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First published online 6 September 2005
doi: 10.1242/jcs.02555

Journal of Cell Science 118, 4343-4352 (2005)
Published by The Company of Biologists 2005
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A unique population of bone marrow cells migrates to skeletal muscle via hepatocyte growth factor/c-met axis

Michael Rosu-Myles1,2, Erin Stewart1, Jennifer Trowbridge1,2, Caryn Y. Ito3, Peter Zandstra3 and Mickie Bhatia1,2,*

1 Robarts Research Institute, Krembil Centre for Stem Cell Biology and Regenerative Medicine, 100 Perth Drive, London, Ontario, N6A 5K8, Canada
2 The University of Western Ontario, Department of Microbiology and Immunology, Toronto, Ontario, Canada
3 University of Toronto, Department of Bioengineering, Toronto, Ontario, Canada



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  		Fig. 1. Bone marrow cells fully replace the skeletal-muscle CD45+-fraction following i.v. transplantation. Whole BM from GFP mice was i.v. transplanted into sub-lethally irradiated NOD/SCID mice (10x106 cells per mouse). After 6 to 8 weeks, skeletal muscle was removed from transplanted mice and sibling mice that had not received GFP BM cells. (A) (i) The proportion of skeletal muscle CD45+ cells expressing GFP was determined using flow cytometry (n=12) and gated based on light side-scatter (SSC) properties. (ii) Skeletal muscle cells were also examined for the presence of CFU (n=2). GFP-expressing hematopoietic colonies were detected by fluorescent microscopy. (B) Skeletal muscle from non-transplanted and GFP BM transplanted mice were further examined by flow cytometry for the frequency of CD45+ cells. (C) The proportion of Sca-1+ cells within the CD45+ fraction of skeletal muscle from transplanted and non-transplanted mice and within the BM-derived CD45+ cells of donor GFP mice was also compared (n=8). (D) NOD/SCID mice were (i.v.) transplanted with varying doses of Sca-1+CD45+ GFP+ cells. The proportion of GFP-expressing CD45+ cells in the skeletal muscle and BM compartments were compared using linear regression analysis.

 


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  		Fig. 2. CD45+ cells residing in the bone marrow and skeletal muscle are intrinsically distinct. Whole BM MNCs (white bars) and skeletal muscle MNCs (black bars) were isolated from GFP mice by collagenase digestion, and CD45+ cells were purified by FACS and (A) analyzed for the (i) frequency and (ii) type of CFU progenitors, and (iii) the frequency of B-cell progenitors. The number of experiments completed (n) is given with each graph. (B) CD45+ cells purified from BM and skeletal muscle were cultured for 2, 3, 4 and 5 weeks on confluent monolayers of MS-5 stroma. (i) At each time point the number of CD45+ cells arising from BM-derived ({diamond}) or skeletal muscle-derived ({blacksquare}) co-cultures was determined by flow-cytometry. (ii) The absolute number of Sca-1+CD45+ cells initially seeded at day 0 and following 2 weeks in culture was also examined and the fold-expansion of (iii) Sca-1+CD45+ cells and (iv) CFUs evaluated in 2-week and 5-week cultures (n=5-8). * indicate statistically significant differences compared with skeletal muscle, with P<0.05.

 


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  		Fig. 3. BM-derived CD45+ cells and progenitors are capable of migrating towards mature muscle cells. BM-MNCs (500,000 per well) derived from GFP mice were loaded into the upper chamber of a transwell apparatus that contained a monolayer of MS-5 stromal cells, C2C12-derived myotubes, primary skeletal muscle cells derived from adult CD-1 mice, or random migration-controls consisting of stroma culture media or myogenic culture media alone in the lower chamber as indicated. After 24 hours, cells were harvested from the lower chambers and the number of CD45+ cells and CFU progenitors was determined by flow-cytometry and methylcellulose assays, respectively. Statistically significant differences from random migration controls are indicated by an * (P≤0.05; n=11, or n=3 when primary muscle cells were used). BFU-E, CFU-M, CFU-G, CFU-GM, CFU-GEMM are defined as Burst-forming-unit erythroid, colony-forming-unit macrophage, colony-forming-unit granulocyte, colony-forming-unit granulocyte-macrophage and colony-forming-unit granulocyte-erythroid-macrophage-megakaryocyte, respectively.

 


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  		Fig. 4. Myofiber and stroma-migrating CD45+ cells and CFUs represent distinct populations of bone marrow hematopoietic cells. (A) Schematic diagram of the procedure used for analysis of the 2° migration potential of migrating and non-migrating cells isolated from 1° myofiber migration assays. (B) Twenty-four hours after loading 1° migrating (i) and non-migrating (ii) cells (100,000 to 500,000 cells per well) into 2° migration assays, the number of CD45+ cells and CFU progenitors migrating to confluent layers of C2C12-derived myotubes (red), MS-5 stroma (blue) or culture media alone was determined. * indicate statistically significant differences in the percentage of migrating cells compared with 2° migration-controls containing stromal cell (white) or myofiber (gray) growth media in the lower chambers (P≤0.05).

 


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  		Fig. 5. Migration of BM cells to mature muscle cells is independent of SDF-1 and CXCR4 but requires c-met expression. GFP-expressing BM cells (500,000 per well) were placed in the upper chamber of transwells. (A) Migration of CD45+ cells and CFU towards MS-5 stroma (i) and C2C12 myotubes (ii) was tested in the absence (stromal cells or muscle cells alone) and presence of SDF-1-neutralizing antibody (white and black bars, respectively), and following treatment of BM with CXCR4-blocking antibody (hatched bars). (B,C) Migration of CD45+ cells and progenitors derived from whole BM cells (untreated) and BM cells treated with c-met-blocking antibody (white and black bars, respectively) was examined in response to HGF or bFGF alone (B or C, respectively). Random migration controls consisted of media without HGF or bFGF as indicated (gray bars). (D) The ability of c-met-blocking antibody to inhibit the migration of BM-derived CD45+ cells and CFU was determined by placing untreated and anti-c-met-treated BM-MNCs (white and black bars, respectively) in the upper chamber of transwell assays containing MS-5 stromal cells (i) or C2C12 myotubes (ii) in the lower chambers. Pooled results of five separate experiments are given as the percentage of CD45+ cells or CFUs that had initially been loaded in the upper chamber (input), and which had then migrated to the lower chamber. (E) Analysis of c-met expression on CD45+ cells (gated) engrafting recipient mice transplanted with donor GFP+ BM cells, n=3. Significant differences of migration in the absence of antibody or of the percentage of cells migrating to media alone were determined by using Student's t-test (*P≤0.05).

 

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