Conventional methods of neuronal differentiation in human induced pluripotent stem cells (iPSCs) are tedious and complicated, involving multistage protocols with complex cocktails of growth factors and small molecules. Artificial extracellular matrices with a defined surface topography and chemistry represent a promising venue to improve neuronal differentiation in vitro. In the present study, we test the impact of a type of colloidal self-assembled patterns (cSAPs) called binary colloidal crystals (BCCs) on neuronal differentiation. We developed a CRISPR activation (CRISPRa) iPSC platform that constitutively expresses the dCas9-VPR system, which allows robust activation of the proneural transcription factor NEUROD1 to rapidly induce neuronal differentiation within 7 days. We show that the combinatorial use of BCCs can further improve this neuronal differentiation system. In particular, our results indicate that fine tuning of silica (Si) and polystyrene (PS) particle size is critical to generate specific topographies to improve neuronal differentiation and branching. BCCs with 5 μm silica and 100 nm carboxylated PS (PSC) have the most prominent effect on increasing neurite outgrowth and more complex ramification, while BCCs with 2 μm Si and 65 nm PSC particles are better at promoting neuronal enrichment. These results indicate that biophysical cues can support rapid differentiation and improve neuronal maturation. In summary, our combinatorial approach of CRISPRa and BCCs provides a robust and rapid pipeline for the in vitro production of human neurons. Specific BCCs can be adapted to the late stages of neuronal differentiation protocols to improve neuronal maturation, which has important implications in tissue engineering, in vitro biological studies, and disease modeling.

Item Type:	Refereed Article
Keywords:	CRISPR activation; artificial extracellular matrix; colloidal self-assembled patterns (cSAPs); induced pluripotent stem cells (iPSCs); neuronal differentiation.
Research Division:	Biomedical and Clinical Sciences
Research Group:	Ophthalmology and optometry
Research Field:	Ophthalmology
Objective Division:	Health
Objective Group:	Clinical health
Objective Field:	Diagnosis of human diseases and conditions
UTAS Author:	Hewitt, AW (Professor Alex Hewitt)
ID Code:	155613
Year Published:	2022
Web of Science® Times Cited:	3
Deposited By:	Menzies Institute for Medical Research
Deposited On:	2023-03-02
Last Modified:	2023-04-20
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