Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana47907, United States.
Departments of Otolaryngology and Plastic and Oral Surgery, F.M. Kirby Neurobiology Center, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts02115, United States.
Biomacromolecules. 2022 Oct 10;23(10):4141-4152. doi: 10.1021/acs.biomac.2c00585. Epub 2022 Sep 8.
Chemically defined hydrogels are increasingly utilized to define the effects of extracellular matrix (ECM) components on cellular fate determination of human embryonic and induced pluripotent stem cell (hESC and hiPSCs). In particular, hydrogels cross-linked by orthogonal click chemistry, including thiol-norbornene photopolymerization and inverse electron demand Diels-Alder (iEDDA) reactions, are explored for 3D culture of hESC/hiPSCs owing to the specificity, efficiency, cytocompatibility, and modularity of the cross-linking reactions. In this work, we exploited the modularity of thiol-norbornene photopolymerization to create a biomimetic hydrogel platform for 3D culture and differentiation of hiPSCs. A cell-adhesive, protease-labile, and cross-linkable gelatin derivative, gelatin-norbornene (GelNB), was used as the backbone polymer for constructing hiPSC-laden biomimetic hydrogels. GelNB was further heparinized via the iEDDA click reaction using tetrazine-modified heparin (HepTz), creating GelNB-Hep. GelNB or GelNB-Hep was modularly cross-linked with either inert macromer poly(ethylene glycol)-tetra-thiol (PEG4SH) or another bioactive macromer-thiolated hyaluronic acid (THA). The formulations of these hydrogels were modularly tuned to afford biomimetic matrices with similar elastic moduli but varying bioactive components, enabling the understanding of each bioactive component on supporting hiPSC growth and ectodermal, mesodermal, and endodermal fate commitment under identical soluble differentiation cues.
化学定义的水凝胶越来越多地被用于确定细胞外基质（ECM）成分对人类胚胎和诱导多能干细胞（hESC 和 hiPSC）细胞命运决定的影响。特别是，通过正交点击化学交联的水凝胶，包括巯基-降冰片烯光聚合和逆电子需求 Diels-Alder（iEDDA）反应，由于交联反应的特异性、效率、细胞相容性和模块化，被探索用于 hESC/hiPSC 的 3D 培养。在这项工作中，我们利用巯基-降冰片烯光聚合的模块化，创建了用于 hiPSC 3D 培养和分化的仿生水凝胶平台。作为构建 hiPSC 负载仿生水凝胶的骨架聚合物，使用了细胞黏附的、蛋白酶不稳定的和可交联的明胶衍生物，明胶-降冰片烯（GelNB）。GelNB 通过使用四嗪修饰的肝素（HepTz）的 iEDDA 点击反应进一步肝素化，生成 GelNB-Hep。GelNB 或 GelNB-Hep 与惰性大分子聚乙二醇-四巯基（PEG4SH）或另一种生物活性大分子巯基化透明质酸（THA）进行模块化交联。这些水凝胶的配方被模块化地调节，以提供具有相似弹性模量但具有不同生物活性成分的仿生基质，从而能够理解每种生物活性成分在相同的可溶性分化信号下对支持 hiPSC 生长和外胚层、中胚层和内胚层命运决定的作用。
