Article Figures & Data
Figures
- Fig. 1.
Immunopanned cells were highly enriched in p75+ EG. Comparisons of Hoechst (HOE) staining (A) with S100 (B) and p75 (C) staining of the same field show that essentially all p75+ cells are also S100+. Similarly, a comparison of Hoechst staining (D) with GFAP (E) and p75 (F) staining of the same field shows that essentially all p75+ cells are also GFAP+. Approximately 95% of the cells were p75+ at 3 d after immunopanning. A small number of p75−GFAP+ cells were found (arrows in D–F), suggesting that astrocytes were a contaminant of the preparation. This staining was performed after immunopanning with p75 antibody and maintenance for 3 d in vitro in DF-10S plus mitogens. Scale bars, 50 μm.
- Fig. 2.
Purified control DRGN cultures occasionally contained SCs. Phase photomicrographs of two DRGN control cultures are shown. A, A culture maintained for 4 weeks in NLA− medium; in this culture, non-neuronal cells were successfully eliminated. B, A DRGN culture maintained in NLA+ medium for the final 2 weeks of the myelination period and containing a colony of cells morphologically similar to SCs. A few SC myelin sheaths were observed in 4 of 12 cultures in this group. Scale bars, 50 μm.
- Fig. 3.
EG failed to form myelin under conditions that enabled SCs to produce myelin. A, C, Bright-field micrographs showing SC/DRGN cocultures in serum-free NLA medium without (A) or with (C) the addition of ascorbate. Myelin sheaths (arrows inC) are found only in cultures receiving ascorbic acid. EG/DRGN cultures are illustrated in B (no ascorbate) andD (with ascorbate); myelin was very rarely observed, and only in cultures with ascorbate. All cultures were fixed at the same time, at 2 weeks after the addition of serum and ascorbic acid.E, F, Bright-field micrographs showing cocultures in NLA medium supplemented with both serum and ascorbic acid. All cultures were fixed at the same time, at 3 weeks after the addition of serum and ascorbic acid. E, An SC/DRGN culture is illustrated; myelin (arrows) was readily observed in this culture group. F, An EG/DRGN culture exhibits no myelin, despite the presence of ascorbate. Scale bar, 25 μm. G, Graph comparing myelination in SC/DRGN and EG/DRGN cultures under serum-free conditions. In the SC/DRGN group, a mean of 99 ± 5.6 myelin segments was counted per culture when fed ascorbate-containing medium. EG/DRGN cultures fed the same medium showed no or rare myelin segments, comparable with control DRGN cultures. Bar heights are the mean number of myelin segments counted per culture for each culture type (n = 12); error bar represents the SEM. *p < 0.001 compared with control DRGN cultures.H, Graph comparing myelination in SC/DRGN and EG/DRGN cultures in the presence of serum; four cultures were counted in each group. Bar heights indicate the mean number of myelin segments counted per culture for each culture type (n = 4). Myelin was only observed in cultures to which SCs were added. A similar result was observed in a repeat experiment. *Error bar indicates SD.
- Fig. 4.
Most purified adult EG maintained a stable p75+GFAP+ phenotype during 5 weeks of coculture with DRGN. SC/DRGN and EG/DRGN cultures were fixed at 3 weeks after switching the cultures to myelination conditions (NLA medium supplemented with serum and ascorbic acid). A–C, A field in an SC/DRGN culture is illustrated. A, In this phase-contrast image, SC nuclei are observed in typical longitudinal arrays. B, C, Whereas individual cell boundaries are not easily observed at this low magnification, both p75 and GFAP staining were found where SCs were located, resulting in a high degree of correspondence between p75 and GFAP staining. D–F, A field in an EG/DRGN culture is illustrated. D, Nuclei of EG are visible in this phase-contrast image. The density of EG achieved after 5 weeks of coculture with neurons typically appears to be lower than that achieved by SCs. E, F, Despite the smaller number of EG, p75+GFAP+ processes cover much of the culture area. As with SCs, there is a strong correspondence between the position of EG nuclei and p75 and GFAP staining. Scale bar, 25 μm.
- Fig. 5.
Myelin formation and ensheathment in an SC/DRGN culture. In an SC/DRGN culture fed ascorbate-containing but serum-free medium, a myelin sheath formed by an SC is illustrated in the center. Surrounding it are nonmyelinated, ensheathed axons; complete encirclement of an axon by SC cytoplasm is indicated by the arrow. Scale bar, 1 μm.
- Fig. 6.
Lack of typical SC-type ensheathment in an EG/DRGN culture. These two panels illustrate two examples of the flat meandering EG processes (arrows, top panel) that only partially encircle axons or partition fascicles of axons but do not provide furrows for each axon as seen in SC/DRGN cultures. The EG cytoplasm contains numerous intermediate filaments and microtubules (best seen in the cell in the top panel). The culture was maintained in ascorbate-containing, serum-free NLA medium. Scale bar, 1 μm.
Tables
- Table 1.
Schwann cells and ensheathing glia exhibit different relationships to axons, as determined by electron microscopy
No glial contact Glial contact Ensheathment Myelin Number of axons counted (% total axons) SCs1-a 66 (7.4) 605 (72.3) 162 (19.5) 8 (0.8) EG1-b 526 (62.6) 302 (35.9) 11 (1.3) 1 (0.1) Mean axonal diameter (μm) ± SD SCs 0.17 ± 0.06 0.21 ± 0.09 0.32 ± 0.13 0.78 ± 0.21 EG 0.19 ± 0.06 0.20 ± 0.10 0.30 ± 0.10 1.48 Axons were considered to be ensheathed when at least 75% of the axolemma was covered by a glial process.
↵F1-a A total of 841 axons was counted throughout five electron micrographs of SC/DRGN cultures at 8900× magnification.
↵F1-b A total of 840 axons was counted throughout two electron micrographs of EG/DRGN cultures at 8900× magnification.
