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. 2011 Dec;29(12):2030-41.
doi: 10.1002/stem.759.

CD34 Promotes Satellite Cell Motility and Entry Into Proliferation to Facilitate Efficient Skeletal Muscle Regeneration

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Free PMC article

CD34 Promotes Satellite Cell Motility and Entry Into Proliferation to Facilitate Efficient Skeletal Muscle Regeneration

Leslie Ann So Alfaro et al. Stem Cells. .
Free PMC article

Abstract

Expression of the cell surface sialomucin CD34 is common to many adult stem cell types, including muscle satellite cells. However, no clear stem cell or regeneration-related phenotype has ever been reported in mice lacking CD34, and its function on these cells remains poorly understood. Here, we assess the functional role of CD34 on satellite cell-mediated muscle regeneration. We show that Cd34(-/-) mice, which have no obvious developmental phenotype, display a defect in muscle regeneration when challenged with either acute or chronic muscle injury. This regenerative defect is caused by impaired entry into proliferation and delayed myogenic progression. Consistent with the reported antiadhesive function of CD34, knockout satellite cells also show decreased motility along their host myofiber. Altogether, our results identify a role for CD34 in the poorly understood early steps of satellite cell activation and provide the first evidence that beyond being a stem cell marker, CD34 may play an important function in modulating stem cell activity.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors declare no conflict of interest, financial or otherwise.

Figures

Figure 1
Figure 1
Impaired skeletal muscle regeneration in Cd34−/− mice. (A–J) H&E staining of WT and Cd34−/− muscles following acute NTX damage. TA muscles were analyzed at days 0 (A, F), 5 (B, G), 10 (C, H), 14 (D, I), and 21 (E, J) post-NTX damage (n=5–6 mice). Scale bar=100 μm. (K) Quantification of necrotic and regenerating areas of damage 5 days after NTX. Error bars represent ± SEM for n=4–6 mice. Scale bar=200 μm. (L) H&E staining of muscle sections showing centrally nucleated myofibers 21 days after NTX damage. Scale bar=50 μm. (M) Myofiber CSA measurements were performed on undamaged (day 0) and damaged (day 21) myofibers. Error bars represent ± SEM for n=5–6 animals with >200 fibers per animal. (N–S) H&E staining of mdx and mdx/Cd34−/− muscle sections at 1 (N, Q), 6 (O, R), and 18 (P, S) months of age. Scale bar=100 μm. (T) CSA measurements performed on regenerating myofibers of mdx and mdx/Cd34−/− muscles at 1, 6, and 18 months of age. Error bars represent ± SEM for n=3–5 mice with > 200 fibers per animal.
Figure 2
Figure 2
CD34 is expressed on myogenic cells and this expression is dynamically regulated during in vivo and in vitro myogenesis. (A) CD34 and CD34+ fractions were isolated from Myf5LacZ animals and stained for LacZ to assess β–galactosidase activity. Quantification of LacZ+ cells was performed. Error bars represent ± SEM for n=3 mice. (B) Representative image of LacZ-stained CD34 and CD34+ MPCs is shown (LacZ+, blue). Scale bar=16 μm. (C) Representative FACS plots showing regulated CD34 expression on MPCs following NTX damage (n=3–5 mice per time-point). An isotype antibody control was used to verify specificity. (D) qRT-PCR analysis of total CD34 expression in purified MPCs after NTX damage. Error bars represent ± SEM for n=3 independent experiments with 10–20 mice per timepoint. (E) End-point RT-PCR analysis of CD34FL and CD34CT isoform expression from purified MPCs. (F–Q) Analysis of CD34 localization (G, J, M, P) in Pax7+ cells (F, I, L, O) on WT fibers at 0, 12, 24, and 42 hours post-culture (Pax7, red; CD34, green; Hoechst, blue). Scale bar=20 μm.
Figure 3
Figure 3
In vitro and in vivo functional assessment of WT and Cd34−/− MPCs. (A) Representative images showing WT and Cd34−/− differentiated MPCs forming multinucleated myotubes (MyHC, green; Hoechst, blue). Scale bar=65 μm. (B) Fusion index (percent of total nuclei found in myotubes) for WT and Cd34−/− myotubes. Error bars represent ± SEM for n=3 mice with 15 random fields of view per animal. (C) Schematic of the transplant experiment. (D) Direct enumeration and comparison of WT LacZ+ donor-derived myofibers used as internal standards in transplantation experiments. Bar graphs displaying the relative amount of LacZ+ fibers injected with WT/GFP+ or Cd34−/−/GFP+ MPCs. Error bars represent ± SEM for n=3 mice. (E) Representative image showing engraftment of WT/GFP+ and Cd34−/−/GFP+ MPCs 3 weeks following injection into non-damaged WT recipients (GFP, green; Laminin, red). Scale bar=95 μm. (F) Quantification of engraftment. GFP+ donor-derived myofibers were counted and normalized to the number of LacZ+ donor fibers. Ratios were then normalized to WT controls. Error bars represent ± SEM for n=3–5. (G) MPCs from WT and Cd34−/− mice on a Myf5LacZ background were sorted, cytospun, and stained for LacZ to assess β-galactosidase activity. Representative images displaying LacZ+ cells in both groups are shown. Scale bar=50 μm. (H) Frequency of LacZ+ cells in WT and Cd34−/− sorted MPCs. Error bars represent ± SEM for n=3 mice.
Figure 4
Figure 4
Inefficient in vivo proliferation of MPCs lacking CD34. (A) Representative FACS plots showing detection of a distinct BrdU+ MPC population at 0, 1, 2, 3, 5, and 7 days following NTX damage in WT and Cd34−/− animals. (B) Graph showing the frequency of BrdU+ WT and Cd34−/− MPCs at 0, 1, 2, 3, 5, and 7 days following NTX damage. Error bars represent ± SEM for n=3–5 mice per time-point. (C) Frequency of BrdU+ WT and Cd34−/− FAPs at 0, 1, 2, 3, 5, 7 days post-NTX damage. Error bars represent ± SEM for n=3–5 mice per time-point.
Figure 5
Figure 5
Defective activation of Cd34−/− satellite cells. (A) Immunofluorescent detection and enumeration of satellite cells on WT and Cd34−/− single fibers (Pax7, red; Hoechst, blue). Scale bar=50 μm. Error bars represent ± standard deviation for n=5–7 animals. (B) Combined Pax7 and MyoD staining on single fibers identify two subpopulations of myogenic progenitors: Pax7+ MyoD+ as activated satellite cells and Pax7 MyoD+ as differentiation-committed myoblasts (Pax7, red; MyoD, green; Hoechst, blue). Scale bar=50 μm. (C) Quantification of activated satellite cells and differentiation committed myoblasts on WT and Cd34−/− cultured fibers. Error bars represent ± standard deviation for n=3–7 animals per time-point. (D) Total myogenic cells counted per fiber. Error bars represent ± SEM for n=3–7 animals per time-point. (E) Number of satellite cell divisions detected using time-lapse microscopy between 24 and 48 hours after fiber culture initiation. Error bars represent ± SEM for n=42–107 satellite cells. (F) Timing of first division of individual satellite cell. Line represents the mean values ± SEM for n=19–22 satellite cells in 5–7 mice.
Figure 6
Figure 6
CD34 is necessary for efficient satellite cell motility. (A) Representative images of WT and Cd34−/− satellite cell tracking on single fibers based on time-lapse microscopy imaging. Each color represents a different cell tracked. Scale bar=50 μm. (B) WT and Cd34−/− satellite cell velocities as determined using time-lapse microscopy. Error bars represent ± SEM for n=42–107 satellite cells. (C) Total distances traveled for WT and Cd34−/− satellite cell were determined. Error bars represent ± SEM for n=42–107 satellite cells. (D) Measurement of frame-by-frame instantaneous velocities for WT and Cd34−/− cells.

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