Engineered quorum sensing and programmed multi-step differentiation of mammalian stem cells into pancreatic beta cells

拟议的研究将使用基因工程干细胞，通过结合人工细胞间通讯和仔细调节的多步分化，能够自动调节其分化为产生胰岛素的β细胞。总体目标是建立一个人工组织稳态系统模型，该系统包含调节元件，使细胞能够检测干细胞和β细胞群，并根据细胞群阈值进行适当分化。实现这一目标的步骤将通过在体外控制 mES 分化为胰腺 β 细胞来实现。该提案整合了合成生物学、计算生物学和发育生物学的多学科领域；生物和组织工程；以及建模/计算和干细胞生物学。信号元件用于分化的第一步（ES细胞分化为内胚层），其中天然甲胎蛋白（AFP）用于调节红色荧光蛋白的表达，并允许在荧光显微镜下观察mES细胞向内胚层的转变。分化的第二步（通过 GFP 表达（来自小鼠胰岛素启动子 (MIP)）可视化为 β，这是由天然胰岛素产生引起的。这些实验观察结果将指导合成自动调节系统的正向工程。

The proposed research will use genetically engineered stem cells capable of autoregulating their differentiation into insulin-producing beta cells by incorporating artificial cell-cell communication and carefully regulated multistep differentiation. The overall goal is to model an artificial tissue homeostasis system which contains regulatory elements that will allow cells to detect stem cell and beta cell populations and differentiate appropriately depending on cell population thresholds. Steps toward this goal will be carried out by controlling the differentiation of mES into pancreatic beta cells in vitro. The proposal integrates the multidisciplinary areas of synthetic, computational, and developmental biology; biological and tissue engineering; and modeling/computational and stem cell biology. Signaling elements are used in the first step of differentiation (ES cells into endoderm) where the native alpha-fetoprotein (AFP) is used to regulate expression of red fluorescent protein and allows for the visualization of mES transition into endoderm by cells) is?nfluorescence microscopy. The second step of differentiation (into beta visualized by GFP expression (from the Mouse Insulin Promoter (MIP)), which is caused by natural insulin production. These experimental observations will direct the forward-engineering of the synthetic autoregulating system.