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

多能干细胞(PSC)可以在三个生殖层分化出不同类型的细胞，是研究体外组织发育和谱系承诺的良好模型。虽然传统的PSCs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并入。我们以前的研究已经证明，地形通过参与和重组局灶性黏附对干细胞命运的决定产生重大影响。然而，PSC在体外生态位中维持多能性和谱系承诺的生物物理调节仍然是一个谜。我们假设，1)生物物理线索，如纳米拓扑学，通过焦点黏附信号通路调节多能性维持；2)合适的生物物理环境和生化因素可以协同作用，增强人类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工具和3D生物反应器。 纳米制造技术使微电子学、光学和体外生物系统在研究细胞-基质相互作用方面取得突破。然而，有两个关键因素限制了纳米拓扑术在干细胞生物学研究和细胞扩增的工业放大中的应用：构图材料的性质和传统的2D图案化培养表面。因此，在拟议的研究计划中，我们的目标是 1)开发机械性能和折射率可调的纳米粒子研究工具，通过超分辨显微镜研究细胞-基质相互作用； 2)研究多能性维持过程中细胞-基质的相互作用，以获得促进PSC扩增的最佳生物物理条件； 3)研制用于PSC扩容的三维纳米生物反应器； 4)应用生物反应器和纳米拓扑学工具扩大PSC的生产用于研究。 我们的创新方法和研究计划将开发新的研究工具来研究干细胞。有了新的工具，该计划将通过提供对PSC谱系承诺的生物物理调节的更深层次的理解，在干细胞生物学研究中带来新的见解。这将是对干细胞和机械生物学研究界的重大贡献。干细胞扩增的最低限度、生物物理和生物化学定义的利基也将提供一种有效的手段，以满足不同应用所需的一致和可复制的细胞的严格需求，如生物研究或药物开发。 最终，该计划将产生新的、可能获得专利的设备和概念，这些设备和概念适用于许多研究领域，并将有助于加拿大生物技术产业的发展。它还将为这些至关重要的学术和工业社区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.