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Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

Joy Rathjen1, Bryan P. Haines1,*, Kathryn M. Hudson1, Antonietta Nesci2, Stephanie Dunn2 and Peter D. Rathjen1,3,{dagger}

1 Department of Molecular Biosciences,
2 Cell Therapy Program and
3 ARC SRC for the Molecular Genetics of Development, The University of Adelaide, Adelaide, South Australia 5005, Australia
* Present address: Section of Gene Function and Regulation, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, UK



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  		Fig. 1. Formation of early primitive ectoderm-like cells in suspension culture. (A-D). 7 µm sections of EBM4 (A,B) and EB4 (C,D) stained with Haemotoxylin and Eosin (A,C) and Hoechst 22358 (B,D) and viewed using brightfield (A,C) and fluorescent (UV2A filter; B,D) microscopy. Scale bar: 170 µm. (E) RT-PCR analysis for the presence of AFP and actin transcripts in EB4 and EB9, two independent populations of EBM4 and EBM9, and 8.5 d.p.c. mouse embryos. A control reaction in which reverse transcriptase was omitted is included (no RT). (F) Northern blot analysis of 20 µg of RNA isolated from EB2-5 and EBM2-5 probed for Oct4 (1.55 kb), Fgf 5 (2.7 kb), brachyury (2.1 kb) and mGAP (1.5 kb). (G-L). Whole-mount in situ hybridisation analysis of EBM4 (G,H,K) and EB4 (I,J,L) probed with digoxigenin-labelled antisense probes to Oct4 (G,I), Fgf5 (H,J) and brachyury (K,L). Scale bar: 85 µm.

 


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  		Fig. 2. Morphology and gene expression in differentiating pluripotent cells in vitro and in vivo. (A) EBM7 and (B) EBM9 viewed using Hoffmann interference contrast microscopy. (C) 7 µm section of an EBM9 aggregate stained with haemotoxylin and viewed using Hoffmann interference contrast microscopy. Size bars: 210 µm. (D) Northern blot analysis of 20 µg RNA isolated from EB4-8 and EBM4-8 probed for Oct4 and mGAP. (E) Whole-mount in situ hybridisation analysis of an EBM7 aggregate seeded and cultured for a further 24 hours and probed with a digoxigenin-labelled antisense probe to Oct4. Size bar: 210 µm. (F,G) 10 µm transverse section of a 7.75 d.p.c. mouse embryo probed with a radiolabelled antisense probe to Oct4 viewed in brightfield (F) and darkfield (G) illumination. A concentration of silver grains can be seen over the neural ectoderm (black arrow). Mesoderm (outlined arrow).

 


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  		Fig. 3. Differentiation of EPL cell aggregates in MEDII results in the formation of homogeneous populations of neurectoderm. (A) 15 µg of RNA isolated from EBM6-9 was analysed for the expression of Sox1 and mGAP by RNase protection. (B) Immunohistochemical analysis for the presence of the neurofilament protein nestin in a seeded EBM7 aggregate after a further 48 hours culture. Size bar: 210 µm. (C,D) EB7 and EBM7 were seeded into individual 2 ml wells and examined on days 8, 10 and 12 for the formation of beating cardiocytes (C) and neural extensions (D). n>48/experiment, 5 experimental repeats represented. (E,F) EBM7 were seeded and cultured for a further 4 days in serum-free medium before analysis for the presence of NeuN (E) and tubulin-ß III (F). (G,H) EBM9 were analysed by whole-mount in situ hybridisation for the expression of Sox1 (G) and Sox2 (H) using digoxigenin-labelled anti-sense probes. After colour development, aggregates were fixed, embedded and cut into 7 µm sections. Sections were viewed under brightfield microscopy. Size bar: 210 µm. (I) EBM10 were disaggregated, probed for the expression of NCAM by immunohistochemistry and analysed by flow cytometry. The bar, which indicates positive fluorescence, was determined experimentally by analysis of cells probed with secondary antibody alone (data not shown).

 


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  		Fig. 4. MEDII reduces the formation of mesoderm and promotes neuron formation from EPL cell embryoid bodies. EPLEB7 and EPLEBM7 aggregates were seeded into individual 2 ml wells and cultured for a further 5 days before scoring for the presence of beating cardiocytes and neural extensions. n>48/experiment, 3 experimental repeats represented.

 


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  		Fig. 5. Temporal expression of Sox1 and Gbx2 in EPL cell-derived neurectoderm. (A-F) Seeded EBM7 were cultured for a further 24 (A,D), 48 (B,E) and 72 (C,F) hours and analysed by in situ hybridisation with digoxigenin-labelled antisense probes directed against Sox1 (A-C) and Gbx2 (D-F). Aggregates were viewed using Hoffmann interference contrast microscopy. Size bar: 210 µm.

 


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  		Fig. 6. Expression of neurectoderm markers in EPL cell-derived neurectoderm. cDNA was synthesised from 1 µg of total RNA isolated from EB9, EPLEB9, EBM9 and 10.5 d.p.c. mouse embryo (used as a positive control), or EB9, EBM8, EBM9 and 8.5 d.p.c. mouse embryo (used as a positive control), and used as a template for PCR analysis of the genes denoted. Expression of actin was used as an example of a gene expressed in all cell types to normalise the PCR reaction. Primer sequences and product sizes can be found in the Materials and Methods.

 


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  		Fig. 7. EPL cell-derived neurectoderm can be directed to neural crest and glial lineages (A-D). EBM9 explants were seeded onto cellular fibronectin-treated tissue culture plasticware in medium supplemented with 25 nM staurosporine/0.1% DMSO (A,C,D) or 0.1% DMSO alone (B). Cultures were examined after 3 (A,B) or 48 hours (C,D). (D) In situ hybridisation analysis of EBM9 explants with digoxigenin-labelled antisense probes for Sox10. (E-H) EBM9 explants were seeded onto poly-L-ornithine-treated tissue culture plasticware in medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 1 µg/ml laminin (E,F) followed by culture in medium supplemented with 10 ng/ml PDGF-AA (G,H). Cultures were examined after 2 (A), 4 (F) and 6 (G,H) days. (H) Immunohistochemistry of EBM9 explants with antibodies directed against glial fibrillary acidic protein (GFAP).

 

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