人类多能干细胞的模板微图案用于探测分化命运的空间组织
Summary
人多能干细胞（hPSCs）具有分化和自我组织成不同的组织型态的内在能力;尽管这需要空间环境梯度的表现。我们提出模版图案化作为一种简单而可靠的方法，以产生生物化学和机械梯度用于控制HPSC分化模式。
Abstract
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.
Introduction
人多能干细胞（hPSCs），包括胚胎干细胞（胚胎干细胞）和诱导多能干细胞（人iPS细胞），被广泛利用于再生医学以及正常和病变器官的实验模型，因为它们的分化潜能的进入所有的细胞谱系三种胚层1,2。 hPSCs分化命运是可以调节通过物理线索3-5介导的自分泌或旁分泌信号1以及机械传导过程当地的环境因素非常敏感。细胞微图案包括一组已发展到在空间上组织的细胞群的几何形状和位置作为平均值来控制本地细胞微环境，如细 胞-细胞相互作用6和细胞-基质相互作用3的技术。在hPSCs的背景下，细胞微已被用来获得显著的认识到如何利基依赖耳鼻喉科自分泌信号调节胚胎干细胞多能性分化的第7和组织成早期胚胎分化的模式6。二维和三维微图案hPSCs已被用来控制多细胞型态，这反过来又影响分化决定成三种胚层8,9的菌落尺寸。我们采用多HPSC微图案来调制HPSC菌落内的细胞-细胞和细胞-基质相互作用的程度来探测整联E-钙粘蛋白的串扰可以如何产生细胞命运异质10。从朝hPSCs作为实验模型用于发育疾病11，要研究的组织或器官发育过程中的生长因子和激素的影响药毒性筛选的多细胞微图案的应用上述报告打开新的途径的示范，并解开的形成组织模式。
细胞micropatter有无数宁技术已被开发作为由Falconnet 等审查。人 12，但只有一小部分，如微接触印刷7,8,13，微孔文化14,15，光图案6和microstencils 16已成功地实施hPSCs。与缩微hPSCs的挑战在于他们的脆弱性和具体的细胞外基质（ECM）和生长条件的细胞附着和生存的严格的要求。对于2D HPSC模式，微接触印刷是最常用的方法，以产生对组织培养和玻璃基板13 HPSC微图案之一。该方法可用于在HPSC培养，包括层粘连蛋白和基底膜基质，如基质胶中使用图案共同的ECM。然而，它通常需要用聚-D-赖氨酸辅助的两步涂层的方法，并且需要特定的惰性大气和湿度条件下，使稳定的ECM微图案为hPSCs到6,13- 附加。每个图案化方法的首要考虑因素是表面修饰制度是否能够产生在所需的几何分辨率HPSC粘性ECM模式，同时尽量减少非特异性的细胞附着到周边地区。
在这里，我们报告的模板使用缩微作为一种简单的方法来产生HPSC微图案无粘结ECM模式产生之前的其他表面改性步骤hPSCs附加上。小区模版包括薄膜， 例如，聚二甲基硅氧烷（PDMS）片材，具有微米到毫米的通孔密封到一个细胞培养基材的物理上包含ECM的涂料，并随后接种hPSCs大小。作为模版图案形成工程通过物理约束，其中HPSC可以访问和连接直接到下面的ECM涂覆的衬底的位置，该方法是与能够支持HPSC培养各种基材相容。唯一requirement是该模版材料的选择可以形成与基材的可逆密封。这些底物包括常规组织培养聚苯乙烯（TCPS）17，配体缀合的基片18，以及与可调刚性的弹性体基底（ 例如 ，PDMS）19。这种方法还允许不同的ECM的涂层，例如玻连蛋白（或VTN蛋白），层粘连蛋白和基底膜基质（ 例如 ，基质胶和Geltrax），以允许适当附着和hPSCs的分化。为特定HPSC线。因此，我们可以传送优化的ECM-基板配置到模版图案化以获得最佳的细胞 – 基质粘附，存活和分化。最近，一个类似的方法也有报道直接通过使用聚甲基丙烯酸甲酯微图案的hESCs（PMMA）微模版阵列16肝分化。
细胞的模板可以由不同的材料制成，包括满足ALS 20,21，聚（对苯二甲基）聚合物22,23，聚甲基丙烯酸甲酯16和最常见的是，PDMS 24-28。硅和聚（对-亚二甲苯）聚合物模具需要具有专门的设备20-23，这限制了它们的可访问生物用户通孔的直接蚀刻。 PDMS模具可以通过根据所需的特征尺寸，其通常从3微米至2,000微米11,26-29范围不同的方法来制造。如果小的功能需要，薄板材制版可通过冲压成型PDMS在包含微图案28的浮雕精细加工硅模板预聚物生产。为特征> 1000微米，CO 2激光切割器提供了一种简单和低成本的方法模版制造期间直接切上的预铸的PDMS片的图案。 PDMS模具的可回收也使得它们具有成本效益的，进行了一系列的具有足够一致性的实验。
<p cl屁股=“jove_content”>在这里，我们介绍的详细方法为PDMS模板由激光切割制造1000微米的特性和人类胚胎干细胞微图案的产生。这些胚胎干细胞微图案被用于调节整联的程度和E-cadherin介导的凝聚力的hESC集落内粘连以便调查细胞粘附的空间偏振如何导致细胞命运异质10。Protocol
Representative Results
Discussion
微图案模板的制作
模板缩微提供生成HPSC微图案调查利基介导的分化图案的理想方法。比其他图案化技术，例如微接触印刷和光图案模版图案形成的主要优点是，它不需要表面改性，可在常规的TCPS衬底来实现。因此，对于不同的HPSC线进行了优化培养基和ECM涂料可以很容易地转移到模版图案化。
有有助于良好微图案的产生的细胞模版的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由新加坡国立大学（R-397-000-192-133）和ETPL峡基金（R-397-000-198-592）支持的启动资助。 GS是新加坡国立大学的研究学者。作者想感谢Jiangwa兴博士为她的细胞微技术支持。
Materials
| 2 mm thick PDMS sheet | Specialty Silicone Products Inc., USA | SSPM823-.005 | Used to form reservoir for stencil |
| 120-150 μm thick PDMS sheet | Specialty Silicone Products Inc., USA | SSPM823-.040 | Used to form stencil |
| 60 mm petri dish | Nunc Nunclon Delta | 150326 | Substrate for micropatterning |
| Accutase | Accutase, Merck Millipore, Singapore | SCR005 | Enzyme to break H9 Cells into single cells |
| Activin | R&D Systems, Singapore | 338-AC-010 | Growth factor for H9 differentiation |
| BMP4 | R&D Systems, Singapore | 338-BP-010 | Growth factor for H9 differentiation |
| Plasma system | Femto Science, Korea | CUTE-MP | For plasma oxidation of stencil |
| Dispase | StemCell™ Technologies, Singapore | 7923 | Enzyme used to weaken the cell-ECM adhesion during passaging |
| DMEM/F12 | GIBCO, USA | 11330032 | Basal medium for H9 cells |
| FGF2 | R&D Systems, Singapore | 233–FB–025 | Growth factor for H9 differentiation |
| H9 Cell line | WiCell Research Institute, Inc., USA | WA09 | Human embryonic stem cells |
| hESC-qualified basement membrane matrix | Matrigel, BD Biosciences, Singapore | 354277 | Extra-cellular matrix coating to support growth of H9 cells |
| Inverted microscope | Leica Microsystems, Singapore | DMi1 | For capturing bright-field images |
| Laser cutter | Epilog Helix 24 Laser System | Used to generate through holes in PDMS sheet | |
| mTeSR™1 medium | StemCell™ Technologies, Singapore | 5850 | Maintainence medium for H9 cells |
| PDMS | SYLGARD® 184, Dow Corning Co., USA | 3097358-1004 | Used for sticking the PDMS stencil and reservior |
| ROCKi Y27632 | Calbiochem, Merck Millipore, Singapore | 688000 | Maintains H9 cells as single cells |
| STEMdiff™ APEL™ medium | StemCell™ Technologies, Singapore | 5210 | Differentiation medium for H9 cells |
| Polyethylene terephthalate film | SureMark Singapore | SQ-6633 | Used to form stencil |
| Cell culture compatible non-ionic surfactant | Pluronic acid F-127, Sigma, Singapore | P2443 | Passivating reagent to repel cell adhesion in non-micropatterned substrates |
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