Sharaf Ahmed, Frimat Jean-Philippe, Accardo Angelo
Department of Precision and Microsystems Engineering, Faculty of Mechanical Engineering, Delft University of Technology, Mekelweg 2, 2628 CD Delft, the Netherlands.
Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands.
Mater Today Bio. 2024 Nov 2;29:101325. doi: 10.1016/j.mtbio.2024.101325. eCollection 2024 Dec.
The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far, and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these, yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA concerning neurite outgrowth in mechanically confined microenvironments. Moreover, our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study, we fabricated, via two-photon polymerization (2PP), 2.5D microgrooves and 3D polymeric microchannels, with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves, both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels, both proteins' N/C ratio exhibited minima in presence of 5 or 10 μm channels, a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion, actin, and nuclear polarization. Further, especially in presence of 2.5D microgrooves, cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally, we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly, YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves, indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments.
到目前为止，多项研究已报道了机械信号对细胞行为的影响，其中一个特别受关注的方面是机械转导蛋白在神经元发育中的作用。在这些蛋白中，Yes相关蛋白（YAP）在神经元发育中发挥多种功能，如神经祖细胞的迁移和分化，而心肌素相关转录因子A（MRTFA）则促进神经突生长和轴突导向。这两种蛋白通过其信号通路有着间接交织的命运。关于YAP和MRTFA在机械受限微环境中对神经突生长的作用，相关文献较少。此外，我们对它们在工程化受限微环境中培养的未成熟神经元中的关系仍缺乏了解。在本研究中，我们通过双光子聚合（2PP）制造了直径范围为5至30μm的2.5D微槽和3D聚合物微通道。我们在2.5D和3D微结构上培养SH-SY5Y细胞，并将它们分化为未成熟的神经元样细胞，以研究机械限制对细胞形态和蛋白质表达的影响。在2.5D微槽中，YAP和MRTFA的核/质（N/C）比在10μm的微槽中均表现出最大值，表明与机械应力诱导的限制有很强的关系。在3D微通道中，两种蛋白的N/C比在5或10μm的通道中表现出最小值，这种行为与在2.5D微槽中观察到的相反，这表明由于粘着斑、肌动蛋白和核极化，3D微环境的几何形状和机械限制与2.5D微环境相比是独特的。此外，特别是在2.5D微槽存在的情况下，细胞的YAP N/C比与平均神经突长度之间呈现反比关系。最后，我们还在微结构上培养了人类诱导多能干细胞（hiPSC），并将它们分化为皮质神经元，培养长达2周。有趣的是，YAP和MRTFA的N/C比在10μm的2.5D微槽周围也显示出最大值，表明我们研究的生理相关性。我们的结果阐明了2.5D和3D受限微环境在神经元发育中可能引起的差异，并为理解机械转导蛋白之间的复杂相互作用及其对工程化细胞微环境中神经细胞命运的影响铺平了道路。
