Combinatorial application of binary colloidal crystals biomaterial and CRISPR activation system to direct pluripotent stem cell differentiation into neurons
Daniel Urrutia-Cabrera, Roxanne Hsiang-Chi Liou, JiaoLin, Kun Liu, Sandy S.C. Hung, Alex W. Hewitt, Peng-Yuan Wang, Raymond Ching-Bong Wong
Abstract
Human induced pluripotent stem cells (iPSCs) represent a promising cellular source for the generation of neurons in vitro, which has fostered the development of better models of the human nervous system and regenerative medicine. However, conventional methods of neuronal differentiation are somewhat tedious and complicated, involving multi-stage protocols with complex cocktails of growth factors and small molecules. Artificial extracellular matrix with defined surface topography and chemistry represent a promising venue to improve the neuronal differentiation in vitro. In the present study, we test the impact of a type of colloidal self-assembled patterns called binary colloidal crystals (BCCs) in neuronal differentiation. We developed a CRISPR activation (CRISPRa) iPSC platform that constitutively expresses the dCas9-VPR system, which allows robust activation of endogenous gene expression to promote cell differentiation. Using this system we showed that the sole activation of proneural transcription factor NEUROD1 can rapidly induce differentiation into neurons within seven days, with the first neuronal morphology observed as early as four days. Furthermore, we provide evidence that the combinatorial use of BCCs allow the generation of enriched neuronal cultures and improved neuronal maturation, including increased neurite outgrowth and more complex ramification. These results indicate that biophysical cues can support rapid differentiation and improve neuronal maturation. Our combinatorial approach of CRISPRa and BCCs provides a robust and rapid pipeline for in vitro production of human neurons, which have important implications in tissue engineering and in vitro biological studies and disease modeling.
Read the publication here.
Comments