cell Stem Cell 2017 20, 648-658.e4DOI: (10.1016/j.stem.2017.01.002)
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3. Figure 1
G-CSF-Induced HSC Mobilization Requires Signals from the Muscarinic Receptor Type-1, Chrm1
(A) HSCs per milliliter following G-CSF mobilization in wild-type mice treated with saline or Scopolamine hydrobromide (Scop 1 mg/kg; n = 5–8). HSCs are defined as Lineage− Sca1+ cKit+ Flt3−.
(B) Schematic of G-CSF-induced mobilization and analyses. Wild-type mice are denoted by black bars and Chrm1−∕− mice are denoted by white bars.
(C) HSCs per milliliter of peripheral blood are shown (Lineage− Sca1+ cKit+ Flt3−; n = 12–16).
(D) Colony-forming units per milliliter of peripheral blood determined in vitro are shown (CFUs-C; n = 6–13).
(E) Lineage− Sca1+ cKit+ (LSK) cells per femur at steady state are shown (n = 9).
(F) Reconstitution after competitive BM transplantation of mobilized blood into irradiated hosts is shown.
(G) Tri-lineage engraftment of donor cells 16 weeks post-transplantation: B220+ cells (B), CD4/CD8+ cells (T), and Mac1+ cells (M) are shown (n = 6–7).
∗p < 0.05 and ∗∗p < 0.01, determined by Student’s t test. Data are represented as mean ± SEM. See also Figure S1.
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4. Figure 2 Central Cholinergic Signals Are Required for Mobilization
(A) G-CSF-induced mobilization after reciprocal BM transplantation (n = 6–13).
(B) The qRT-PCR for Chrm1 mRNA transcripts in sorted BM cell populations. The mRNA was undetectable in the BM (n = 3–13).
(C) HSCs mobilized to peripheral blood by G-CSF in mice treated peripherally with pirenzepine (PZP) are shown (n = 9).
(D) Experimental design and results of G-CSF-induced HSC mobilization when pirenzepine is administered centrally are shown (n = 7–8).
∗∗p > 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < , determined by Student’s t test. Data are represented as mean ± SEM.
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5. Figure 3
Glucocorticoids Are Necessary for Enforced HSC Mobilization by G-CSF
(A) Schematic of parabiosis experimental design.
(B) HSCs per milliliter in blood of G-CSF-mobilized parabionts are shown (n = 6–8).
(C) Plasma corticosterone levels measured by ELISA are shown (n = 3–5).
(D) Bone marrow extracellular fluid (BMEF) corticosterone levels measured by ELISA are shown (n = 4–5).
(E) HSCs per milliliter in G-CSF-mobilized blood of metyrapone-treated and control mice are shown (n = 10).
(F) HSCs per milliliter in G-CSF-mobilized blood of vehicle-treated (Veh. 1.5% ETOH) or CORT-treated mice are shown (n = 5–7).
(G) Immunofluorescence images of sorted Lin−Sca1+c-Kit+Flt3− LSKF BM HSCs stained with anti-Nr3c1 antibody and DAPI. DAPI-stained nuclear border is denoted by yellow dashed outlines. Scale bar, 10 μm.
(H) Quantification of cytoplasmic staining in HSCs is shown (n = 23–32).
∗p < 0.05 and ∗∗p < 0.01, determined by Student’s t test. Data are represented as mean ± SEM.
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6. Figure 4
Hypothalamic Cholinergic Signals Are Required for Mobilization
(A) In situ hybridization images of Chrm1 expression in wild-type brain. Hybridization probe is directed at Chrm1 mRNA, and it binds to cells localized in the paraventricular nucleus (PVN) and the arcuate nucleus (ARC) of the hypothalamus (i, 10× magnification; ii, 20× magnification; ∗third ventricle). White dashed outline indicates cells contained in the PVN or ARC nuclei.
(B) The qRT-PCR of dissected hypothalamic regions of brains for Chrm1 transcripts is shown (n = 7).
(C) Experimental design for ex vivo analysis of Crh production in wild-type hypothalamus tissue explants and Crh mRNA quantification relative to Gapdh is shown (n = 7).
(D) Hypothalamic denervation is shown by neonatal injection of MSG or control saline followed by the mobilization by G-CSF at adulthood (n = 8–10).
∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, determined by Student’s t test. Data are represented as mean ± SEM.
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7. Figure 5
Altered Expression of Genes Involved in Actin Polymerization and Microtubule Assembly in Chrm1−∕− HSCs
(A) Heatmap shows differentially expressed genes (by >2.0 fold and p < 0.05) between wild-type- and Chrm1−∕−-sorted HSCs.
(B) Gene set enrichment analyses show a significant alteration of gene sets involved in actin and microtubule assembly.
(C) The qRT-PCR from sorted HSCs validating microarray data is shown (n = 4–5).
(D) Transwell assay experimental design is shown.
(E) CFUs-C per well of migrated stem progenitor cells with and without CORT are shown (n = 6).
(F) CFUs-C per well of migrated stem progenitor cells with and without CORT plus RU486 or vehicle (PEG) are shown (n = 8).
∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < , determined by Student’s t test. Data are represented as mean ± SEM.
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8. Figure 6
Nr3c1 Expression on Hematopoietic Cells Is Required for Enforced G-CSF-Induced Migration
(A) Representative images show leukocytes stained for phalloidin (polymerized actin) and CD45.2 (donor cells), obtained from radiation chimeras generated by the transplantation of control (Nr3c1fl/fl) or Nr3c1-deficient (Nr3c1fl/fl; Mx1-cre+) BM into lethally irradiated C57BL/6 mice. Quantification of phalloidin staining normalized to control mean fluorescence intensity (MFI) also is shown (n = 3).
(B) Quantification of phalloidin staining in Chrm1−/− cells and Chrm1−/− cells taken from animals treated with corticosterone normalized to control MFI is shown (n = 3).
(C) Schematic shows the experimental design for G-CSF-induced mobilization.
(D) G-CSF mobilized HSCs in control (Nr3c1fl/fl) and Nr3c1-deficient (Nr3c1fl/fl; Mx1-cre+) mice (n = 6–11).
(E) Model whereby finely tuned GC levels regulate HSC migration. States associated with low GCs (Chrm1−/− mice, metyrapone administration, Nr3c1-deficient HSCs) or high GCs (administration of ACTH, dexamethasone, or corticosterone) lead to reduced mobilization efficiency.
∗p < 0.05 and ∗∗p < 0.01, determined by Student’s t test. Data are represented as mean ± SEM.
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