Optimisation of long term culture conditions for human induced neurons

Induced neurons (iNs) are reprogrammed somatic cells that are forced to change their fate without passing through a pluripotent state, shortening the process of neuronal generation compared to the use of human pluripotent stem cells. The scientific interest in these cells is explained by their suitable use for disease modelling, drug screening and cell therapy for diseases such as Parkinson Disease (PD).  Recently, we have developed an all-in-one vector that delivers two factors, Brn2a and Ascl1, together with a REST (RE1-silencing transcription factor) inhibition to successfully generate neurons from both PD patient and control fibroblasts at high efficiencies.

An important characteristic of the reprogrammed cells that has to be taken into account to confirm their neuronal identities is their electrophysiological profile. Here, we aim at studying the electrical activity of iNs using the whole cell patch-clamp technique. We first evaluate different culture conditions by testing different coatings to promote an optimal long-term maturation of the iNs, neccessary for electrophysiological maturity. More specifically, we compare Gelatin, Matrigel, laminin 111 and different polyornithine, laminin and fibronectin triple coatings, and found that the later condition provides better plating and long-term survival (>80 days). We further compare our best coating condition with the co-culturing of iNs with either primary rodent astrocytes or neurons. These experiments will allow further assessment of the long-term adult iNs electrical activity, which constitutes an important step towards using iNs for disease modelling of patient-derived cells.