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

描述(申请人提供)：水凝胶是亲水性聚合物基质，可以支持细胞和组织的三维生长。经过精心设计，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，该系统最初支持细胞增殖，但在稍后阶段可以“切换”到促进细胞/组织分化为特定细胞类型的微环境。我们的中心假设是，细胞的扩张和分化可以在单一的水凝胶基质中实现，并结合动态的生物物理和生化线索。我们将通过开发一种多功能的水凝胶系统来验证这一假设，该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-烯凝胶来促进胰腺导管上皮细胞系PANC-1的增殖(目标1)。我们将结合基质金属蛋白酶2(MMP2)特异性多肽底物作为水凝胶交联剂。这使得凝胶可以通过PANC-1细胞分泌的基质金属蛋白酶-2在局部降解(从而为细胞增殖创造额外的空间)。通过调节水凝胶中细胞-细胞外基质和细胞-细胞间的相互作用，以及为被包裹的细胞提供可扩散的可溶性生长因子，将促进被包裹的细胞的增殖。接下来，我们将“切换”细胞水凝胶从“促进增殖”到“促进分化”的微环境(目标2a)。我们将通过进行第二次硫醇-烯点击反应，在细胞负载的水凝胶中引入促进分化的信号，从而促进胰岛样、胰岛素分泌细胞团的形成。分化过程将是使用无血清培养液、基于亲和力的碱性成纤维细胞生长因子(BFGF)和适时结合胰高血糖素样肽1(GLP-1)的组合结果。分化的细胞团可以从可腐蚀的凝胶中提取(由于特定的酶活性)，用于进一步的表征和生物/临床应用(目标2b)。由于强烈的细胞-细胞相互作用，分化的簇有望形成紧密的聚集体。总而言之，我们的策略有助于形成具有自然细胞-细胞相互作用和正常胰岛素分泌特征的胰岛样细胞团。 与公共卫生相关：这项提议旨在设计生物材料设备，其中包含促进上皮细胞增殖的信号，并加强这些细胞向胰岛素产生细胞的分化。如果成功，这一策略将提供替代细胞来源，使1型糖尿病患者受益。

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.