The Impact of sub-µm Surface Topography on Pluripotent Stem Cells

诱导多能干细胞（iPSCs）可以分化成人体的每种细胞类型，但关于特定的表面模式如何影响多能状态或引导谱系特异性细胞分化知之甚少。我们最近使用多束激光技术在聚酰亚胺中产生具有亚微米范围内的周期性的槽脊结构，其诱导iPSC集落的伸长，引导顶端肌动蛋白纤维的取向，并指导细胞分裂的极性。接下来，我们希望探索使用该技术调节iPSC功能的可能性，具体目标如下：（1）定制亚微米结构的生物材料。我们将研究如何多光束干涉可以应用于生成更复杂和均匀的表面图案。为此，将研究超短脉冲激光和改进的光学装置，以在不同材料上产生合适的表面纹理，使材料降解最小化，包括聚苯乙烯（PS）-这也可以应用于结构传统的PS组织培养板。(2)表面图案对多能性的影响。我们将进一步确定亚微米模式如何影响iPSC集落的形态、运动性、细胞分裂和空间异质性。此外，将分析对重编程效率、细胞骨架组织和基因表达谱的影响。(3)地形对iPSC分化的影响。我们将遵循亚微米结构可以影响谱系特异性分化的假设。为此，我们将用形态发生剂处理iPSC以研究iPSC集落内的早期分化事件和空间重组，并且我们将在基质上诱导无偏的多谱系分化或谱系特异性分化。(4)雅普/TAZ在地形线索识别中的作用雅普/TAZ通路在机械力传导中起着重要作用。我们将用CRISPR-Cas9 n技术调节雅普和TAZ的表达，以确定细胞对表面形貌反应的后果。此外，对相关信号级联的影响将通过单细胞RNA测序来解决。这项建议结合了激光技术和干细胞研究的专业知识。它提供了新的视角来解开表面形貌对多能干细胞的影响，这可能最终支持再生医学和药物筛选的定向分化。

Induced pluripotent stem cells (iPSCs) can differentiate into every cell type of the human body, but little is known about how specific surface patterns impact on pluripotent state or guide lineage specific cellular differentiation. We have recently used multi-beam laser technology to generate groove-ridge structures in polyimide with a periodicity in the submicrometer range that induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. In continuation we want to explore the possibility of using this technology to modulate the function of iPSCs with the following specific aims: (1) Tailored sub-micrometer structured biomaterials. We will investigate how multi-beam interference can be applied to generate more complex and homogeneous surface patterns. To this end ultrashort pulsed lasers and modified optical setups will be investigated for the generation of suitable surface textures on different materials with minimized material degradation, including polystyrene (PS) – that could also be applied to structure conventional PS tissue culture plates. (2) Impact of surface patterns on pluripotency. We will further determine how morphology, motility, cell divisions and spatial heterogeneity of iPSC colonies are affected by sub-micrometer patterns. Furthermore, effects on reprogramming efficiency, cytoskeletal organization, and gene expression profiles will be analyzed. (3) Impact of topography on differentiation of iPSCs. We will follow the hypothesis that sub-µm structures can influence lineage-specific differentiation. To this end, we will treat iPSCs with morphogens to study early differentiation events and spatial reorganization within iPSC colonies, and we will induce unbiased multi-lineage differentiation or lineage-specific differentiation on substrates. (4) The role of YAP/TAZ in recognizing topographic cues. The YAP/TAZ pathway plays a central role in mechanotransduction. We will modulate expression of YAP and TAZ with CRISPR-Cas9n technology to determine the sequel on cellular response to surface topography. Furthermore, the impact on relevant signal cascades will be addressed by single-cell RNA-sequencing. This proposal combines expertise of laser technology and stem cell research. It provides new perspectives to unravel effects of surface topography on pluripotent stem cells, which may ultimately support directed differentiation for regenerative medicine and drug screening.