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First published online 8 April 2008
doi: 10.1242/jcs.021675

Journal of Cell Science 121, 1426-1434 (2008)
Published by The Company of Biologists 2008
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Bone marrow side population cells are enriched for progenitors capable of myogenic differentiation

Eric S. Luth1,*, Susan J. Jun2, McKenzie K. Wessen1, Kalliopi Liadaki1,3,{ddagger}, Emanuela Gussoni1 and Louis M. Kunkel1,3,§

1 Program in Genomics, Division of Genetics, Children's Hospital Boston, Boston, MA 02115, USA
2 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA
3 Howard Hughes Medical Institute, Children's Hospital Boston, Boston, MA 02115, USA


Figure 1
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  		Fig. 1. Experimental set-up and methods of analysis. ROSA26 mice were lethally irradiated and transplanted with either GFP+ WBM or bone marrow SP cells. Seven weeks after transplantation, recipient muscles were damaged with intramuscular cardiotoxin injections. Five and 14 days later, bone marrow and cardiotoxin-damaged muscles were harvested for various analyses (shaded boxes). Recipient bone marrow was analyzed for lineage reconstitution by transplanted cells via FACS. FACS was also used to analyze mononuclear muscle cells for donor cell contribution and for the markers CD45 and Sca-1. Cells from this muscle analysis were sorted based on GFP and CD45 expression, and transferred to slides via cytospin where they were stained for myogenic markers Desmin, MyoD and Pax7. Whole TA muscles were also frozen, sectioned for immunohistochemistry, stained for β-galactosidase and assayed for engraftment of donor-derived cells. Overall, at least seven rounds of irradiation/transplantation were carried out with at least three mice.

 

Figure 2
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  		Fig. 2. WBM and SP transplantations result in equivalent hematopoietic lineage reconstitution. Markers for B cell (B220), T lymphocyte (CD3), mature granulocyte (Gr-1), monocyte/macrophage (Mac-1) and erythroid (Ter-119) lineages were tested using a mouse lineage panel. For each marker, the percentage of lineage-positive cells as determined by FACS in WBM-transplanted mice was normalized to 1, and the corresponding percentage in bone marrow SP transplanted mice was adjusted accordingly. Transplantation of WBM and SP cells resulted in equivalent reconstitution of these lineages as no significant differences in the percent of lineage-positive cells for a given marker were seen between the two transplant types. A t-test was used for statistical analysis and error bars represent the s.d. from three independent experiments.

 

Figure 3
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  		Fig. 3. Regenerating muscles of SP-transplanted mice contain a population of GFP+/CD45–/Sca-1+ cells. (A) Analysis of muscle mononuclear cells 5 days post-CTX of both transplant types revealed a large population of infiltrating immune cells (upper right quadrant) as well as a smaller population of muscle-resident CD45+ cells (upper left quadrant). SP-transplanted mice also contained a population of donor-derived cells that have lost CD45 (lower right quadrant). (B) 14 days after CTX damage, the percentages of GFP+/CD45– and GFP–/CD45+ populations (in both transplant types), as well as GFP+CD45– cells from the SP-transplanted mice, were reduced. In WBM transplants, the percentage of GFP+/CD45– cells was stable. (C) Comparing the abundance of this population of donor-derived CD45– cells across the two transplant types, SP-transplanted mice contained significantly more GFP+/CD45– cells than did their WBM-transplanted counterparts 5 days after damage (P=0.1, Wilcoxon ranksum test, n=4). We believe that these donor-derived cells are not an inflammatory population because the cells do not express the hematopoietic marker CD45. Analysis carried out 14 days post-CTX did not show any significant difference between the two transplant types. (D) GFP+/CD45– cells isolated from SP-transplanted mice sacrificed 5 days post-CTX were analyzed for Sca-1 expression and were 97.3% positive for this marker.

 

Figure 4
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  		Fig. 4. GFP+/CD45– cells from regenerating muscle appear myogenic in vitro. (A,B) Following FACS sorting, GFP+/CD45– cells from SP-transplanted mice analyzed 5 days after CTX damage were cultured alone in proliferation medium. (A) Observation by phase contrast microscopy showed that these cells displayed a myogenic morphology (arrows), and (B) visualization by fluorescence microscopy revealed that cells with this morphology were, indeed, donor-derived, GFP+ (arrows). (C,D) Expression of myogenic markers by isolated GFP+/CD45– cells from multiple time points co-cultured in 1:1 ratio with C2C12 myoblasts. (C) MyoD staining (red) performed on GFP+/CD45– cells from a WBM-transplanted mouse 5 days post-CTX injury revealed that these cells can express this muscle marker when cultured in a myogenic environment (arrows). (D) Desmin staining (red) showed that even 7 weeks after CTX injection, the regenerated muscle still contains mononuclear cells capable of adopting myogenic markers. DAPI stained in blue.

 

Figure 5
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  		Fig. 5. Bone marrow-derived cells can form de novo myofibers. (A) Examples of GFP+ myofibers in WBM- and SP-transplanted mice at 5 and 14 days following CTX are shown. Three different myofiber types were visible in each condition: `red' GFP–/β-Gal+ (asterisk), `yellow' GFP+/β-Gal+ (arrowhead) and `green' GFP+/β-Gal– (arrow). (B) Serial sectioning was used to track certain `green' fibers throughout the length of the muscle. This particular fiber (found in an SP-transplanted mouse 5 days post-CTX) stayed `green' for its entire length (>290 µm) and contained at least four distinct nuclei. As the bottom panel illustrates, in the following section (slide 14), the surrounding `red' fibers were observed, but the `green' fiber in question was gone (arrow). The last section in which this `green' fiber was visible (slide 13) showed many GFP+ mononuclear cells in the immediate vicinity of the `green' fiber (arrowhead). (A) Scale bars: 65 µm in the first and second rows, 20 µm in the third row and 31 µm in the fourth row. (B) Scale bars, 20 µm.

 

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