Thiol-Ene Click Hydrogels for in situ Cell Expansion and Differentiation

描述（由申请人提供）：水凝胶是亲水性聚合物基质，可支持细胞和组织三维生长。通过精心设计，水凝胶不仅可以作为研究细胞行为的3D平台，还可以作为培养和分化干/祖细胞的支架。我们的目标是开发一种多功能水凝胶系统，最初支持细胞增殖，但在后期可以“切换”到一个微环境，促进细胞/组织分化成特定的细胞类型。我们的中心假设是，细胞扩增和分化可以实现在一个单一的水凝胶基质与动态生物物理和生化线索。我们将测试这一假设，开发一个通用的水凝胶系统，使用多种细胞相容的硫醇烯“点击”反应。在初始阶段，我们将设计局部可降解的硫醇-烯凝胶，以促进PANC-1细胞（胰腺导管上皮细胞系）的增殖（目的1）。我们将基质金属蛋白酶2（MMP-2）特异性肽底物作为水凝胶交联剂。这允许凝胶通过PANC-1细胞分泌的MMP-2局部降解（从而为细胞增殖创造额外的空间）。通过调节水凝胶中的细胞-ECM和细胞-细胞相互作用，以及为包囊细胞提供可扩散的可溶性生长因子，将促进包囊细胞的增殖。接下来，我们将把载有细胞的水凝胶从“促增殖”微环境“切换”到“促分化”微环境（目标2a）。我们将通过进行第二次硫醇-烯点击反应来实现这一点，以在载有细胞的水凝胶内引入促分化因子，从而促进胰岛样胰岛素分泌细胞簇的形成。分化过程将是使用无血清培养基、基于亲和力的碱性成纤维细胞生长因子（bFGF）募集和胰高血糖素样肽1（GLP-1）及时偶联的组合结果。分化的细胞簇可以从可侵蚀的凝胶中回收（由于特定的酶活性），用于进一步表征和生物学/临床应用（目的2b）。分化的簇预期由于强的细胞-细胞相互作用而形成紧密的聚集体。总之，我们的策略促进了具有天然细胞间相互作用和正常胰岛素分泌特征的胰岛样细胞簇的形成。 公共卫生相关性：该提案旨在设计生物材料装置，其包含促进上皮细胞增殖的信号，并增强这些细胞分化为胰岛素产生细胞。如果成功，这一策略将为1型糖尿病患者提供替代细胞来源。

DESCRIPTION (provided by applicant): Hydrogels are hydrophilic polymeric matrices that can support cell and tissue growth in three-dimension. With careful design, hydrogels serve not only as a 3D platform for studying cell behaviors, but also as scaffolds for culturing and differentiating stem/progenitor cells. We aim to develop a multifunctional hydrogel system that initially supports cell proliferation, but at a later stage can be "switched" into a microenvironment that promotes cell/tissue differentiation into a specific cell type. Our central hypothesis is that cell expansion and differentiation can be achieved in a single hydrogel matrix incorporated with dynamic biophysical and biochemical cues. We will test this hypothesis by developing a versatile hydrogel system using multiple cytocompatible thiol-ene "click" reactions. In the initial stage, we will design locally degradable thiol-ene gels to promote proliferation of PANC-1 cells (a pancreatic ductal epithelial cell line) (Aim 1). We will incorporate matrix metalloproteinase 2 (MMP-2) specific peptide substrates as hydrogel crosslinkers. This allows the gels to degrade locally (thus creating additional space for cell proliferation) by MMP-2 secreted from PANC-1 cells. The proliferation of encapsulated cells will be promoted by tuning cell-ECM and cell-cell interactions in hydrogels, as well as providing the encapsulated cells with diffusible soluble growth factors. Next, we will "switch" the cell-laden hydrogels from a "pro-proliferation" to a "pro-differentiation" microenvironment (Aim 2a). We will achieve this by performing a second thiol-ene click reaction to introduce pro-differentiation cues within the cell-laden hydrogels, thus promoting the formation of islet-like, insulin secreting cell clusters. The differentiation process will be a combinatorial result of using serum-free culture media, affinity-based recruitment of basic fibroblast growth factor (bFGF), and timely conjugation of glucagon-like peptide 1 (GLP-1). The differentiated cell clusters can be retrieved from the erodible gels (due to specific enzyme activity) for further characterization and biological/clinical applications (Aim 2b). The differentiated clusters are expected to form tight aggregates due to strong cell-cell interactions. Together, our strategies facilitate the formation of islet-like cell clusters with natural cell-cell interactions and normal insulin secretion profiles. PUBLIC HEALTH RELEVANCE: This proposal aims to design biomaterial devices that incorporate signals to promote the proliferation of epithelial cells and to enhance the differentiation of these cells into insulin-producing cells. If successful, this strategy will provide alternative cell sources to benefit type 1 diabetic patients.