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First published online 30 March 2004
doi: 10.1242/jcs.01051

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Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations

François Rivier^1,*,, Ozan Alkan^2,, Alan F. Flint¹, Kristina Muskiewicz¹, Paul D. Allen³, Philippe Leboulch³ and Emanuela Gussoni^1,

¹ Division of Genetics, Children's Hospital Boston, MA 02115, USA
² Massachusetts Institute of Technology, Harvard-MIT Division of Health Sciences and Technology, 45 Carleton Street, Building E25, Room 529, Cambridge, MA 02139, USA
³ Department of Anesthesia, Brigham and Women's Hospital, Boston, MA 02115, USA

View larger version (32K): [in a new window]   Fig. 1. Cell trafficking from the BM to the skeletal muscle 3 weeks after lethal irradiation and BMT. Detection via FACS analysis of donor-derived CD45.2+ cells within the MP and SP cell populations in the BM (A-D) and the skeletal muscle (E-H) of recipient females. BM cells stained with 5 µg ml-1 Hoechst in the presence (A) or absence (B) of verapamil. Over 99% of BM MP cells (C) and SP cells (D) are CD45.2+ or of donor origin. Skeletal muscle mononuclear cells stained with 12.5 µg ml-1 Hoechst dye in the presence (E) or absence (F) of verapamil. Approximately 18% of muscle MP cells (G) are CD45.2+ donor-derived, whereas all muscle SP cells are CD45.2- (H).

View larger version (34K): [in a new window]   Fig. 2. Cell trafficking and detection of donor-derived CD45.2+ cells within the muscle MP and SP cell populations of recipient females 10 weeks (A-D) and 30 weeks (E-H) after lethal irradiation and BMT. Muscle cells were stained with 12.5 µg ml-1 Hoechst dye in the presence (A,E) or absence (B,F) of verapamil. Approximately 17% (C) and 11% (G) of muscle MP cells at 10 and 30 weeks after BMT, respectively, are CD45.2+ donor-derived. By contrast, 1% (D) and 0% (H) of muscle SP cells are CD45.2+ at 10 and 30 weeks after BMT.

View larger version (36K): [in a new window]   Fig. 3. Analysis of host-derived CD45.1+ cells in the BM (A-C) and skeletal muscle (D-F) of MP and SP cell populations 30 weeks after BMT. As expected, very few CD45.1+ host-derived cells are detected in the MP (B) or SP population (C) of the BM after lethal irradiation and BMT, because the BM was replaced by CD45.2+ donor cells. In the skeletal muscle, host-derived CD45.1+ cells constitute 4% (E) of the MP population, whereas no host-derived CD45.1+ cells are detected within the muscle SP population (F).

View larger version (35K): [in a new window]   Fig. 4. Phenotypical differences of muscle SP cells detected using varying Hoechst dye concentrations. Parallel samples stained with 5 µg ml-1 (A) or with 12.5 µg ml-1 (D) of Hoechst dye show different percentages of SP cells. As expected, the total percentage of CD45+ cells within the samples does not vary (B,E). However, when CD45+ cells are analyzed for Hoechst exclusion properties, 26% of CD45+ cells fall within the SP cell gate when the sample is stained with 5 µg ml-1 of Hoechst dye (C), whereas no CD45+ cells are present in the SP gate when the sample is stained with 12.5 µg ml-1 Hoechst dye (F).

View larger version (37K): [in a new window]   Fig. 5. (A) CD45.2-PE+ BM cells purified by FACS and hybridized with the Y-chromosome probe by FISH (red). A red-orange outline at the periphery of the nuclei indicates the CD45.2-PE signal that persisted after FISH. Nuclei are counter-stained with DAPI (blue). (B) CD45.2-PE-negative muscle cells sorted by FACS and hybridized with the Y-probe by FISH. No hybridization signals were observed. (C) FACS analysis of a muscle sample, which was taken 8 weeks after BMT from a mouse injected with e-GFP plus BM. SP (2.2%) and MP (71.4%) cell gates are shown. (D) Percentage of GFP+ BM-derived cells in muscle MP cells and in muscle SP cells (E). (F) More than 98% of muscle SP cells from an e-GFP transgenic mouse are positive for e-GFP.

View larger version (41K): [in a new window]   Fig. 6. Analysis of BM-cell trafficking to skeletal muscle MP and SP cells in the absence (A-C) or in the presence (D-F) of cardiotoxin injury (Mouse B in Table 2). FACS profiles and percentages of SP and MP cells in control (A) or cardiotoxin-injected muscles (D). Percentage of CD45.2+ donor-derived cells in muscle MP cells in the absence (B) or presence (E) of cardiotoxin injury. A near 5-fold increase of donor-derived cells is observed upon cardiotoxin injury in the MP population. Percentage of CD45.2+ donor-derived cells in muscle SP cell population in control (C) or in cardiotoxin-injured (F) muscles.

View larger version (44K): [in a new window]   Fig. 7. Combined immunohistochemistry/FISH analysis on muscle-tissue sections of recipient female mice injected with donor male BM cells. Tissue sections were collected from samples harvested at 11 w (A), 19 w (B) and 34 w (C) after BMT. Samples (D) and (E) are muscles analyzed 18 w after transplantation and 2 w after cardiotoxin injury. (A-E) The green signal represents the expression of CD45.2 (donor-derived cells antigen). Nuclei are stained in blue by DAPI. Red dots within nuclei represent FISH hybridization using the Y-chromosome probe to confirm the donor-cell origin. (F) Immunohistochemistry using anti-CD45.2 (green) and anti-desmin (red) antibodies. A host-derived satellite cells (red) and a donor-derived CD45.2+ BM cell (green) that has migrated to the muscle are shown.

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