Establishment of an inducible human pluripotent stem cell-based 3D model of Alzheimer's disease

Abstract

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the aged population. The vast majority of cases are sporadic, however, various mutations may cause early-onset, familial AD (FAD) variants. AD is characterized by deposition of amyloid β (Aβ) plaques in the extracellular space and hyperphosphorylated tau (p-tau) within neurons. This project aimed to implement a human stem cell-based model in a 3D setting in order to more authentically model extracellular Aβ accumulation and its downstream pathogenic effects. To maximize production of aggregation-prone Aβ42, the safe-harbor locus AAVS1 of human induced pluripotent stem cells (iPSCs) was modified to conditionally overexpress FAD mutations in the genes encoding the amyloid precursor protein (APPSwe/Lon) and Presenilin-1 (PS-1ΔE9). Transgenic iPSCs were differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, and embedded in a 200-300 µm thick Geltrex matrix for long-term differentiation in 3D. Induction with doxycycline caused 40-fold increased Aβ42 secretion in 2D cultures, whereas Aβ was efficiently entrapped in the gel matrix in 3D cultures. After 8 weeks, induced 3D cultures displayed Thioflavin T-positive Aβ deposits that strongly resemble amyloid plaques in size, structure and specific autofluorescence. After 16 weeks, induced 3D cultures contained TBS-insoluble Aβ. P-tau was present in induced 3D cultures from week 6, and fibril-specific tau epitopes could be detected after 16 weeks. As expected, p-tau pathology did not develop in the presence of γ-secretase inhibitors. Cellular energy metabolism was impaired in induced cultures as p-tau-positive neurons displayed hallmarks of mitochondrial fragmentation, alongside a reduction in essential respiratory chain complexes and lowered respiratory capacity. Induced cultures also showed neuronal degeneration compared to uninduced and γ-secretase-inhibited controls. It follows that 3D matrix cultures of lt-NES neurons represent a valid model to study intra- and extracellular AD pathology in an authentic human system.