光駆動型分化に向けた金ナノ粒子含有ヒドロゲルへの胚様体包接

本课题旨在建立三维球形核壳结构的水凝胶诱导多能干细胞培养平台，实现多能干细胞的可控增殖和分化。我们的系统允许在水凝胶珠的中心（核心）原位生成胚体（EB），并通过调节海藻酸盐内核组成来控制细胞外环境，从而以受控的、体内模拟的方式指导EB的分化。该系统有望成为一个更有效、高效、仿生的iPS细胞分化平台，最终将有利于再生医学研究界，并为发育生物学家更好地理解胚胎发育提供有价值的工具。在2013财年，开展了两方面的研究：1)开发用于iPS细胞包封的改性水凝胶基质，可以控制EB的形成和分化；2)表征EB的形成，细胞增殖和分化。具体而言，在第一部分中，我们通过将RGD和YIGSR肽等ECM蛋白基序共价连接到藻酸盐分子链上，合成了改性藻酸盐凝胶。我们成功地获得了内核成分经过修饰的核壳结构水凝胶珠。对于第二部分，我们发现通过改变内核组成，我们能够控制珠子内部形成的EB的大小。0.5%海藻酸盐芯内的细胞形成最大~600um（直径）的EBs， 1%海藻酸盐与0.5% ha按1:1比例混合芯内的细胞形成最小~ 250um（直径）的EBs。内核液化和改性影响EB的分化形态和轨迹。PCR结果表明，液化内芯可促进EB细胞向内胚层分化，将海藻酸盐浓度从1%降低至0.5%可抑制hiPS细胞分化，用RGD肽修饰海藻酸盐可诱导细胞向外胚层分化。少

The goal of this project is to develop a 3D spherical core-shell structure hydrogel-based induced pluripotent stem (iPS) cell culture platform for controlled iPS propagation and diflerentiation. Our system allows the in situ generation of embryo body (EB) at the center (core) of hydrogel beads and the control of extracellular environment by adjusting alginate inner core composition, in such way todirect the differentiation of EB in a controlled, and in-vivo mimic manner. This proposed system is expected to become a more effective, efficient, and biomimetic platform for iPS cell differentiation, which might ultimately benefit the regenerative medicine research community, and serve as a valuabletool for developmental biologist for better understanding of the development of embryo.ln 2013 fiscal year, two folds of studies have been carried out : 1) development of modified hydrogel matrixes for iPS cells encapsulation, which allow controlling EB formation and differentiation, 2) characteri … More ze EB formation, cell Proliferation and differentiation.Specifically, for part 1, we have achieved synthesizing modified alginate gel by covalently attaching oxtracollular matrix (ECM) protein motifs such as RGD and YIGSR peptide to the chain of alginate molecules. We were able to successfully got core shell structure hydrogel beads with modified inner core composition. For the second part, we have found that by changing the inner core composition, we were able to control the size of EB fomed inside our beads. Cells inside 0.5% alginate core formed the biggest~600um (diameter) EBs and cells inside 1% alginate mixed with 0.5%HA at 1:1 ratio core formed smallest~250 um (diameter) EBs. The inner core liquefying and modification can affect EB differentiation morphology and trajectory. PCR results suggested that liquefying inner core enhanced EB differentiation towards endodermal lineage, decreasing alginate concentration from 1 percent to 0.5 peroent suppressed hiPS cell differentiation, and modifying alginate with RGD peptide induced differentiation towards ectodermal lineage. Less