Sahni Geetika, Yuan Jun, Toh Yi-Chin
Department of Biomedical Engineering, National University of Singapore.
Department of Biomedical Engineering, National University of Singapore; Singapore Institute of Neurotechnology (SINAPSE), National University of Singapore;
J Vis Exp. 2016 Jun 17(112):54097. doi: 10.3791/54097.
Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.
人类多能干细胞（hPSC），包括胚胎干细胞和诱导多能干细胞，具有分化为所有三个胚层的内在能力。这使其成为再生医学以及正常和患病器官发生实验建模中极具吸引力的细胞来源。然而，hPSC在体外的分化是异质性的且空间无序。细胞微图案化技术有可能提供空间控制干细胞微环境并组织最终分化命运的方法。对hPSC进行微图案化需要考虑hPSC存活和维持的严格要求。在此，我们描述模板微图案化作为一种与hPSC高度兼容的方法。hPSC微图案由细胞模板通孔的几何形状确定，这些通孔物理限制了hPSC能够接触并附着到下层细胞外基质包被底物的位置。由于这种操作模式，与其他细胞微图案化方法相比，在使用能够充分支持hPSC的底物方面具有更大的灵活性。我们还强调了成功生成hPSC微图案的关键步骤。例如，我们证明hPSC的模板微图案化可用于调节细胞 - 细胞和细胞 - 基质粘附的空间极化，进而决定中内胚层分化模式。这种对hPSC进行微图案化的简单且稳健的方法拓宽了建立实验模型以研究早期胚胎发育过程中组织组织和图案形成的前景。
