| Summary: | Pluripotent cells are defined by their ability to both self-renew and to differentiation into any cell type within the human body. As such, pluripotent cell lines are of great interest as starting material for drug screening and cell therapies for regenerative treatment of diseased tissues. Pluripotent cell lines were originally derived from germ cell tumors (embryonal carcinoma cells; EC), but have since been isolated and expanded from the inner cell mass of an early embryo (human embryonic stem cells; hESCs). This project set out to investigate the relative ability of the pluripotent NTERA2 (EC) cell line and hESC lines: Shef3, HUES7 and RH5, to differentiate into neurons, using mechanical and enzymatic culture methods. Focus was placed on monitoring differentiation efficiency and function between the different lines. The tumour origin, in addition to the poor reproducibility, low yield and reduced functionality of NTERA2 derived neurons, compared to primary neurons, makes their incorporation into regenerative therapies unlikely. As such, an enhanced neuronal differentiation protocol was developed for use in hESCs. Cell populations were monitored for relative changes in gene and protein expression at selected time points throughout differentiation using standard RT-PCR, Q-PCR and immuno fluorescence analysis. End stage neurons were screened for functionality using patch clamping and calcium imaging techniques. Monitoring of cellular behavior through differentiation was aided by the concurrent development of a portable microscope incubator stage in collaboration with Linkam scientific Ltd. These data demonstrate a variation in the ability to generate neurons from pluripotent cell lines, and suggests a predetermined, preferential cell fate within each line, even at the level of pluripotency. This study also characterises in detail neuronal differentiation from pluripotent cells, adding to the understanding which is essential for translation into therapies for neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntingdon’s disease.
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