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. 2002 May 13;157(4):571-7.
doi: 10.1083/jcb.200112106. Epub 2002 May 6.

Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle

Tetsuro Tamaki  1 , Akira Akatsuka, Kiyoshi Ando, Yoshihiko Nakamura, Hideyuki Matsuzawa, Tomomitsu Hotta, Roland R Roy, V Reggie Edgerton
Affiliations

Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle

Tetsuro Tamaki et al. J Cell Biol. .

Abstract

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.

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Figures

Figure 1.
Figure 1.
Localization of CD34 + cells in the interstitial spaces of skeletal muscles of 3-wk-old mice. Small mononucleated CD34+ cells were observed in the interstitial spaces (surrounding brown reaction, arrows in A–D). The CD34+ cells are located outside of the basal lamina (laminin staining, dark purple). The location, outside of the basal lamina was also confirmed by immunoelectron micrograph (E and inset). The CD34+ cell shows lower nuclear/cytoplasmic ratio representing immature cell. Reaction products of DAB can be seen in surface of CD34+ cell as well as in the lumina of endothelial cell (arrowheads in E). However, note that the satellite cells shows CD34−, whereas CD34+ cell can be seen within the interstitial spaces (F). Inset in F shows higher magnifications of the corresponding square areas on the photograph. Cell membrane of both satellite cell and muscle cell is evident (arrowheads). Immunofluorescent costaining with CD34 (G, Rhodamine-red) and CD45 (J, FITC-green) shows that CD34+ cells are CD45− (G and J, arrows), and they are located in the interstitial spaces (H and I, arrows). Vasculature related CD34+ reactions are also observed in enclosed portions in G and H. Staining in the marrow of the tibia on the same sections with A–C and with G–J are shown in D (CD34, brown) and K (CD34, red and CD45, green) as a positive control. The mean frequencies of CD34+ and CD34− mononuclear cells appearing in a unit area of 3-wk-old mice muscles were 1.9 (±0.3, SE) and 2.9 (±0.4), respectively. The data were obtained from four to six unit areas (175 × 130 μm per unit area) in 20 sections taken from three mice, i.e., corresponding to ∼40% of the total interstitial cells in the unit area. Note that there were no CD34+ cells inside the basal lamina. Green reactions in A–D, nuclear stain with methyl green. Blue staining in G and J, nuclear stain with DAPI. HE, hematoxylin-eosin staining; TV, transmission view of confocal laser scanning microscope; 34+, CD34 positive cell; EC, endothelial cell; Cap, capillary; SC, satellite cell; MN, myonuclear. Bars, (A–D and G–J) 10 μm; (E and F) 1 μm; (K) 25 μm.
Figure 2.
Figure 2.
Isolation and characterization by flow cytometry of EECs and Sk-34 cells from skeletal muscles of the hind limbs of 3-wk-old mice. The EEC was divided into four subpopulations: CD34−/45+, 34+/45+, 34+/45− (Sk-34) and 34−/45−(C). The mean frequencies of each population were 19.8 ± 2.8%, 11.6 ± 1.7%, 49.7 ± 4.1%, and 17.7 ± 4.0% (n = 20 mice), respectively. The frequencies of coexpression of other markers on the Sk-34 cells (n = 5) were: 2.6 ± 1.3% for CD14 (D); 1.7 ± 1.2% for CD31 (E); 93.7 ± 1.3% for Sca-1 (F); 1.0 ± 0.4% for CD49 (G); 2.4 ± 2.0% for c-kit (H); 0.2 ± 0.3% for FLK-1 (I); and 0.9 ± 0.8% for CD144 (J). (A and B) Isotype controls. Values, mean ± S.E.
Figure 3.
Figure 3.
Growth and differentiation of Sk-34 cells in vitro and in vivo. Colonies derived from the Sk-34 cells after 4 (A), 6 (B), 10 (C), and 14 (D) days of culture. Typically, colonies were composed of floating and/or weakly attaching large round cells and adherent spread cells, and the frequency of these mixed colonies was 75 ± 58 out of 1 × 104 cells (n = 10 culture dishes). (E) An adherent cell colony at the bottom of the plate after washing-out the semisolid medium with the weakly attached cell populations. (F) Uptake of DiI-Ac-LDL in the adherent cells populations shown in E (E and F, arrows). (G) MyoD-positive cells in the cytospin of weakly attaching cell populations as shown in B. (H) Fat cells in a colony after 10 d of culture stained with oil red O (arrows). (I and J) Sphere and floating colony in a semisolid medium culture on a soft agar medium. (K) Engrafted Sk-34 cells prepared from GFP transgenic mice forming vascular endothelial cells (brown reactions, arrows) and myofibers (brown reactions, arrowheads) in the muscle of NOD/SCID mice. (L) High magnification of the inset in K. (M) Positive control using muscle tissue section from a GFP transgenic mouse. (N) Negative control stained with the secondary antibody alone. Bars, 10 μm.
Figure 4.
Figure 4.
Expression of the MyoD family gene mRNAs, M-cadherin (M-cad), c-met, Pax-7, and Pax-3 in EEC, Sk-34, and CD34 − /45 − cells (A). Bcrp-1 in CD34−/45−, Sk-34, and CFU-Sk-34 cells (B). N.C., negative control without template. d-0: immediately after sorting or enzymatic extraction; d-3 and d-4: after 3 and 4 d of culture. The RT-PCR product for β-actin in the same samples was used as a control.
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References

    1. Asahara, T., T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li, B. Witzenbichler, G. Schatteman, and J.M. Isner. 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 275:964–967. - PubMed
    1. Asahara, T., T. Takahashi, H. Masuda, C. Kalka, D. Chen, H. Iwaguro, Y. Inai, M. Silver, and J.M. Isner. 1999. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J. 18:3964–3972. - PMC - PubMed
    1. Beauchamp, J.R., L. Heslop, D.S. Yu, S. Tajbakhsh, R.G. Kelly, A. Wernig, M.E. Buckingham, T.A. Partridge, and P.S. Zammit. 2000. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J. Cell Biol. 151:1221–1234. - PMC - PubMed
    1. Birchmeier, C., and E. Gherardi. 1998. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase. Trends Cell Biol. 8:404–410. - PubMed
    1. Bischoff, R. 1986. Proliferation of muscle satellite cells on intact myofibers in culture. Dev. Biol. 115:129–139. - PubMed

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