School of Medicine
Cancer Cell Biology
Area of interest
I have had a long-standing scientific interest in the genetic mechanisms regulating stem cell behavior. Throughout my career I have focused on conducting research using in vivo approaches as demonstrated by my publications. These studies have required the identification and isolation of various cell populations using fluorescence activated cell sorting (FACS). To facilitate FACS separation of cells of interest, I have developed several fluorescent reporter gene transgenic mice from which tissue is dissociated and primary cells are sorted based in the fluorescent signature of these cells. Additionally, my lab has developed novel cell isolation techniques for both the small and large intestinal epithelium, and have optimized whole crypt and single cell dissociation techniques. We have recently used these techniques to demonstrate that the transcription factor, Sox9, is a biomarker of functional intestinal epithelial cells (IESCs) using a three-dimensional culture system.
To our knowledge my lab was the first to validate this in vitro culture system defined by Sato et al, and since then we have made several modifications to the protocol that have increased efficiency and reproducibility. The ‘enteroids’ (small intestine) or ‘colonoids’ (colon) derived from single IESCs or whole crypts contain multiple crypt-like units that are quite small (~50 – 100 um in diameter) and difficult to work with for downstream applications. We developed several strategies that enable whole mount staining or efficient embedding and sectioning of the cryptoids for immunostaining. We have developed tissue dissociation methods that allow for efficient FACS to isolate very rare populations of cells, which we have used for downstream applications including western blotting, real-time PCR gene expression analysis and cell cycle analysis. Importantly, we have devised novel methods for 3-dimensional confocal reconstruction of ‘enteroids’ or ‘colonoids’ in microfabricated devices. We currently conduct these procedures on a weekly basis and have trained several labs on how to efficiently conduct these experiments. We are confident that our expertise in primary tissue and cell isolation, FACS, quantitative crypt culture, immunostaining techniques and micromanipulation will facilitate the experiments described in the aims of this grant proposal. Additionally, we have just published a manuscript detailing critical reagents and methods that can be used to assess cell lineages and to culture intestinal
epithelium from pig as the first step towards a translational model for intestinal tissue engineering.