A Single Progenitor Population Switches Behavior to Maintain and Repair Esophageal Epithelium

Abstract

Diseases of esophageal epithelium (EE) such as reflux esophagitis and cancer are rising in incidence. Despite this, the cellular behaviors underlying EE homeostasis and repair remain controversial. Here we show that in mice, EE is maintained by a single population of cells that divide stochastically to generate proliferating and differentiating daughters with equal probability. In response to challenge with all-trans Retinoic Acid (atRA) the balance of daughter cell fate is unaltered but the rate of cell division increases. However, following wounding, cells reversibly switch to producing an excess of proliferating daughters until the wound has closed. Such fate switching enables a single progenitor population to both maintain and repair tissue without the need for a "reserve" slow-cycling stem cell pool.Murine EE consists of layers of keratinocytes. These tissue lacks structures such as crypts or glands which form stem cell niches in other epithelia . Proliferation is confined to cells in the basal layer (6). On commitment to terminal differentiation, basal cells exit the cell cycle and subsequently migrate to the tissue surface from which they are shed. Early studies suggested all proliferating cells were functionally equivalent, but recent reports propose that a discrete population of slow-cycling stem cells is responsible for both maintenance and wound healing (7)(8)(9)(10)(11). This controversy and the importance of EE in disease motivated us to resolve the proliferative cell behavior in homeostatic EE and in tissue challenged by systemic treatment with the vitamin A metabolite all-trans Retinoic Acid (atRA) or acute local wounding (12-13).To investigate cell division rates in EE we used a transgenic label retaining cell (LRC) assay ( Fig. 1C) (1, 14-15). Doxycycline (DOX) induction of Histone-2B EGFP fusion protein (HGFP) expression in Rosa26 M2rtTA /TetO-HGFP mice resulted in nuclear fluorescent labeling throughout the EE (Fig. 1D and fig. S1A). When DOX is withdrawn, HGFP is diluted by cell division, leaving 0.4% basal layer cells (561 out of 140000) retaining label after a 4 week chase (Figs. 1E and S1B). 3D imaging showed these label retaining cells * To whom correspondence should be addressed. phj20@cam.ac.uk. 5 These authors contributed equally to this work Supplementary Materials: Materials and Methods Figures S1-S13 However, 99.9% (2457 out of 2459) of LRC were positive for the pan leukocyte marker CD45 (Fig. 1E inset), comprising of a mixture of Langerhan's cells and lymphocytes (Figs. S1E and F). These findings lead to the surprising conclusion that, unlike tissues such as the epidermis, there are no slow-cycling or quiescent epithelial stem cells in EE (1, 17). Indeed, HGFP dilution in basal cells was strikingly homogeneous, suggesting that all cells divide at a similar rate of approximately twice per week (Fig. S1G).Although epithelial cells have the same rate of division, they may still differ in their ability to generate cycling and differentiated progeny. We therefore used inducible ...

Identification of Lineage-Uncommitted, Long-Lived, Label-Retaining Cells in Healthy Human Esophagus and Stomach, and in Metaplastic Esophagus

“…Antibodies to musashi-1 have been shown to bind to parietal cells 39,40 and no lineage tracing from a musashi-1-positve cell has been published and studies using CD133 have only shown lineage tracing in the intestine not the upper gastrointestinal tract 41 . While a recent paper in a murine model has suggested a 'reserve' slow cycling stem cell pool is not needed to maintain and repair tissue in the esophagus, it is unclear whether slower cycling cells exist and where they could be located in man 12,42 .…”

“…1,[12][13] Analysis for human LGR5 mRNA showed that expression was indeed located at the base of the Barrett's glands ( Figure 5E (light field) (dark field) F)). However no expression was seen in the normal squamous esophagus or in the submucosal glands/ducts.…”

“…These confusing data are particularly troublesome in the case of the esophagus and the common premalignant lesion Barrett's Esophagus (BE). First there have been reports of fast cycling cells with stem cell capacity in animal models and some of them indicate a residual stem cell population is needed while others do not [11][12][13][14][15] . Second some additional reports have implicated endodermal (including epithelial) embryonic markers in man but their relevance to human stem cells remains unclear 16 .…”

“…Despite these problems other techniques which trace lineage by genetic analyses strongly favour the likelihood of common stem cells between squamous and columnar esophageal mucosa [19][20][21][22][23][24] . The site of stem cells in BE has been more problematic [10][11][12][13][14][15] as well as the availability of true 'biomarkers' of stem cells [16][17][18] .…”

Tissue Engineering Functional Gastrointestinal Regions: The Importance of Stem and Progenitor Cells

“…The esophagus is comprised of a proliferative basal layer supporting multiple strata of keratinocytes (Doupe et al 2012). These proliferative cells previously appeared to be a uniform population capable of homeostasis, repair, and return to homeostasis after injury.…”