Background Pluripotent stem cell technology is allowing the study of disease-relevant cell types of human origin, and is accompanied by much interest in their application for the screening of disease phenotypes and potential therapeutics. Here, we have generated and characterised induced pluripotent stem cells (iPSCs) from HD gene carriers differentiated into disease-specific striatal medium spiny neurons (MSNs) by high content imaging to investigate the effects of increasing HTT CAG repeat length on neuronal function.

Aims To adapt a protocol for the directed differentiation of iPSCs to MSNs to a high throughput format, in order to carry out a broad phenotypic screen of HD-related phenotypes using high content imaging.

Methods The iPSC lines we used were derived from closely related individuals, three of whom were suffering from juvenile HD and their unaffected mother. This created a unique familial allelic series with increasing HTT CAG repeat lengths of 20, 56, 67 and 73 CAGs. High content imaging was carried out at differentiation day 36 using the Opera Phenix High Content Screening System and data analysed using Columbus software (Perkin Elmer).

Results We report here significant phenotypic differences in endpoint total cell number and culture composition between control and HD lines with the latter accumulating fewer cells over time and a reduction in the proliferative precursor pool. Interestingly, there were no significant differences in the proportions of CTIP2+ cells, neurons or MSNs generated. However, in terms of pathology, we find evidence of enhanced cell death, reduced DARPP-32 expression within MSNs and mHTT inclusion formation in the longest (73 CAG) length line.

Conclusions We find no differences in holistic measurements of neuronal differentiation between control and HD lines suggesting that the pathway to neuronal development is unaffected at this stage. However, differences in endpoint cell number and HD-related pathologies were found which could be used as potential endpoints for high throughput drug screening.

Support Takeda, Cerevance Ltd, UCLH Biomedical Research Centre, Medical Research Council