Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

Abstract

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems.

Aging, clonal hematopoiesis and preleukemia: not just bad luck?

“…Accordingly, tissues with greater stem cell replication will carry more mutations, and consequently will have a higher incidence of cancer. While the authors have provided evidence for such a correlation, several questions remain: first, the number of stem cell replications in a tissue is the product of the number of tissue stem cells and how many times each of them divides [40,41]. As we will next demonstrate, the accumulation of mutations is influenced not only by the total number of stem cell divisions, but also by the topology of the lineage tree [40][41][42].…”

“…While the authors have provided evidence for such a correlation, several questions remain: first, the number of stem cell replications in a tissue is the product of the number of tissue stem cells and how many times each of them divides [40,41]. As we will next demonstrate, the accumulation of mutations is influenced not only by the total number of stem cell divisions, but also by the topology of the lineage tree [40][41][42]. A 'shallow' tree topology with many stem cells that divide at a low rate has a significantly lower probability of yielding an oncogenic clone than does a 'deep' tree containing a few rapidly dividing stem cells (Fig.…”

A generic, cost-effective, and scalable cell lineage analysis platform

“…Most of the MS panel is designed for (A) MSs of type AC on the X Chromosome to allow for monoallelic MS calling Wasserstrom et al 2008;Reizel et al 2011;Segev et al 2011;Shlush et al 2012) and for (B) the longest MS loci possible, which exhibit a higher mutation rate in vivo (Ellegren 2004;Supplemental Table S2). Notably, primers were designed such that the entire MS will be covered within a 150-bp Examples of single-cell analyses to reconstruct clonal/lineage analysis Wang et al 2012;Xu et al 2012;Gawad et al 2014;Lohr et al 2014;Lodato et al 2015) (Navin et al 2011;Cai et al 2014;Wang et al 2014) ) Salipante et al 2010;Reizel et al 2011Reizel et al , 2012Shlush et al 2012;Evrony et al 2015) read (see Methods). (2) We modified the second PCR to apply a sample barcode by utilizing combinations of forward and reverse PCR primer combinations, resulting in a dual indexed NGS library (Supplemental Fig.…”

“…Our laboratory has demonstrated (Frumkin et al 2005Wasserstrom et al 2008;Reizel et al 2011Reizel et al , 2012Segev et al 2011;Shlush et al 2012) the feasibility of cell lineage analysis utilizing a low-resolution capillary electrophoresis (CE)-based system (∼100 loci per cell). We analyzed microsatellite (MS) loci, which have high mutation rates in vivo (Table 1) and are considered neutral (Ellegren 2004).…”

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

“…These are high yield methods, yet changes in intercellular signaling have been reported under these conditions that may not represent the native environment 6,7 . Laser capture microdissection allows for the collection of spatial distinct material, but achieves low molecular yield 8,9 . In human colonic tissue, serial horizontal cryosectioning methods have been developed 10 .…”