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First published online 6 October 2004
doi: 10.1242/dev.01395

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Muscle reconstitution by muscle satellite cell descendants with stem cell-like properties

¹ Tissue Stem Cell Research Team, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
² Fine Structure Analysis Section, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
³ Kitasato University, School of Science, Department of Biosciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan

View larger version (81K): [in a new window]   Fig. 1. Two distinct subpopulations appear in satellite cell-derived clones. (A-D) Single myofibers were isolated from EDL (A,B) or soleus (C,D) muscle and cultured for 4 (A,C) or 6 days (B,D). Two distinct subpopulations, including cells designated round cells (white arrowheads) with a rounded shape and more flattened cells designated thick cells (black arrowheads), appeared in either EDL- or soleus-fiber culture. Images were obtained by phase-contrast microscopy. Scale bars: 50 µm. (E) The ability to undergo myogenic differentiation of satellite cell clones derived from EDL (white column) and soleus muscles (black column). Single myofiber cultures were fixed on day 4 or 8, and then subjected to immunofluorescence for MHC. Expression of MHC was determined in 45-80 colonies. (F) Ultra-microscopic view of round cells. Round cells derived from gastrocnemius muscle were observed under a scanning electron microscope. The arrow indicates a filopodium. Scale bar: 10 µm.

View larger version (144K): [in a new window]   Fig. 2. Round cells are converted to thick cells prior to myogenic terminal differentiation. The sequence of events that occur during myogenic terminal differentiation of satellite cell-descendants was observed by phase-contrast, time-lapse microscopy. (A-L) The images were taken at the indicated time points. Arrowheads indicate the position near the nucleus of the same cell in A through L. Scale bar: 10 µm.

View larger version (102K): [in a new window]   Fig. 3. Thick cells, but not round cells, express myogenin. Myofibers obtained from gastrocnemius muscle were cultured in pmGM for 7 days. Then, satellite cell-derived colonies were subjected to immunostaining with anti-myogenin antibody. (A,B) A fraction of thick cells (arrowheads) around a myotube (asterisk) expressed myogenin (green in B). Scale bar: 50 µm. (C,D) One of the thick cells (arrowhead), but none of the round cells (within the square), expressed myogenin (D). Scale bar: 20 µm. Nuclei were visualized with DAPI (blue in B and D). (A,C) Phase contrast microscopy images of the same fields as those shown in B and D.

View larger version (45K): [in a new window]   Fig. 4. A distinct subpopulation responds to BMP2 and undergoes osteogenic differentiation. (A,B) Satellite cell-derived colonies obtained in EDL-fiber culture were exposed to BMP2 on days 2-4 (A) or days 7-9 (B). They were then subjected to staining for ALP. Scale bar: 20 µm. (C,D) Analysis of the ability to respond to BMP2 by satellite cell-descendants derived from EDL- (C) or soleus- (D) fiber cultures. Satellite cell-derived colonies were exposed to BMP2 on days 2-4 (BMP d2-4) or days 7-9 (BMP d7-9), or were cultured for the same periods in the absence of BMP2. Light blue indicates colonies that contain neither thick cells nor ALP-positive cells (ALP–; Thick–). Yellow indicates colonies that contain thick cells but no ALP-positive cells (ALP–; Thick cell+). Dark red indicates colonies that contain ALP-positive cells but no thick cells (ALP+; Thick cell–). Dark blue indicates colonies that contain both ALP-positive cells and thick cells (ALP+; Thick cell+). The numbers of colonies examined were 52-109 in control cultures without exposure to BMP2 and 107-199 in cultures exposed to BMP2.

View larger version (47K): [in a new window]   Fig. 5. Basic FGF and LIF synergistically suppress the expression of MHC. (A-D) Single myofibers isolated from gastrocnemius muscle were cultured in pmGM (A), or in pmGM supplemented with bFGF alone (B), LIF alone (C), or bFGF plus LIF (D), for 6 days. Cells were then subjected to immunostaining for MHC (red). Nuclei were visualized by DAPI. Scale bars: 100 µm. (E) Quantitative analysis of inhibition of MHC expression by growth factors. Percentages of nuclei in MHC-expressing cells to total nuclei (bars) and total numbers of nuclei in each field (0.55 mm2) (open circles) were calculated in four independent samples. Averages and standard errors are shown.

View larger version (151K): [in a new window]   Fig. 6. Basic FGF and LIF synergistically enhance the clonal growth of round cells. Myogenic cell clones derived from satellite cells in gastrocnemius muscle were cultured in pmGM alone (B,C), or in pmGM supplemented with bFGF plus LIF (A,D), for 7 days. Images were obtained by phase-contrast microscopy. Scale bars: 1 mm in A,B, 100 µm in C,D.

View larger version (79K): [in a new window]   Fig. 7. Round cells enhance muscle regeneration more efficiently than thick cells. Five thousand round cells (A,B) or thick cells (C), or 1,000,000 thick cells (D) derived from gastrocnemius muscles of GFP-transgenic mice were transplanted into gastrocnemius muscles of congenic C57Bl/6 mice pre-treated with CTX. The muscles were removed and cryosectioned 28 days after transplantation. Images in A-D were obtained by epifluorescence microscopy with a GFP filter. Scale bars: 100 µm in A,C,D; 50 µm in B.

View larger version (26K): [in a new window]   Fig. 8. Round cells retain the capacity to restore dystrophin in myofibers of mdx nude mice. Five thousand round cells derived from GFP-transgenic mice were transplanted into gastrocnemius muscles of mdx nude mice pre-treated with CTX. The muscles were removed 28 days after transplantation and subjected to immunofluorescence analysis with antibodies to GFP (B) and dystrophin (C). Immunofluorescence analysis with antibodies to dystrophin revealed the absence of revertant myofibers in muscles that were treated with CTX alone (A). Scale bars: 20 µm.

View larger version (46K): [in a new window]   Fig. 9. Round cells divide slowly and generate new round cells. (A) The behavior of round cells on day 6, 7 or 8 of myofiber cultures (black symbols), and thick cells at early passages (white symbols), was recorded by time-lapse microscopy. Cell numbers in the same fields were counted every 6 hours. Three independent cultures of round cells or thick cells were analyzed. (B) A round cell on day 6 of the fiber culture (arrowhead) generated two daughter cells (asterisks) displaying a rounded shape. The images were taken at the indicated time points. Scale bar: 10 µm.

View larger version (90K): [in a new window]   Fig. 10. Round cells express Pax7 at high levels. Myofibers from EDL muscle were cultured in pmGM for 6 days. (A,B) Round cells expressed Pax7 at high levels. (C,D) Undifferentiated thick cells expressed Pax7 at a reduced level. (E,F) Differentiating thick cells (asterisks) expressed myogenin (green in F) but not Pax7 (red in F). A round cell (arrow) expressed Pax7 but not myogenin. (A,C,E) Phase contrast microscopy images of the same fields as those shown in B, D and F. Scale bars: 20 µm.

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