Research on human tissue with informed consent was approved by the Research Ethics Committee of Lothian Health Board. Frozen supernumerary human embryos were donated for research under licence R0132 issued by the Human Fertilisation and Embryology Authority. Inner cell masses were isolated by immunosurgery and cultured on human foreskin fibroblasts in medium supplemented with 15% serum replacement (Invitrogen) plus human leukaemia inhibitory factor and FGF-2 [73 (link)]. After three to four passages, cells with ES cell morphology differentiated into rosettes of neuroepithelial-like cells. These colonies were passaged into NS expansion medium without feeders or serum replacement. Human foetal tissue was obtained following elective termination with consent for research according to the Polkinghorne guidelines [74 ]. Cortex was dissected from Carnegie stage 19–20 foetuses and processed as described for mouse foetal tissue. In some cases, LIF (100 U/ml) was added to the expansion medium [75 (link)].
To induce neuronal differentiation, a similar protocol was followed as for mouse NS cells but with addition of brain derived neurotrophic factor (R&D Systems; 10 ng/ml) after the first 7 d without EGF, and retention of FGF-2 (5 ng/ml) until 14 d.
To induce neuronal differentiation, a similar protocol was followed as for mouse NS cells but with addition of brain derived neurotrophic factor (R&D Systems; 10 ng/ml) after the first 7 d without EGF, and retention of FGF-2 (5 ng/ml) until 14 d.
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