Development of an iPSCs-derived macrophage model of Gaucher disease to enable high-throughput screening and drug discovery

Authors

• D Dabral
• Y Chen
• Y Fang
• Y Lin
• T Poudel
• D Tao
• J Marugan
• E Sidransky
• M Henderson

Abstract

Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, which encodes the glycolipid hydrolase glucocerebrosidase (GCase). Most pathogenic GBA1 variants cause misfolding of GCase, leading to its retention in the endoplasmic reticulum and destruction through the ER-associated degradation pathway. While various cell types are affected in GD, macrophages (MØ) are particularly impacted, leading to enlarged spleen in patients. Indeed, MØ packed with glucosylceramide and glucosylsphingosine (GluSph) are called ‘Gaucher Cells’ and are hallmark of GD. Towards the development of chaperones that restore GCase function, we have developed isogenic iPSC lines (WT/WT, N370S/N370S, L444P/L444P, KO/KO), and differentiated them into mature MØ, as confirmed by flow cytometry using panel of surface markers. Lipid measurement by mass spectrometry revealed significant accumulation of GluSph. To assess the lysosomal GCase activity, we utilized a novel in-house GCase activity probe and high-content imaging from 384-well plates. We have optimized a lipid-based assay using Bravo liquid handling system in tandem to high-throughput RapidFire multiple reaction monitoring mass spectrometer, to measure GluSph from 384-well plates. High MØ production in combination with the RapidFire-MS and high content imaging enable high-throughput screening and will serve as powerful tool to facilitate the development of therapeutic chaperones for GD and GBA1-associated Parkinson’s disease.
Funding: This work was supported by the intramural research programs at NCATS and NHGRI and by a Michael J. Fox Foundation award.

Scientific Focus Area: Stem Cell Biology

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