Nanopatterning of advanced research tools to harness the mechanobiology of cell-matrix interaction for stem cell expansion

多能干细胞（PSC）可以产生所有三种胚层的不同细胞类型，是体外研究组织发育和谱系承诺的良好模型。虽然PSCs的传统培养条件侧重于生化定义，但干细胞生态位中存在的生物物理线索并未在体外纳入。我们之前的研究已经证明了地形通过参与和重组局灶黏附对干细胞命运的决定有重要影响。然而，体外生态位中PSC多能性维持和谱系承诺的生物物理调控仍然是一个谜。我们假设1)纳米形貌等生物物理线索通过黏附信号通路调控多能性维持；2)适宜的生物物理环境和生物化学因子协同作用，促进人体PSC的维持；3)先进的纳米制造技术可以制造用于干细胞扩增的纳米化工具和3D生物反应器。纳米制造技术使微电子、光学和体外生物系统研究细胞-基质相互作用取得突破。然而，两个关键因素限制了纳米形貌在干细胞生物学研究和细胞扩增工业规模中的应用：分别是图像化材料的特性和传统的二维图像化培养表面。因此，在我们提出的研究计划中，我们的目标是***1)开发具有可调力学性能和折射率的纳米研究工具，以便通过超分辨率显微镜研究细胞-基质相互作用；***2)研究多能性维持过程中细胞与基质的相互作用，寻找促进PSC扩增的最佳生物物理条件；***3)开发用于PSC膨胀的三维纳米生物反应器；***4)应用生物反应器和纳米形貌工具扩大PSC的生产规模。我们的创新方法和研究项目将开发新的研究工具来研究干细胞。有了这些新工具，该项目将通过更深入地了解PSC谱系承诺的生物物理调控，为干细胞生物学研究提供新的见解。这将是对干细胞和机械生物学研究界的重大贡献。一个简约的、生物物理和生物化学定义的干细胞扩展生态位也将提供一种有效的手段，以满足生物研究或药物开发等不同应用对一致和可再生细胞的严格要求。***最终，该计划将产生新的，潜在的专利设备和概念，适用于许多研究领域，并将有助于发展加拿大的生物技术产业。它还将为这些重要的学术和工业团体培养下一代生物工程师。

Pluripotent stem cells (PSC), which can give rise to different cell types of all three germ layers, are excellent models to study tissue development and lineage commitment in vitro. While conventional culture conditions for PSCs focus on biochemical definition, biophysical cues that are present in the stem cell niche are not incorporated in vitro. Our previous studies have demonstrated the significant influence of topography on stem cell fate determination through the involvement and restructuring of focal adhesion. Yet the biophysical regulation of PSC pluripotency maintenance and lineage commitment in the in vitro niche remains a mystery. We hypothesize that 1) biophysical cues such as nanotopography regulate pluripotency maintenance via a focal adhesion-signaling pathway; 2) a suitable biophysical environment and biochemical factor can work synergistically to enhance human PSC maintenance; and 3) advanced nanofabrication techniques can fabricate nanopatterned tools and 3D bioreactors for stem cell expansion.***Nanofabrication technologies enable breakthroughs in microelectronics, optics, and in vitro biological systems to study cell-matrix interaction. However, 2 key factors limit the application of nanotopography for stem cell-biology studies and industrial scale-up of cell expansion: the properties of the patterning materials, and conventional 2D patterned culture surfaces, respectively. Therefore, in the proposed research program, we aim to ***1) develop nanopatterned research tools with tunable mechanical properties and refractive index to enable the study of cell-matrix interactions through super-resolution microscopy; ***2) investigate the cell-matrix interaction during pluripotency maintenance to harness the optimal biophysical conditions that enhance PSC expansion; ***3) develop a 3D nanopatterned bioreactor for PSC expansion; and ***4) apply bioreactor and nanotopography tools in scaling up the production of PSC for research.***Our innovative approaches and research program will develop new research tools to study stem cells. With the new tools, the program will lead to new insights in stem cell biology research by providing a deeper understanding of the biophysical regulation of PSC lineage commitment. This will be a significant contribution to the stem cell and mechanobiology research communities. A minimalistic, biophysically- and biochemically-defined niche for stem cell expansion will also provide an effective means to meet the rigorous demand for consistent and reproducible cells needed for different applications such as biological studies or drug development. ***Ultimately, this program will yield new, potentially patentable devices and concepts that are applicable to many research areas and will help grow Canada's biotechnology industries. It will also produce the next generation of bioengineers for these vital academic and industrial communities.