Transcript Supplementary Figure 4 - PowerPoint (2.5 MB )

Figure S4
b
eMHC/Hoechst
c
Aged (WT)
-GFP
GFP
eMHC/Hoechst
Young (GFP)
GFP/eMHC/Hoechst
a
Figure S4 Muscle regeneration in parabionts is mediated by endogenous, resident muscle stem cells,
not by circulating progenitor cells.
a, GFP- mice were parabiosed to GFP+ partners for 5 weeks. Muscles of each partner were injured,
allowed to heal for 5 days, and then dissected and cryosectioned. Embryonic myosin heavy chain
(eMHC) immunostaining (red) indicates regenerating myofibers. Hoechst dye (blue) labels all nuclei.
In more than 99% of the fields examined, there was no evidence of GFP expression in regenerating
fibers of the GFP- parabionts. The GFP transgene has been shown not only to be robustly expressed in
mature myofibers of the transgenic strain, but cells isolated from this strain clearly express GFP after
incorporation into a GFP- recipient1-3.
b, In muscles of GFP- mice connected parabiotically to GFP+ mice, occasional GFP+ cells could be
seen in the interstitial spaces between fibers in areas of regeneration (arrows), most likely being
infiltrating leukocytes as has been previously described1,3.
c, Muscles of GFP- mice connected parabiotically to GFP+ mice were also analyzed for GFP
expression using an antibody to GFP (red). Even by this more sensitive detection, there was no
evidence of GFP expression in more than 99% of the fields of regenerating (eMHC+) myotubes
(green) examined in the GFP- parabionts (two separate fields shown). Very rare single GFP+, eMHC+
regenerating fibers were detected. A single such fiber (arrow) is shown in the panels on the right.
1. Wagers,A.J., Sherwood,R.I., Christensen,J.L. & Weissman,I.L. Little evidence for developmental
plasticity of adult hematopoietic stem cells. Science 297, 2256-2259 (2002).
2. Sherwood,R.I. et al. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of
cells within and engrafting skeletal muscle. Cell 119, 543-554 (2004).
3. Sherwood,R.I., Christensen,J.L., Weissman,I.L. & Wagers,A.J. Determinants skeletal muscle
contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem Cells in
press, (2004).