The lumen of the mammalian intestine is lined by a single layer of epithelial cells, which are renewed every 4-5 days. This process is fueled by intestinal stem cells located in invaginations of the epithelial layer called crypts. Preliminary data demonstrate that single mRNA molecules of the intestinal stem cell marker Lgr5 can be detected in single cells.
In Aim 1 and 2 we plan to use single-cell transcript counting to quantitatively map the transcriptional program of stem cells, transit amplifying cells, and differentiated cells in the intestine at the single-cell level. We will use murine model systems for intestinal cancer to explore the rapid formation of macroscopic adenomas fueled by Apc-deficient stem cells. We will develop new model systems for intestinal cancer in which combinations of inactivated tumor suppressor genes and activated oncogenes will be expressed in stem cells. These systems will allow us to explore the potential of different adenoma cells in initiating the adenoma-to-carcinoma transition. Single-cell mRNA counting will be used to quantify the transcriptional program in the human intestine samples obtained from patients with colorectal cancer. These quantitative data will be used to validate quantitative models of transcriptional networks that control cell fate commitment in normal gut and to understand how these networks are altered when a stem cell undergoes malignant transformation.
In Aim 3 we will use single-cell transcript counting integrated with theoretical modeling to quantify the stochastic dynamics of reprogramming of differentiated somatic cells to the pluripotent embryonic state. Two hypotheses may explain how forced expression of the transcription factors initiates reprogramming: First, a stochastic series of molecular events such as chromatin or transcriptional changes may occur in no particular sequence inducing the pluripotent state in a small fraction of the infected cells. Alternatively, a series of consecutive molecular events such as a destined pattern of transcriptional and epigenetic changes have to occur sequentially to eventually convert the differentiated cell to the pluripotent state. The main objectives are to molecularly define the steps in reprogramming in single cells and to distinguish between the two hypotheses raised above.
The data generated in this project will be used to better understand how regulatory networks are altered when a stem cell undergoes malignant transformation leading to intestinal cancer. We will also monitor the precise timing, quantity and patterns of the ES cell- and/or differentiated cell-specific gene expression in individual cells during reprogramming which could overcome current barriers for clinical applications.
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