EGF Enhances Oligodendrogenesis from Glial Progenitor Cells

Abstract

Emerging evidence indicates that epidermal growth factor (EGF) signaling plays a positive role in myelin development and repair, but little is known about its biological effects on the early generation and differentiation of oligodendrocyte (OL) lineage cells. In this study, we investigated the role of EGF in early OL development with isolated glial restricted precursor (GRP) cells. It was found that EGF collaborated with Platelet Derived Growth Factor-AA (PDGFaa) to promote the survival and self-renewal of GRP cells, but predisposed GRP cells to develop into O4^- early-stage oligodendrocyte precursor cells (OPCs) in the absence of or PDGFaa. In OPCs, EGF synergized with PDGFaa to maintain their O4 negative antigenic phenotype. Upon PDGFaa withdrawal, EGF promoted the terminal differentiation of OPCs by reducing apoptosis and increasing the number of mature OLs. Together, these data revealed that EGF is an important mitogen to enhance oligodendroglial development.

Keywords: GRP cell; OPC; oligodendrocyte lineage; self-renewal; synergistic effect.

Figure 1

The antigenic phenotypes of E13.5 spinal cord derived A2B5⁺ cells. (A–P) Immunostaining of A2B5⁺ cells with PDGFRα, epidermal growth factor receptor (EGFR), Olig2 and Nestin antibodies, respectively, and the antigenic phenotypes were confirmed by Western blotting (D,H,L,P). (Q) Quantification of PDGFRα⁺, EGFR⁺, Olig2⁺, Nestin⁺, GFAP⁺ and NF-1⁺ cells in A2B5⁺ cell cultures, n = 3. Scale bars: 50 μm.

Figure 2

Effect of EGF on A2B5⁺ cells. (A) Histogram of the number of BrdU⁺ cells found in A2B5⁺ cell cultures after exposure to different doses of EGF. *Indicates differences between groups 10, 20 and 40 ng/ml EGF vs. the low-dose groups, P < 0.05. No differences were found among the groups of 10, 20 and 40 ng/ml. n = 3. (B,C) Quantification of BrdU⁺ and TUNEL⁺ cells in A2B5⁺ cell cultures after EGF, EGF + Erlotinib-HCl, basic fibroblast growth factor (bFGF), PDGFaa, T3, bFGF + PDGFaa and EGF + PDGFaa treatments for various time lengths, Ctrl refers to the groups without any supplemented factor, n = 3. (D) Histogram of the number of secondary clones in A2B5⁺ cell cultures at clonal density after exposure to EGF, PDGFaa and EGF + PDGFaa, respectively. n = 3. Statistical analyses are presented as mean ± SD. *P < 0.05, **P < 0.01.

Figure 3

Expanded clones of A2B5⁺ cell expressed typical differentiation phenotype of tripotential glial restricted precursor (GRP) cells. (A) An example of an expanded clone in presence of EGF + PDGFaa. (B) A2B5⁺ clones were digested and replated into separate wells of 24-well plates and induced to differentiate for 5 days in the presence of bFGF + CNTF or FBS, A2B5⁺/GFAP⁺ and A2B5⁻/GFAP⁺ astrocytes were obtained, respectively. (C) Quantification of A2B5⁺/GFAP⁺ and A2B5⁻/GFAP⁺ astrocytes in the differentiation cultures exposed to bFGF + CNTF and FBS, n = 3. (D) A2B5⁺ cells were cultured in T3 for 5 days and MBP⁺ Oligodendrocytes (OLs) can be detected. (E) The culture described in (D) was confirmed by western blotting with MBP antibody. Abbreviation: OL, oligodendrocyte. **P < 0.01. Scale bars: (A) 100 μm; (B,D) 50 μm.

Figure 4

EGF enhanced the formation of OPCs from GRP clones. (A) The single cell tracking of a GRP cell generating O4⁺/MBP⁻ daughter cells in EGF for 5 days. With the progression of cell division, a fibroblast-like GRP cell was gradually converted to typical OPCs of bipolar or tripolar morphology, well separated from each other, and expressed O4 antigen but not MBP. Daughter cells of the same progenitor were indicated by arrows of different color. (B) Quantification of the clones containing O4⁺/MBP⁻ cells in the GRP cell cultures exposed to various combinations of factors for 5 days, respectively, n = 3. (C) OPC-like cells from (B) differentiated into MBP⁺ cells after 5 days of culture in glial basal medium without supplemented factors. Left: phase image; right: anti-MBP immunostaining. (D) Quantification of the clones containing MBP⁺ cells in OL differentiation cultures described in (C), n = 3. (E) OPCs derived from EGF + PDGFaa + T3 treament differentiated into A2B5⁺/GFAP⁺ astrocytes in presence of FBS. *P < 0.05. Scale bars: (A,C) 75 μm; (E) 50 μm.

Figure 5

Synergistic effect of PDGFaa and EGF in promoting the self-renewal of O4⁻ OPCs. (A) Representative images of O4⁺ and/or BrdU⁺ cells cultured in PDGFaa or PDGFaa + EGF for 5 days, cell proliferation was analyzed by BrdU incorporation for 24 h before fixation. (B) Quantification of O4⁺ cells in PDGFaa and PDGFaa + EGF cultures. (C) Quantification of BrdU⁺ cells in PDGFaa and PDGFaa + EGF cultures. Statistical analyses are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, Scale bars: 100 μm.

Figure 6

EGF promotes OPC differentiation in cooperation with T3. (A) Quantification of BrdU⁺ cells for OPCs cultures with EGF or not for various time lengths. (B) Quantification of MBP⁺ cells in OPCs differentiation cultures treated with T3, EGF or T3 + EGF for various time lengths. (C) Quantification of TUNEL⁺ cells in OPCs differentiation cultures on day 4. (D,E) Western blotting and quantitative analysis of EGFR protein expression in O4⁻ OPCs and MBP⁺ OLs, histograms express results in arbitrary units, taking GRP cells values as 100%. Statistical analyses are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01.

Figure 7

Biological effects of EGF, PDGFaa, bFGF and T3 in the progression of OL lineage. EGF has multiple biological effects on oligodendrogenesis, and its functional output is influenced by other signal molecules, such as PDGFaa and T3. In GRP cells, EGF and bFGF collaborate with PDGFaa to promote the self-renewal of GRP cells. When EGF is present alone, it favors the development of GRP cells to OPCs, and this progress is accelerated by supplementing PDGFaa and T3 simultaneously. In OPCs, EGF and bFGF enhance their responsiveness to PDGFaa and thus maintains their O4 negative phenotype as well as bipolar or tripolar cell morphology. When EGF is present alone, it synergizes with T3 to promote the terminal differentiation of OPCs, whereas bFGF and PDGFaa inhibit this differentiation process.