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

Sherida M De Leeuw,Stephanie Davaz,Debora Wanner,Vincent Milleret,Martin Ehrbar,Anton Gietl,Christian Tackenberg

doi:10.1016/j.jneumeth.2021.109254

Sherida M De Leeuw, Stephanie Davaz + Show 5 more

Open Access

https://doi.org/10.1016/j.jneumeth.2021.109254

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Abstract

BackgroundInduced 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 methodWe cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. Comparison with existing methodsIn 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. ResultsIPSC-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. ConclusionsWe 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.

Highlights

Induced pluripotent stem cell technology has induced a paradigm shift in biomedical research, as it enables the use of human cells for disease modeling and drug discovery
We further provided evidence for the increased maturation of Induced pluripotent stem cell (iPSC) neurons in a 3D microenvironment
This differentiation technique requires sequential seeding, as primary mouse glial cells need to be added in a 1:1 ratio to neurons in 2D and 3D, 3 d after initial iPSC plating, as previously described (Birnbaum et al, 2018) (Fig. 1A)

Summary

Introduction

Induced pluripotent stem cell (iPSC) technology has induced a paradigm shift in biomedical research, as it enables the use of human cells for disease modeling and drug discovery. Some limitations exist that reduce the value of iPSCs for modeling age-related diseases, such as the immature nature of differentiated iPSCs and their resemblance to fetal tissue (de Leeuw and Tackenberg, 2019). Elevated synaptic density was observed in iPSC-derived neurons embedded in a layered, hyaluronic acid-based hydrogel (Zhang et al, 2016). New method: We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. 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. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment

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