Lineage-tracing approaches, widely used to characterize stem cell populations, rely on the specificity and stability of individual markers for accurate results. We present a method in which genetic ...labeling in the intestinal epithelium is acquired as a mutation-induced clonal mark during DNA replication. By determining the rate of mutation in vivo and combining this data with the known neutral-drift dynamics that describe intestinal stem cell replacement, we quantify the number of functional stem cells in crypts and adenomas. Contrary to previous reports, we find that significantly lower numbers of “working” stem cells are present in the intestinal epithelium (five to seven per crypt) and in adenomas (nine per gland), and that those stem cells are also replaced at a significantly lower rate. These findings suggest that the bulk of tumor stem cell divisions serve only to replace stem cell loss, with rare clonal victors driving gland repopulation and tumor growth.
•Replication-dependent clonal label applied to infer stem cell dynamics•Stem cell numbers and replacement rates are lower than previous estimates•Stem cell dynamics are unchanging with age•Low stem cell numbers but high replacement rates follow oncogenic transformation
A replication-dependent clonal-label approach reveals significantly lower than expected numbers of “working” stem cells in the intestinal epithelium and in adenomas and slower rates of stem cell replacement.
The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly ...cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.
With the capacity for rapid self-renewal and regeneration, the intestinal epithelium is stereotypical of stem cell-supported tissues. Yet the pattern of stem cell turnover remains in question. ...Applying analytical methods from population dynamics and statistical physics to an inducible genetic labeling system, we showed that clone size distributions conform to a distinctive scaling behavior at short times. This result demonstrates that intestinal stem cells form an equipotent population in which the loss of a stem cell is compensated by the multiplication of a neighbor, leading to neutral drift dynamics in which clones expand and contract at random until they either take over the crypt or they are lost. Combined with long-term clonal fate data, we show that the rate of stem cell replacement is comparable to the cell division rate, implying that neutral drift and symmetrical cell divisions are central to stem cell homeostasis.
Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently ...occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Ape loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.
The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using ...lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium.
The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism ...of, and requirement for, progenitor plasticity in mediating pathological response remain unknown. Here we show that phosphorylation of the transcription factor Atoh1 is required for both the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. As confirmed by lineage tracing, Atoh1+ cells (Atoh1(WT)CreERT2 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant Atoh1(9S/T-A)CreERT2 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis, Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenicity that affects overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium, and the balance between stem and progenitor fate is directly coordinated by ATOH1 multisite phosphorylation.
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•Atoh1+ progenitors contribute to the stem cell pool in homeostasis and regeneration•Multisite phosphorylation of ATOH1 regulates the plasticity of secretory progenitors•Loss of phosphorylation of ATOH1 reduces clonogenic capacity of Atoh1+ cells•Phosphomutant ATOH1 mice are more susceptible to chemical colitis
Tomic et al. report that multisite phosphorylation of ATOH1 regulates the contribution of secretory progenitors to stem cell self-renewal in the small intestine and colon. With damage, the enhanced role of Atoh1+ progenitors in mediating tissue repair is ablated in mice expressing phosphomutant ATOH1 and overall tissue regeneration is impaired.
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the ...identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.
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•Mex3a is expressed in a subset of Lgr5+ cells that proliferate slowly•Mex3a+ cells are multipotent and can generate all intestinal lineages•Mex3a+ cells can convert to fast-dividing ISCs to maintain homeostasis•Mex3a+ cells regenerate the epithelium after chemotherapeutic insult
Lgr5+ intestinal stem cells are considered to be a homogeneous and rapidly proliferating population. Barriga et al. show that the RNA binding protein Mex3a defines a subset of slowly proliferating Lgr5+ cells that contribute to all intestinal lineages with slow kinetics, are resistant to chemotherapy, and support intestinal regeneration.
The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three ...different classes of secretory cells--mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3--a marker of enteroendocrine fate--or Gfi1--a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.
We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. ...First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.
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•Colonic stem cell dynamics predict lifetime mutant allele frequencies•Mutant clone fixation in colonic crypts takes years due to slow stem cell turnover•Crypt fission enables lateral expansion of mutant clones•Biases in both fixation and expansion increase age-related pro-oncogenic burden
Winton and colleagues describe stem cell dynamics in normal human colon to identify the efficiency of clone fixation within the epithelium and the rate of subsequent lateral expansion. Against these benchmarks biased stem cell behaviors advantaged in both fixation and expansion can be quantified to predict the age-related burden of pro-oncogenic mutation.
Cellular dormancy and heterogeneity in cell cycle length provide important explanations for treatment failure after adjuvant therapy with S-phase cytotoxics in colorectal cancer (CRC), yet the ...molecular control of the dormant versus cycling state remains unknown. We sought to understand the molecular features of dormant CRC cells to facilitate rationale identification of compounds to target both dormant and cycling tumor cells. Unexpectedly, we demonstrate that dormant CRC cells are differentiated, yet retain clonogenic capacity. Mouse organoid drug screening identifies that itraconazole generates spheroid collapse and loss of dormancy. Human CRC cell dormancy and tumor growth can also be perturbed by itraconazole, which is found to inhibit Wnt signaling through noncanonical hedgehog signaling. Preclinical validation shows itraconazole to be effective in multiple assays through Wnt inhibition, causing both cycling and dormant cells to switch to global senescence. These data provide preclinical evidence to support an early phase trial of itraconazole in CRC.
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