Midbrain Dopamine Neurons Derived from Induced Pluripotent Stem Cells from Patients with Lesch Nyhan Disease and Gene-Edited Knock-In HPRT1 Mutations
Fatemeh Seifar1, Diane Sutcliffe1, H. Jinnah1
1Department of Neurology, Emory University
Objective:
To create a novel in-vitro model to study molecular pathogenesis of midbrain dopamine abnormalities during neuronal development in Lesch Nyhan disease (LND). 
Background:

LND is a neurodevelopmental disease which manifests with neurobehavior abnormalities starting in early childhood. It is linked to abnormal development of nigrostriatal dopamine neurons. However, the molecular pathogenesis of impaired development is not well understood. One of the main reasons has been lack of a good model to study midbrain development.

Design/Methods:

We generated 24 induced pluripotent stem cell (iPSC) lines; 12 from 4 individuals with LND, and another 12 from 4 normal individuals. We also generated 12 gene-edited lines, all containing a c.C508>T stop codon in the HPRT1 gene.  All 24 lines were assessed for pluripotency markers and several of the lines were differentiated into midbrain dopamine neurons using a floor-plate induction protocol. Differentiation was assessed at 3 stages of development in vitro: day 11 (neuroprogenitor), day 25 (early neuron) and day 60 (late neuron). The percentages of dopamine neurons were determined by tyrosine hydroxylase (TH) immunostaining with flow cytometry. The expression of FOXA2, TH and DAT were also measured by quantitative qPCR.  Dopamine and its derivatives were measured using HPLC.

Results:

The differentiation protocol yielded a high percentage of TH-positive neurons (50-80%) that produced a high quantity of dopamine and its metabolites across all cell lines. TH and related markers all varied significantly among the different cell lines, and during differentiation.  Results are being interrogated to identify any systematic changes between the LND and control lines.

Conclusions:
We have generated a large bank of iPSCs from individuals with LND, HPRT1 gene-edited lines, and parallel control lines.  We have shown that these lines can be used to generate developing dopamine neurons, providing a new model to investigate the molecular pathogenesis of LND. 
10.1212/WNL.0000000000203414