Quantifying intestinal stem cell dynamics using microsatellite sequencing

The intestinal epithelium is rapidly renewing throughout life. A population of stem cells exist within the intestinal crypt that drive rapid cell renewal and replace each other by a pattern of neutral drift. Perturbation of these dynamics through oncogenic mutation can predispose the epithelium to neoplastic transformation. Understanding the factors that govern these dynamics will give insight into the early stages of oncogenesis. Continuous clonal labelling, whereby DNA strand slippage leading to the contraction or expansion of a microsatellite during mitotic replication, can be employed to enable the detection of a single clone. Previous studies have shown that quantification of clone size over time allows inference of the functional stem cell number and stem cell replacement rate within intestinal crypts. Current continuous labelling techniques require the introduction of a transgenic microsatellite into a model system genome, such as mouse, that leads to reporter expression following mutation. This obviously precludes human studies. Alternative somatic alterations techniques used for continuous labelling in humans requires spontaneous loss of a protein, or change in methylation status, within a single clone that can act as a clonal mark. Though these techniques have given insight into the spread of mutations within the intestinal epithelium and enabled inference of adenoma clonality, the true neutrality of these changes are currently unknown. We propose that the small changes in endogenous microsatellite length will act as a neutral clonal mark within the intestinal epithelium and allow an unbiased approach to quantifying intestinal stem cell dynamics in human intestinal tissues. To overcome the many technical challenges associated with accurate measurement of microsatellite length, a stepwise approach was taken to develop a technique for the multiplexed high throughput sequencing of up to 21 native dinucleotide repeats in hundreds of single crypts. Furthermore, a novel method was developed for the quantification of clone size from data generated from the targeted re-sequencing of microsatellites in single crypts. This protocol was validated in vitro and in vivo in mouse. Furthermore, proof of principle sequencing in human crypts was performed to show that this method is suitable for larger scale quantification of intra-cryptal clone size in human tissue. This, and similar approaches, may be the only way to quantify intestinal stem cell dynamics within the healthy human colon or, dysplastic or adenomatous patient tissue. These measurements should give a unique insight into the dynamics of healthy, pre-neoplastic and neoplastic human intestinal stem cells.