David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Biomaterials. 2011 Nov;32(31):7793-800. doi: 10.1016/j.biomaterials.2011.06.057. Epub 2011 Aug 11.
Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.
人类胚胎干细胞（hESCs）作为再生医学的资源具有巨大的潜力。在实现治疗相关性之前，必须开发方法来控制干细胞分化。很明显，干细胞可以响应遗传信号，例如核酸赋予的信号，以促进谱系特异性分化。在这里，我们使用类脂材料开发了一种在 3D 培养基质中有效递送至 hESC 的 siRNA 的方法。我们表明，在 3D 支架中非病毒 siRNA 的递呈可以有效地敲低 GFP-hESCs 中 GFP 表达的 90%。我们进一步表明，该系统可以用作指导 hESC 分化的平台。通过 KDR 受体基因的 siRNA 沉默，我们实现了内胚层代表基因的同时下调（60-90%）和中胚层代表基因的显著上调（27-90 倍）。这表明 siRNA 可以通过阻断一个胚层的代表性基因来指导干细胞分化，并且还提供了一种特别有效的方法来将内胚层从中胚层和外胚层中分离出来。这种抑制内胚层分化的能力可以允许对 hESC 分化为所需细胞类型进行更好的控制。
