Crypt fusion as a homeostatic mechanism in the human colon.

OBJECTIVE: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. DESIGN: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. RESULTS: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. CONCLUSIONS: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.wellcome trust royal societ

Quantitative Measurement of Clonal Evolution in Human Colon

PhD thesisThe clonal history of a cell is recorded within its (epi)genome via the accumulation of heritable changes. Studying the patterns of these heritable changes, termed lineage tracing markers, allows for the reconstruction of a tissue’s clonal architecture and the dynamics of clone replacements. In this thesis, I attempt to quantitatively measure clonal dynamics within normal human tissue from a single time-point, uncovering new homeostatic mechanisms in the intestine, and developing a new technique for quantifying somatic cell evolutionary dynamics at high temporal resolution across human tissues. Intestinal crypt fission provides a mechanism for mutations fixed in a single crypt to colonise the colon. In this thesis, evidence is presented that human colonic crypts also undergo fusion, a hitherto unknown process in human by which two crypts fuse to form a single daughter crypt. The existence of this balancing homeostatic process upon the distribution of mutant patch sizes was explored, allowing the estimate of the fission rate to be updated. The spatial distribution of crypt fission/fusion events exhibited spatial clustering, further emphasising the complex nature of the human gut epithelium. I present evidence that fluctuating DNA methylation can be used as molecular clocks in cells, where ongoing (de)methylation cause repeated “flip-flops” between methylated and unmethylated states. Endogenous fluctuating CpG sites were identified using standard methylation arrays, and a mathematical model was developed to quantitatively measure human adult stem cell dynamics from individual colon, small intestine and endometrial glands. The mathematical framework developed above is inappropriate for studying large polyclonal systems, such as haematopoietic stem cells. A flexible, stochastic modelling approach was developed that allows for the quantification of clonal dynamics for both fixed and growing populations of arbitrary size. This thesis demonstrates that mathematical interpretation of clone size data reveals clonal dynamics in human tissues without requiring longitudinal data

Lineage Tracing in Human Tissues

The dynamical process of cell division that underpins homeostasis in the human body cannot be directly observed in vivo, but instead is measurable from the pattern of somatic genetic or epigenetic mutations that accrue in tissues over an individual's lifetime. Because somatic mutations are heritable, they serve as natural lineage tracing markers that delineate clonal expansions. Mathematical analysis of the distribution of somatic clone sizes gives a quantitative readout of the rates of cell birth, death, and replacement. In this review we explore the broad range of somatic mutation types that have been used for lineage tracing in human tissues, introduce the mathematical concepts used to infer dynamical information from these clone size data, and discuss the insights of this lineage tracing approach for our understanding of homeostasis and cancer development. We use the human colon as a particularly instructive exemplar tissue. There is a rich history of human somatic cell dynamics surreptitiously written into the cell genomes that is being uncovered by advances in sequencing and careful mathematical analysis lineage of tracing data. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland