CHARACTERISATION OF RENCELL VM: A HUMAN DOPAMINERGIC STEM CELL LINE.

Erik A. Miljan, Roberta Donato*, Kenny Pollock, Sara Patel, Sumita Majumdar, Marian Kapiri, Frances Edwards* and John Sinden. ReNeuron Ltd., Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF. * Department of Physiology, University College London, Gower Street, London, WC1E 6BT

Neural stem cells from the human fetal midbrain represent a promising cell-replacement therapy for the treatment of Parkinson's disease. The key to success in developing this therapy centers on a readily expandable stable cell line that is capable of differentiating in vivo into functional dopaminergic neurons. As a precursor to in vivo studies, we have characterized a fetal neural stem cell line, ReNcell VM, in vitro for differentiation into active dopaminergic neurons. ReNcell VM was derived from 10-week ventral mesecephalon fetal brain tissue immortalized by retroviral transduction with the v-myc oncogene. This cell line grows rapidly as a monolayer on laminin with a doubling time of 20-30hr, and has a normal diploid karyotype in culture for over one year (>45 passages). The undifferentiated cells are positive for nestin by immunocytochemistry, western blot and RT-PCR, indicating they are indeed neural stem cells.

Differentiation of ReNcell VM into dopaminergic neurons was investigated using two protocols developed in 96 well plates to facilitate higher throughput: namely 'standard' and 'pre-aggregation'. In brief, the 'standard' protocol involves expanding the cells to confluency on laminin in the presence of bFGF and EGF for 3-4 days and then initiating differentiation by simply removing the growth factors from the media for a further 5-7 days. The 'pre-aggregation' protocol is essentially the same as the standard protocol except that prior to seeding the cells on laminin for expansion and subsequent differentiation, the cells are first grown as adherent aggregates on uncoated plastic for 5-7 days. Both protocols produced morphologically mature neural cells with the majority of the cells positive for the neuronal marker III-tubulin and many co-labelled with tyrosine hydroxylase (TH) supporting a dopaminergic neuronal phenotype. The percentage of TH immunoreactive (TH-ir) cells using the standard protocol was estimated to be 5%, whereas, the pre-aggregation protocol produced up to 10-fold higher numbers of TH-ir cells. Also the standard protocol produced evenly distributed TH-ir cells whereas the pre-aggregation protocol develops foci of cells with radial fasciculations bridging areas of low cell density. As a further refinement, addition of 1mM dibutryl-cAMP and 2ng/ml GDNF to differentiation media discouraged the formation of foci and produced a more even distribution of TH-ir cells. Using Image-Pro plus software, the relative number of cells co-labelled with TH and III-tubulin was enriched from approximately 10% to 50% in the presence of cAMP and GDNF.

With respect to electrophysiology, undifferentiated ReNcell VM cells have low resting membrane potentials but do not display action potentials (-60 10mV, n=5). By contrast, of the cells tested by whole cell patch clamping, 80% (20/25) of the cells differentiated by the pre-aggregation protocol fired action potentials whilst only 25% (4/15) fired when differentiated using the standard protocol. The action potentials were completely blocked by 0.6 M tetrodotoxin (TTX) confirming that Na+ channels carry the inward current driving the action potentials. The differentiated cells had good long-term survival in culture as action potentials were measured 31 days after initiation of differentiation.

In summary, these in vitro studies provide insights into the candidacy of a cell line for in vivo research. Furthermore, ReNcell VM provides a renewable source of cells that can be differentiated into a high population of dopaminergic neurons, which can be an equally valuable research tool in the study and drug development of neurodegenerative disease. ReNcell VM also suggests a prototype approach to development of a reproducible source of dopaminergic neurons for treatment of Parkinson's Disease.

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