Objective

In order to study neurodegenerative disease in vitro on a human physiological background the current iPSC-derived neuronal model is widely utilized. Conventional two-dimensional (2D) in vitro culturing methods poorly recapitulate the tissue microenvironment and for this reason, three-dimensional (3D) in vitro systems are of high interest. Synaptic maturation, and expression of pathological markers in disease models were shown to be enhanced in 3D hydrogel-based systems compared to conventional 2D systems. Setting up hydrogel-based culture systems by encapsulation methods can be laborious and the reproducibility can be limited. In addition, standard encapsulation protocols do not enable the seeding of neuronal and glial cells at different time points to establish 3D co-cultures [1].

Here we have developed a protocol for the neuronal differentiation of human iPSCs in the 3DProSeed hydrogel well plate and established co-cultures with glia cells by sequential seeding. The 3DProSeed well plate features a high-content analysis-compatible format (96-wells) and synthetic PEG-based hydrogels pre-assembled in the plate and stored hydrated, ready for cell seeding. The engineering surface of the 3DProSeed hydrogels enhances cell growth and migration inside the hydrogel [2].

iPSCs were seeded onto the 3DProSeed gel and directed to neurons by the forced expression of NGN2 [3]. After NGN2-positive cell selection, glial cells were subsequently seeded onto the gel to promote cell survival and maturation. After 21 days the iPSCs were successfully differentiated into neurons with comparable cell survival to conventional 2D co-cultures. Synaptic and neuronal maturation were analyzed by immunocytochemical and Western blotting methods. Preliminary results point to a difference in neuronal maturation markers in 3D cultures compared to 2D.  In the near future we aim to establish Alzheimer’s disease relevant cultures, in order to establish an in vitro human model compatible with pharmacological screening.

References

[1] Choi et al. Nature, 2014 ; Kim et al. Nat. Prot., 2015

[2] Ning Zhang et al. , SLAS Discovery, 2017.

[3] Yingsha Zhang et al., Neuron, 2013.