Abstract
In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O2, demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O2 can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O2 environment results in significant cell death, while maintenance in 3% O2 is protective. Together these findings support 3% O2 as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.
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Abbreviations
- BDNF:
-
brain derived neurotrophic factor
- CDM:
-
chemically defined medium
- CNS:
-
central nervous system
- FGF-2:
-
fibroblast growth factor-2
- GDNF:
-
glial derived neurotrophic factor
- hESCs:
-
human embryonic stem cells
- hESC-NPCs:
-
human embryonic stem cell derived neural precursor cells
- HIF:
-
hypoxia inducible factor
- IGF:
-
insulin-like growth factor
- MBP:
-
myelin basic protein
- NAC:
-
N-acetyl-l-cysteine
- NPC:
-
neural precursor cell
- NSC:
-
neural stem cell
- O2:
-
oxygen
- pO2:
-
partial pressure of oxygen
- RA:
-
retinoic acid
- ROS:
-
reactive oxygen species
- TH:
-
tyrosine hydroxylase
- TTX:
-
tetrodotoxin
- VMAT:
-
vesicular monoamine transporter
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Acknowledgements
We thank James Raleigh for his generous gift of pimonidazole (Hypoxyprobe) and antibody, Ludovic Vallier for his kind provision of feeder-free H9 ES cultures, Roger Barker for use of the low oxygen incubator and Rickie Patani, Kristine Westmore and David Story for valuable technical assistance. This work was supported by the MS Society UK, Evelyn Trust, MRC, Wellcome Trust (AL) and Royal Society (RK). SS is supported by a Sir David Walker Fellowship, a joint Medical Research Council and Multiple Sclerosis Society Clinical Research Training Fellowship (no. G0800487) and a Raymond and Beverly Sackler Studentship.
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Stacpoole, S., Bilican, B., Webber, D. et al. 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. Cell Death Differ 18, 1016–1023 (2011). https://doi.org/10.1038/cdd.2010.171
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DOI: https://doi.org/10.1038/cdd.2010.171
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