Neuronal progenitors from feeder-free human embryonic stem cell culture
View Patent ↗The present invention relates to methods for producing feeder cell-free neuroprogenitor cells (preferably adherent) from embryonic stems cells, preferably human embryonic stem cells, the feeder cell-free neuroprogenitor cells, preferably human cells themselves, as well as methods for producing feeder cell-free samples of neuronal cells, preferably adherent human neuronal cells and the feeder cell-free neuronal cells themselves. Pharmaceutical compositions and methods of treating neurodegenerative diseases as well as the use of the described cells in assay systems is also described.
1. A method for producing feeder cell free neuroprogenitor cells, said method comprising the steps of:
a) Providing a population of adherent primate pluripotent stem cells expressing; Sox2, SSEA-4 and OCT4;
b) Contacting said adherent primate pluripotent stem cells with a serum free differentiation medium comprising the components of a minimum essential medium and basic fibroblast growth factor and optionally, non-essential amino acids, leukaemia inhibitory factor (LIF), an antibiotic, mercaptoethanol or another cell medium selected from B27, N2 or mixtures thereof, with an effective amount of at least one pluripotent stem cell differentiation protein to differentiate said adherent primate pluripotent stem cells into neuroprogenitor cells without embryoid body formation or neurosphere transition; and
c) Passaging said neuroprogenitor cells.
2. The method of claim 1 in which greater than 90% of the neuroprogenitor cells are nestin positive.
3. The method according to claim 1 wherein the neuroprogenitor cells are PSNCAM and A2B5 negative.
4. The method according to claim 1 wherein said primate pluripotent stem cells are human embryonic stem cells.
5. The method of claim 1 wherein the antibiotic comprises penicillin and streptomycin.
6. The method of claim 1 wherein the nestin positive neuroprogenitor cells can directly produce all neural lineages.