Disease Modeling Using Patient iPS Cell-Derived Cells and Organoids

National Center For Advancing Translational Sciences

Investigators

Abstract

We have generated nine iPS cell lines from patient samples for three genetic diseases. These lines, developed in collaboration with the NHLBI Stem Cell Core, are fully characterized and ready for use in disease modeling applications. Batten disease. It is a rare, severe, autosomal recessive pediatric neurodegenerative disorder. It manifests with progressive vision impairment, cognitive decline, behavioral dysfunction, seizures, and loss of motor function, leading to a significantly reduced lifespan. The disease is caused by mutations in the CLN3 gene, which result in the pathological accumulation of glycerophosphodiesters within lysosomes and subsequent lysosomal dysfunction. In collaboration with Dr. Ann Dang Do (NICHD/NIH), we generated six induced pluripotent stem cell (iPSC) lines from patient fibroblasts. These lines were extensively passaged and characterized to confirm pluripotency using immunocytochemistry, flow cytometry, and teratoma formation assays (Dwojak E. et al., 2024. Stem Cell Res. 81: 103563). Currently, we are leveraging these patient-derived iPSC lines for assay development, aiming to establish a robust platform for a high-throughput drug repurposing screen to identify compounds that can ameliorate the disease phenotype in patient iPSC-derived cells. Sanfilippo syndrome. This disease, also known as Mucopolysaccharidosis type IIIB (MPS IIIB), is a severe autosomal recessive lysosomal storage disorder. It is caused by mutations in the NAGLU gene, leading to progressive neurodegeneration, significant behavioral problems, and motor dysfunction. We have generated a human iPSC line from patient fibroblasts carrying a homozygous p.R626X (c.1876C > T) mutation in the NAGLU gene. This iPSC line has been thoroughly characterized and is suitable for modeling MPS IIIB disease (Rodriguez-Lopez A et al., 2024. Stem Cell Res. 81: 103612). Alagille Syndrome (ALGS). It is an autosomal dominant disorder primarily caused by mutations in JAG1 (Jagged1), with a smaller percentage (~2–3%) attributed to NOTCH2 mutations. Both gene mutations impair the critical Notch signaling pathway. Clinically, ALGS presents multi-systemic manifestations including bile duct paucity and cholestasis, characteristic cardiac defects (predominantly affecting pulmonary arteries), and skeletal abnormalities of the vertebrae and facial bones. We have established two human iPSC lines, TRNDi037-A (from a female patient) and TRNDi038-A (from a male patient), derived from their respective lymphoblastoid cell lines (LCLs). TRNDi037-A carries a heterozygous p.Val422Lysfs6 mutation in exon 10 of JAG1, while TRNDi038-A harbors a heterozygous c.1057G >T (p.Glu353) mutation in exon 8 of JAG1. These iPSC lines were generated using episomal reprogramming with six transcription factors: Oct4, Sox2, Nanog, Lin28, Klf4, and L-Myc (Evans EF et al., 2025. Stem Cell Res. 82: 103634). Both lines have been thoroughly characterized and are prepared for differentiation into relevant cell types, such as hepatocytes, to facilitate robust disease modeling of ALGS. Midbrain organoids model for Parkinson’s disease (PD). Animal models have been used for Parkinson’s disease for many years which could not recapitulate all the human disease phenotypes. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopamine-producing neurons and the accumulation of Lewy bodies containing misfolded α-synuclein (α-syn). We have employed midbrain organoids, a type of brain organoids enriched in dopaminergic neurons, that exhibits the degeneration of dopaminergic neurons and the accumulation of Lewy bodies containing misfolded α-synuclein (α-syn). We tested and observed promising therapeutic effect of Tilorone, an antiviral drug identified from previous drug repurposing project in this organoids model of Parkinson’s diseases. The study highlights the utility of 3D brain organoid models in evaluation of drug efficacy for neurodegenerative diseases.