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

诱导多能干细胞(IPSCs)可以分化为人体每种类型的细胞，但具体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的机制尚不清楚。我们最近利用多光束激光技术在聚酰亚胺中产生了周期性在亚微米范围内的沟槽-脊状结构，这种结构可以诱导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，我们希望探索利用这项技术来调节IPSCs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。我们将研究如何应用多光束干涉来产生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，并将材料降解降至最低，包括聚苯乙烯(PS)-这也可以用于构建传统的PS组织培养板。(2)表面模式对多能性的影响。我们将进一步确定IPSC集落的形态、运动性、细胞分裂和空间异质性如何受到亚微米模式的影响。此外，还将分析对重新编程效率、细胞骨架组织和基因表达谱的影响。(3)地形对IPSCs分化的影响。我们将遵循以下假设，即亚微米结构可以影响特定血统的分化。为此，我们将用形态因子处理IPSC，研究IPSC群体内的早期分化事件和空间重组，并在底物上诱导无偏见的多谱系分化或谱系特异性分化。(4)YAP/TAZ在识别地形线索中的作用。YAP/TAZ通路在机械信号转导中起着核心作用。我们将利用CRISPR-Cas9n技术调节YAP和TAZ的表达，以确定细胞对表面形貌的反应。此外，对相关信号级联的影响将通过单细胞RNA测序来解决。这项提议结合了激光技术和干细胞研究的专业知识。它为揭示表面形貌对多能干细胞的影响提供了新的视角，最终可能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.