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doi: 10.1242/10.1242/jcs.00049

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Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells

¹ MRC Laboratory for Molecular Cell Biology and Cell Biology Unit and the Biology Department, University College London, London WC1E 6BT, UK
² Centre for Genome Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

View larger version (26K): [in a new window]   Fig. 1. Protocol for studying oligodendrocyte development from genetically engineered ES cells.

View larger version (14K): [in a new window]   Fig. 2. Neuroepithelial cells and residual ES cells in non-selected ES-cell-derived cultures. ES cells were cultured as shown in Fig. 1 but without selection. At various times, the cells were dissociated, allowed to settle on a coverslip for 6 hours and stained for SSEA-1 or nestin by immunohistochemistry. The proportion of stained cells was determined, and the results are shown as mean±s.d. of three independent experiments.

View larger version (42K): [in a new window]   Fig. 3. Effect of combined positive and negative selection on engineered ES cells. Engineered ES cells were treated and selected as described in Fig. 1 to enrich for neuroepithelial cells and to eliminate residual undifferentiated ES cells. At various times, the cells were dissociated, and either stained for SSEA-1 or nestin by immunohistochemistry (A) or processed for RT-PCR analysis using Oct4, Sox1, Sox2 or G3PDH probes (B). The results in (A) are shown as mean±s.d. of four independent experiments.

View larger version (69K): [in a new window]   Fig. 4. Effect of Shh on the development of OPCs. Engineered ES cells were treated and selected as described in Fig. 1. On day 10, after 4 days of selection, Shh was added to some of the cultures, all of which were maintained in FGF-2, without G418 or Ganciclovir. After 5 more days, OPCs were identified either by in situ hybridization to detect Olig1, Olig2 and PDGFR mRNAs or by immunohistochemistry to detect NG2 (A). The nuclei in the bottom panels were stained with bisbenzimide. The scale bar is 20 µm. (B) Table showing the proportion of labelled cells. Randomly selected fields were counted in two experiments.

View larger version (19K): [in a new window]   Fig. 6. Timing of neural cell development and marker expression in ES-cell-derived cultures. Engineered ES cells were treated and selected as described in Fig. 1. At various times after the end of the selection (day 10), Olig2 and PDGFR expression were analysed by in situ hybridization; neurofilaments, NG2, O4 and GC were analysed by immunohistochemistry and MBP expression was assessed by RT-PCR. (A) Expression of neuronal and OPC markers. (B) Expression of neuronal and oligodendrocyte markers. (C) Expression of MBP mRNA. Results of typical experiments are shown, but the experiments were repeated three times with similar results.

View larger version (57K): [in a new window]   Fig. 5. Development of oligodendrocytes from ES-cell-derived OPCs. Engineered ES cells were treated and selected as described in Fig. 1. On day 10, the cells were exposed to FGF-2 and Shh for 5 days. Both FGF-2 and Shh were then removed, and the cells were cultured in PDGF-AA and TH for another 9 days (A) or 3 days (B). (A) The cells were stained with anti-GC antibody to identify oligodendrocytes and bisbenzimide to identify cell nuclei. (B) The cultures were stained with anti-neurofilament antibodies to identify neurons (green), with O4 antibody to identify oligodendrocyte lineage cells (red) and with bisbenzimide to identify cell nulei (blue). Bar, 50 µm.