doi: 10.1242/10.1242/jcs.00110
The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells
Ron J. Jankowski1,2,
Bridget M. Deasy1,2,
Baohong Cao1,
Charley Gates1 and
Johnny Huard1,2,3,*
1 Growth and Development Laboratory, Children's Hospital of Pittsburgh, 4151 Rangos Research Center, Pittsburgh, PA 15213, USA
2 Bioengineering Department, University of Pittsburgh, Pittsburgh, PA 15213, USA
3 Departments of Orthopaedic Surgery and Molecular Genetics and Biochemistry, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA 15260, USA
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Fig. 1. Overview of experimental procedure. The preplate isolation technique was used to obtain two cell populations used for experimentation, found within early adhering preplates (EP) and late adhering preplates (LP). Myogenic (desmin-expressing) cells are enriched with each passage, thus the EP population was re-plated prior to their utilization, in order to further purify the myogenic cells. LP cells were sorted based on either Sca-1 or CD34 expression, in separate experiments, prior to their utilization. Representative dot plots of sorted cell populations, analyzed by flow cytometry, following magnetic antibody cell sorting are shown. Following their isolation, each cell population was injected into mdx skeletal muscle and monitored for their ability to restore dystrophin. They were also examined in terms of various in vitro cell characteristics.
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Fig. 2. Immunofluorescent characterization of MRF protein expression by EP and LP myogenic populations. According to current proposed models of myogenic precursor hierarchy, these patterns of MRF expression indicate that the EP cells are more myogenically committed, and thus closer to terminal differentiation, compared with the LP cells.
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Fig. 3. (A) Efficiency of dystrophin restoration in mdx gastrocnemius muscle by myogenic precursor populations at 7 days following injection. CD34 and Sca-1 sorted LP subpopulations were obtained from separate isolations. A regenerative index, comparing restoration of dystrophin on a per cell basis (desmin-positive cells), was used for a more effective comparison of the various populations. CD34-positive LP cells demonstrated the highest dystrophin restoration (*P<0.05, CD34-positive vs. EP and CD34-negative). Cryosections taken from mdx muscle following transplantation and immunochemically stained to reveal dystrophin expression following injection of EP (B; 220,000 myogenic cells) and CD34-negative and - positive cells (C,D; approximately 95,000 myogenic cells each) are shown (20x objective).
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Fig. 4. Fiber diameter (A) and area (B) distributions of restored dystrophin-expressing myofibers generated by the various myogenic populations. A larger percentage of small, newly formed fibers is seen with the LP population, and the overall range of distribution of the EP-generated fibers suggests that they may be more likely to fuse with larger host myofibers. Mdx myofiber analysis of non-injected areas was used as a control.
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Fig. 5. Relationship between the number of LP cells injected and the number of dystrophin-positive myofibers observed at 7 days post-injection. The greater slope of the CD34+ population's relationship re-emphasizes its higher level of regeneration efficiency. Relatively linear and predictable increases in number of dystrophin-positive myofibers generated with respect to injected cell number were observed for all populations (R2: 0.676, 0.758, 0.736, and 0.497 for Sca-1-, Sca-1+, CD34-, and CD34+, respectively).
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Fig. 6. Images obtained from the bioinformatic cell culture system used for temporal evaluation of cell fusion (A, 20x objective). (B-D) The EP population displayed a higher percentage of cells fusing into myotubes at all time points beyond 48 hours compared with both LP subpopulations (P<0.05 vs. CD34- and CD34+). The CD34-positive LP population displayed lower fusion compared with the CD34-negative LP population at 72 and 84 hours (P<0.05).
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Fig. 7. Cultures exposed to low serum fusing conditions were evaluated by immunofluorescence to determine the number of myogenic nuclei associated with fused and myosin-heavy-chain-expressing (MHC) myotubes at 96 hours (representative images of EP and LP CD34-positive populations are shown in A and B). Significant differences in the degree of fusion, defined by the fusion index (C), and ratio of mononuclear cells to myotubes formed (D) were observed between EP and LP populations (fusion index, *P<0.05 for CD34-positive vs. EP and CD34-negative; ratio of mononuclear to myotubes, *P<0.05 for CD34-positive vs. EP only).
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Fig. 8. (A) Establishment of a CD34-positive subpopulation is observed when culturing CD34-depleted cultures under high serum culturing conditions. Cells enriched for CD34 expression maintain their phenotype over the same time period (representative dot plots are shown along with averaged percentages of CD34-negative and -positive cells at each time point). (B,C) Immunofluorescence and brightfield images of mononuclear myogenic cells found within cultures initially depleted of a CD34-expressing population and exposed to low serum fusion medium. The majority of the undifferentiated myogenic cells were found to express CD34 (B), although some CD34-negative cells are present (C, arrows; 20x objective).
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© The Company of Biologists Ltd 2002
