A Scaleable and Defined System for Generating Neural Stem Cells from Human Embryonic Stem Cells

Abstract

The ability to differentiate human ESCs (hESCs) to defined lineages in a totally controlled manner is fundamental to developing cell-based therapies and studying human developmental mechanisms. We report a novel, scaleable, and widely applicable system for deriving and propagating neural stem cells from hESCs without the use of animal products, proprietary formulations, or genetic manipulation. This system provides a definitive platform for studying human neural development and has potential therapeutic implications. STEM CELLS 2007;25:731-737

Derivation of neural precursor cells from human ES cells at 3% O2 is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

“…37 Previous reports that HIF-1a interacts with Notch, 21 and that HIF-2a regulates Oct4, 22 might suggest that low O 2 would maintain pluripotency in ESCs and present a barrier to efficient neural induction and terminal differentiation. However, we observed that at 3% O 2 , efficient neuralisation was completed by 14 days, a timescale comparable to that at 20% O 2 , 14,33 indicating that a low O 2 environment does not adversely affect acquisition of a neural fate. Additionally, under basal conditions, significantly more NPCs were generated at 3% than at 20% O 2 , with a greater proportion of viable cells; a finding consistent with a previous study based on feeder and matrigel-maintained human ES cells, reporting a decrease in parthanatic cell death in neurectoderm derived at 3% O 2 .…”

“…There was no significant difference between the efficiency of neural conversion at 3 and 20% O 2 , with a neural identity acquired by D14 in both instances (Figure 1c), consistent with previous reports of a 2-week timescale for neural conversion of hESCs at 20% O 2 . 14,33,35,36 Neural conversion at 3% O 2 was robust, highly reliable and reproducible across two independent hESC Figure 1A). Growth curves confirmed a significant increase in numbers of NPCs generated at 3% compared with 20% O 2 , under basal culture conditions (Figure 2a Figure 1B).…”

“…To address whether hESC-NPCs could be efficiently derived in low oxygen conditions, feeder-free hESCs, grown in a chemically defined medium (CDM) [31][32][33] at 20% O 2 , were enzymatically detached and transferred to suspension culture at 3% O 2 , along with removal of activin and FGF-2. A pimonidazole-binding assay was used to biochemically confirm growth of cells at low oxygen; pimonidazole adducts on the surface of hypoxic cells, binding most efficiently at a pO 2 o10 mm Hg (Figure 1d).…”

Therapeutic Potentials of Human Embryonic Stem Cells in Parkinson’s Disease

“…In fact, all animal products must be removed from the culturing condition, and there must be defined culture and media conditions in order to comply with GMP. Fortunately, both researchers 87,128,129 and biotechnology companies are producing media and supplements that are meeting this requirement. Unfortunately, the ingredients are proprietary, none of the companies will provide the ingredients (although the media are for sale), and very few of the scientists are willing at this point to share all of their knowledge.…”

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

“…Moreover, from a clinical point of view, protocols which emphasize on defined/human derived products for cell preparations prevail over methods using animal products. It has been shown that animal products increase the risk of non-human antigens expression and xeno infections (Joannides et al 2007). Our findings establish a simple and efficient protocol to enrich the adult rat SCs without the use of serum or other animal-derived products.…”