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.
The goal of this research is to demonstrate a paradigm shift in tissue engineering and diabetes treatment: Genetically engineered stem cells do autoregulate their differentiation into insulin- producing β cells based on artificial cell-cell communication and programmed multistep differentiation. Additionally, we explore the computational design and modeling of an artificial tissue homeostasis system where transplanted engineered cells are programmed to regenerate and sustain the β cell population in a diabetic patient. We made good experimental progress towards optimizing an artificial cell-cell communication based on a small diffusible molecule in mammalian cells, a critical step for engineering autoregulated growth. Additionally, we laid the theoretical (design and modeling) foundation for a complete autoregulatory system. In parallel, we are engineering and characterizing the modules eeded for the complete homeostatic system (genetic toggle switch, relaxation oscillator, quorum sensing...). Engineering mouse embryonic stem cells with multiple variants of our genetic differentiation programs were brought to differentiate over endoderm-like stages to cells positive for insulin secretion by-products. By investigating these differentiated cells (e.g. glucose-dependent secretion, pancreatic beta cells markers) we are currently deciding on the best genetic program to generate such cells. Initially using lentiviruses to deliver the transgenes, we improved the fidelity andprecision of the chromosomal integrations drastically by developing a novel system to assemble and integrate site-specifically large gene circuits into mammalian cells. Such a system allows for a safe and controlled insertion of therapeutic genetic circuits into patient stem cells, with the potential of being able to generate large quantities of well-defined specialized cells needed for therapies. We could demonstrate that a single site-specifically integrated gene circuit is able to induce the differentiation of embryonic stem (ES) cells to endoderm. We plan to disseminate this new DNA assembly and integration as we believe that it will be of high value to researchers working in mammalian synthesis biology in particular, or anyone performing mammalian (therapeutic) genetic engineering in general.
