Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture

Abstract

Background: Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming.

New method: We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures.

Comparison with existing methods: In contrast to other hydrogel-based 3D culture techniques, which require full encapsulation of cells, our hydrogel allows the seeded iPSCs and iPSC neurons to simply infiltrate the gel.

Results: IPSC-neurons grew to a depth of 500 µm into the hydrogel. Cell viability was comparable to 2D cultures over the course of three weeks, with even better neuronal survival in 3D cultures at the one-week time point. Levels of neuronal and synaptic maturation markers, namely, neural cell adhesion molecule 1 (NCAM1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2, were strongly increased in 3D cultures. Furthermore, we identified 4-repeat (4R) tau in 3D cultures, which was not detectable in 2D cultures.

Conclusions: We describe a simple, hydrogel-based method for 3D iPSC culture that can serve as a fast and drug-screening-compatible platform to identify new mechanisms and therapeutic targets for brain diseases. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment.

Keywords: 3D cell culture; Hydrogel; IPSC-derived neurons; IPSCs; Neuronal maturation.