Generating a novel and reliable human iPSC‐derived midbrain organoid model of sporadic progressive supranuclear palsy

Background The accumulation of abnormal tau protein in neurons and glia in the human brain is the defining feature of neurodegenerative diseases known as tauopathies. Progressive supranuclear palsy (PSP), the most common primary tauopathy, is typified by selective vulnerability of dopaminergic neurons and glia in the midbrain leading to an atypical parkinsonian movement disorder. To investigate candidate disease mechanisms underlying PSP, there is a critical need for model systems that more accurately recapitulate the cellular and molecular environment in the human brain. Human induced pluripotent stem cell (hiPSC)‐derived organoid models have emerged as a powerful tool to address this gap. Method Skin biopsies were collected from living clinically diagnosed PSP patients or during autopsy. Fibroblasts were cultured and reprogrammed into hiPSCs using Sendai virus. HiPSCs were maintained with StemCultures FGF2 Discs to improve pluripotency and FACS was performed to confirm pluripotency marker expression. To generate midbrain organoids, hiPSCs were seeded into suspension spinner flasks, patterned using pharmacological directed differentiation, and grown for four months. Reliable patterning was confirmed with qRT‐PCR, immunohistochemistry and immunoblot using a panel of cell‐type specific markers. Astrocytes were extracted from mature organoids, cultured, and screened for astrocyte‐specific markers. Result Fibroblasts have been banked from twenty‐two PSP patients and seven reprogrammed into hiPSCs. Sporadic case status was determined by Sanger sequencing confirming the absence of a MAPT mutation. We found that hiPSCs grown with controlled‐release FGF2 discs were 90‐100% positive for pluripotency markers and negative for off‐target genes. During patterning, organoids displayed morphological and cytoarchitectural patterns consistent with developing neuroectoderm and midbrain. Midbrain neural progenitor and dopaminergic markers such as FOXA2, LMX1A, and TH were positive in a time‐dependent manner, with mature dopaminergic neurons expressing NURR1 and GIRK2 detected by day 30. GFAP‐positive astrocytes appeared around day 100. Astrocytes extracted from mature organoids were positive for multiple astrocyte markers. Conclusion Sporadic PSP patient hiPSCs reliably differentiate into midbrain dopaminergic organoids and astrocytes, resulting in a sporadic tauopathy model containing key cell types affected in PSP. This cell collection is a valuable resource to investigat