Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C86
Human embryonic stem cell derived functional astrocytes are neuroprotective through glutathione dependent and independent mechanisms
K. Gupta1,2, R. Patani1, P. Baxter2, A. Serio2, D. Story2, J. D. Pickard1, R. A. Pedersen1, G. E. Hardingham2, S. Chandran2
1. University of Cambridge, Cambridge, United Kingdom. 2. University of Edinburgh, Edinburgh, United Kingdom.
Neurodegenerative diseases represent a major healthcare burden, are incurable and without effective treatments. While rodent research has demonstrated the importance of glial:neuronal interaction, inter-species differences limit translation. Human embryonic stem cells (HESC) offer an opportunity to model human injury in vitro and allow clinical extrapolation. Experiments were performed across two independent HESC lines (H9/Hues9); values are mean ± SEM; n=3; p<0.05 was held significant; data were analysed by t-test and ANOVA with Newman-Keuls post-hoc analysis; Cell viability was assayed by CellTiter-Glo (Promega) and confirmed by TuJ1/activated caspase 3 co-staining; cell markers were quantified by immunohistochemistry. HESC were cultured and neuralised by established means (1) and propagated as adherent monolayers. At 30d a homogeneous neural precursor cell (NPC) population was obtained, expressing typical markers (nestin: 93.9±1.0%; 3CB2: 82.7±2.1%). Highly enriched astrocyte populations were derived by differentiation of late NPCs in BMP4/LIF (GFAP: 95.7±3.1%; S100b: 90.1±2.0%). Their functional nature was verified by H3-glutamate uptake. Enriched populations of HESC derived neurons (TuJ1: 95.5±1.4%), were challenged with 50µM H2O2, which caused 42.2±1.4% cell death. Treatment of HESC neurons with astrocyte conditioned medium (ACM) reduced cell death (31.0±1.8% p<0.05), which was unchanged upon astrocyte pretreatment with buthionine sulfoximine (BSO), a glutamate-cysteine ligase (GCLc) inhibitor (34.8±0.8%). These data suggest that ACM is protective by glutathione independent mechanisms, such as GDNF production (2). There is growing evidence for synthetic triterpenoids as potential therapeutic agents in mouse models of neurodegenerative disorders (3). In order to extend these observations to a human system, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid trifluoroethylamide (CDDO-TFEA) was tested. As direct treatment of neurons had no protective effect, induction of NFE2L2 and GCLC, established triterpenoid target genes, was examined by qRT-PCR: Neurons showed no induction with treatment (CDDO-TFEA: 1.25±0.2, 1.23±0.1 fold change), astrocytes showed higher baseline and inducible levels (baseline: 11.9±0.6, 4.8±0.2; CDDO-TFEA: 21.4±0.4, 9.6±0.1 fold change p<0.05). Moreover, CDDO-TFEA pretreated ACM had additional neuroprotective ability above untreated ACM (19.6±1.7% p<0.05), amenable to BSO mediated inhibition (31.6±1.4%). Therefore the additive protective effect of CDDO-TFEA is mediated indirectly by the astrocyte by a glutathione dependent mechanism. These data provide important proof of concept that HESC derived neurons and astrocytes can be used to model neurological injury and protection, and in addition provide a key platform for studies emerging using human iPS cells.
Where applicable, experiments conform with Society ethical requirements