Midbrain Dopamine Neurons Derived from Induced Pluripotent Stem Cells from Patients with Lesch Nyhan Disease and Gene-Edited Knock-In HPRT1 Mutations (P10-9.007)

Abstract

<h3>Objective:</h3> To create a novel in-vitro model to study molecular pathogenesis of midbrain dopamine abnormalities during neuronal development in Lesch Nyhan disease (LND). <h3>Background:</h3> 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. <h3>Design/Methods:</h3> 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&gt;T stop codon in the <i>HPRT1</i> 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. <h3>Results:</h3> 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. <h3>Conclusions:</h3> We have generated a large bank of iPSCs from individuals with LND, <i>HPRT1</i> 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. <b>Disclosure:</b> Ms. Seifar has nothing to disclose. Ms. Sutcliffe has nothing to disclose. Dr. Jinnah has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Takaha/Ene. Dr. Jinnah has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Ipsen. Dr. Jinnah has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Ipsen. Dr. Jinnah has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Abbvie. The institution of Dr. Jinnah has received research support from Addex. The institution of Dr. Jinnah has received research support from Aeon. The institution of Dr. Jinnah has received research support from Revance. The institution of Dr. Jinnah has received research support from Jazz.