Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia.
Ophthalmology, Department of Surgery, University of Melbourne, Parkville 3010, Australia.
ACS Appl Mater Interfaces. 2022 Feb 23;14(7):8669-8679. doi: 10.1021/acsami.1c17975. Epub 2022 Feb 15.
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 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 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 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, biological studies, and disease modeling.
传统的人类诱导多能干细胞（iPSC）神经元分化方法繁琐复杂，涉及多阶段方案和复杂的生长因子及小分子鸡尾酒。具有明确表面形貌和化学性质的人工细胞外基质为改善神经元分化提供了一个很有前途的途径。在本研究中，我们测试了一种称为二元胶体晶体（BCC）的胶体自组装图案（cSAP）对神经元分化的影响。我们开发了一种 CRISPR 激活（CRISPRa）iPSC 平台，该平台持续表达 dCas9-VPR 系统，可快速诱导神经元分化，在 7 天内诱导神经元分化。我们表明，BCC 的组合使用可以进一步改善这种神经元分化系统。特别是，我们的结果表明，精细调整二氧化硅（Si）和聚苯乙烯（PS）颗粒的大小对于产生特定的形貌以改善神经元分化和分支至关重要。5 μm 的二氧化硅和 100 nm 的羧基化 PS（PSC）的 BCC 对增加神经突生长和更复杂的分支具有最显著的效果，而 2 μm 的 Si 和 65 nm 的 PSC 颗粒的 BCC 更有利于促进神经元富集。这些结果表明，生物物理线索可以支持快速分化并改善神经元成熟。总之，我们的 CRISPRa 和 BCC 组合方法为产生人类神经元提供了一种强大而快速的途径。特定的 BCC 可以适应神经元分化方案的后期阶段，以改善神经元成熟，这在组织工程、生物学研究和疾病建模方面具有重要意义。
