Research

Renewing tissues and many cancers are maintained by a small number of long-lived stem cells and this explains interest in trying to define their distinguishing properties. Most models of stem cell organisation take account of their longevity and the fact that they self-renew, and also assume that they are stable populations carrying unique identifying characteristics. For decades the assays used to test different cell populations for their 'stemness' have appeared consistent with such deterministic models. These assays commonly challenge the ability of cells, separated into discrete populations based on the expression of cell surface antigens, to undergo growth when cultured or engrafted. Cells that are able to support long-term growth are taken as being synonymous with stem cells, and it is assumed that the differential expression of transcription factors underpins the fate of stem cell populations.

However, this interpretation of stem cell organisation now seems too simplistic. For example: cell fate is likely determined by small changes in the expression of regulatory transcription factors in the context of transcriptional networks; the cell surface signatures of stem cells may not be as stable over time as previously thought; the success of stem cell engraftment may be partly determined by properties of the recipient rather than the transplanted cells (Chang et al., Nature 2008; 453: 544; Quintana et al., Nature 2008; 456: 593). Stem cell biology may be driven by stochastic switching between different states in response to variations in the balance of signals coming from complex transcriptional networks. In accordance with this view we have recently demonstrated, by following the dynamics of clonal growth in situ, that intestinal stem cell turnover is a constant and rapid stochastic process that follows a pattern of neutral drift (Lopez-Garcia et al., Science 2010; 330: 822).

Given the above our approach is pragmatic: to identify novel ways of assaying stem cells in situ with respect to the functional endpoints that are integral to their biology.

What is the multi-potentiality of stem-like cells in intestinal cancers?

Our long-term objective is to determine the repertoire of differentiation options available to cancer stem cells, how this differs from normal stem cells, and thereby identify unique opportunities for therapies. To measure potentiality we are exploiting the known differences between cell types in the timing of DNA replication during the cell cycle. Genes associated with maintaining pluripotency are replicated early in S-phase, while those associated with neural lineages are replicated late in S-phase (Azuara et al., Nat Cell Biol 2006; 8: 532). The pattern of replication timing for key transcription factors has been described as a barcode of potentiality, indicative of the accessibility of the chromatin for subsequent expression.

We are attempting to devise such a barcode for intestinal stem cells to identify changes in potentiality during carcinogenesis. S-phase cells can be isolated and sorted by DNA content into four fractions. Immunoprecipitation for BrdU allows newly synthesised DNA to be analysed. To date we have shown reproducible differences in replication timing between different loci. For example, the neural transcription factor Mash1 is replicated late, while the transcription factor Ngn3, expressed in the intestine, is replicated early. Currently, the amount of material obtained on pull-down is restrictive. We aim to increase genomic coverage by amplification to generate a comprehensive characterisation of replication timing. The effect of deleting the APC tumour suppressor gene on replication timing patterns is also being determined - deleting this gene also results in dramatic changes in cell type (loss of secretory cell lineages) and differentiation.

Role of quiescent stem cells

Label retaining cells, identified by their ability to sequester and retain label, have long been thought to be synonymous with quiescent stem cells. Using inducible expression of nuclear-localised fluorescent protein (Histone H2B-YFP) we have identified a population of crypt-base cells that appear to divide either very slowly or to be quiescent. Conventional views of stem cell organisation would place these cells as potential long-lived cells acting at the apex of a proliferative hierarchy. However, such an interpretation is not compatible with the dynamics that we have documented: rapid stem cell turnover with neutral drift. We aim to characterise these cells and define both their normal fate and whether they can be tumour initiating in a cancer setting. To this end we are currently performing detailed transcriptional analysis of purified label-retaining cells.

Figure 1
Pictorial representation of the frequency of insertional mutagenesis at specific loci by transposon mobilisation in intestinal tumours arising due to Apc-deficiency.

Cancer models and tumour progression

At a molecular level the development of intestinal cancers is well characterised, with the most common genetic changes incorporated into a paradigm of progression for colorectal cancers in which loss of APC is a central early event (as described by Bert Vogelstein's lab at Johns Hopkins University). Despite this it has been shown that many other gene specific mutations can also be associated with the disease (Sjoblom et al., Science 2006; 314: 268). APC has been deleted in animal models by a variety of strategies that usually lead to the development of benign adenomas. Introduction of additional mutational events in candidate genes has only been partly successful in creating the full carcinomatous (cancer-like) disease. Our ability to induce deletion of APC in the intestinal epithelium lends itself to investigating the nature of other gene mutations that might interact with APC and contribute to the formation of malignant disease. Therefore as an alternative unbiased approach to identifying such genes we are using our Cre models to mobilise a Sleeping-Beauty activated transposable element in mice predisposed to intestinal tumorigenesis by virtue of APC deficiency (Collier et al., Nature 2005; 436: 272). Cloning and sequencing of the insertion sites in tumours allows affected genes to be identified and associated with tumour pathology. Currently we have identified around 900 such genes (Figure 1). Their validation will be a priority for the coming year.