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• W2024876545 abstract "•3D esophageal organoids generated in vitro reflect in vivo tissue architecture•Cell-surface markers separate subpopulations of mouse esophageal basal epithelium•Subpopulations have distinct differentiation and proliferation properties•A nonquiescent stem cell population resides in the basal layer of the esophagus Because the esophageal epithelium lacks a defined stem cell niche, it is unclear whether all basal epithelial cells in the adult esophagus are functionally equivalent. In this study, we showed that basal cells in the mouse esophagus contained a heterogeneous population of epithelial cells, similar to other rapidly cycling tissues such as the intestine or skin. Using a combination of cell-surface markers, we separated primary esophageal tissue into distinct cell populations that harbored differences in stem cell potential. We also used an in vitro 3D organoid assay to demonstrate that Sox2, Wnt, and bone morphogenetic protein signaling regulate esophageal self-renewal. Finally, we labeled proliferating basal epithelial cells in vivo to show differing cell-cycle profiles and proliferation kinetics. Based on our results, we propose that a nonquiescent stem cell population resides in the basal epithelium of the mouse esophagus. Because the esophageal epithelium lacks a defined stem cell niche, it is unclear whether all basal epithelial cells in the adult esophagus are functionally equivalent. In this study, we showed that basal cells in the mouse esophagus contained a heterogeneous population of epithelial cells, similar to other rapidly cycling tissues such as the intestine or skin. Using a combination of cell-surface markers, we separated primary esophageal tissue into distinct cell populations that harbored differences in stem cell potential. We also used an in vitro 3D organoid assay to demonstrate that Sox2, Wnt, and bone morphogenetic protein signaling regulate esophageal self-renewal. Finally, we labeled proliferating basal epithelial cells in vivo to show differing cell-cycle profiles and proliferation kinetics. Based on our results, we propose that a nonquiescent stem cell population resides in the basal epithelium of the mouse esophagus. The esophageal epithelium is a rapidly self-renewing tissue comprised of a basal cell layer and more differentiated suprabasal layers (Messier and Leblond, 1960Messier B. Leblond C.P. Cell proliferation and migration as revealed by radioautography after injection of thymidine-H3 into male rats and mice.Am. J. Anat. 1960; 106: 247-285Crossref PubMed Scopus (463) Google Scholar). Proliferation is restricted to the basal cell layer, which contains cells that self-renew and differentiate over the lifespan of the tissue (Marques-Pereira and Leblond, 1965Marques-Pereira J.P. Leblond C.P. Mitosis and differentiation in the stratified squamous epithelium of the rat esophagus.Am. J. Anat. 1965; 117: 73-87Crossref PubMed Scopus (99) Google Scholar). To maintain tissue homeostasis, esophageal basal cells divide approximately twice per week to replace the differentiated cells that are shed into the lumen (Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar). However, conflicting reports have made it difficult to determine if there is a separate subpopulation of slower-cycling stem cells that give rise to more differentiated cells in the basal layer, or if all basal cells represent a single progenitor population (Croagh et al., 2007Croagh D. Phillips W.A. Redvers R. Thomas R.J. Kaur P. Identification of candidate murine esophageal stem cells using a combination of cell kinetic studies and cell surface markers.Stem Cells. 2007; 25: 313-318Crossref PubMed Scopus (73) Google Scholar, Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar, Kalabis et al., 2008Kalabis J. Oyama K. Okawa T. Nakagawa H. Michaylira C.Z. Stairs D.B. Figueiredo J.L. Mahmood U. Diehl J.A. Herlyn M. Rustgi A.K. A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.J. Clin. Invest. 2008; 118: 3860-3869PubMed Google Scholar, Marques-Pereira and Leblond, 1965Marques-Pereira J.P. Leblond C.P. Mitosis and differentiation in the stratified squamous epithelium of the rat esophagus.Am. J. Anat. 1965; 117: 73-87Crossref PubMed Scopus (99) Google Scholar, Seery, 2002Seery J.P. Stem cells of the oesophageal epithelium.J. Cell Sci. 2002; 115: 1783-1789Crossref PubMed Google Scholar). In the intestine, multipotent LGR5+ stem cells are found in readily identifiable structures called crypts and regenerate all epithelial lineages of the intestine (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. Clevers H. Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (4045) Google Scholar). Conversely, the basal epithelium of the esophagus is morphologically more uniform and gives rise to a single cell lineage that forms the suprabasal layer. This simple structure has led to questions about the presence or necessity of a separate stem cell population in the basal epithelium, similar to the questions that have arisen regarding the interfollicular epidermis (Clayton et al., 2007Clayton E. Doupé D.P. Klein A.M. Winton D.J. Simons B.D. Jones P.H. A single type of progenitor cell maintains normal epidermis.Nature. 2007; 446: 185-189Crossref PubMed Scopus (646) Google Scholar, Doupé and Jones, 2013Doupé D.P. Jones P.H. Cycling progenitors maintain epithelia while diverse cell types contribute to repair.BioEssays. 2013; 35: 443-451Crossref PubMed Scopus (24) Google Scholar, Kaur and Potten, 2011Kaur P. Potten C.S. The interfollicular epidermal stem cell saga: sensationalism versus reality check.Exp. Dermatol. 2011; 20: 697-702Crossref PubMed Scopus (27) Google Scholar, Lim et al., 2013Lim X. Tan S.H. Koh W.L. Chau R.M. Yan K.S. Kuo C.J. van Amerongen R. Klein A.M. Nusse R. Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling.Science. 2013; 342: 1226-1230Crossref PubMed Scopus (254) Google Scholar, Mascré et al., 2012Mascré G. Dekoninck S. Drogat B. Youssef K.K. Broheé S. Sotiropoulou P.A. Simons B.D. Blanpain C. Distinct contribution of stem and progenitor cells to epidermal maintenance.Nature. 2012; 489: 257-262Crossref PubMed Scopus (406) Google Scholar). Our results indicate that the basal epithelium of the mouse esophagus contains both proliferating stem and transit-amplifying cells. During development, both the Wnt and transforming growth factor β cell signaling pathways play an important role to properly form the adult esophagus as well as other endoderm-derived organs such as the trachea, stomach, and intestine (Barker et al., 2010Barker N. Huch M. Kujala P. van de Wetering M. Snippert H.J. van Es J.H. Sato T. Stange D.E. Begthel H. van den Born M. et al.Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Cell Stem Cell. 2010; 6: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, Jacobs et al., 2012Jacobs I.J. Ku W.Y. Que J. Genetic and cellular mechanisms regulating anterior foregut and esophageal development.Dev. Biol. 2012; 369: 54-64Crossref PubMed Scopus (63) Google Scholar, Que et al., 2006Que J. Choi M. Ziel J.W. Klingensmith J. Hogan B.L. Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps.Differentiation. 2006; 74: 422-437Crossref PubMed Scopus (192) Google Scholar, van der Flier and Clevers, 2009van der Flier L.G. Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium.Annu. Rev. Physiol. 2009; 71: 241-260Crossref PubMed Scopus (1245) Google Scholar). These signaling pathways were shown to control the intestinal stem cell niche in a 3D in vitro assay, in which intestinal stem cells generated organoids containing crypt structures (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4290) Google Scholar, Sato et al., 2011Sato T. Stange D.E. Ferrante M. Vries R.G. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2185) Google Scholar). Related 3D assays have been used to characterize stem cells in the brain and breast, among other tissues (Maslov et al., 2004Maslov A.Y. Barone T.A. Plunkett R.J. Pruitt S.C. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice.J. Neurosci. 2004; 24: 1726-1733Crossref PubMed Scopus (474) Google Scholar, Stingl et al., 2006Stingl J. Eirew P. Ricketson I. Shackleton M. Vaillant F. Choi D. Li H.I. Eaves C.J. Purification and unique properties of mammary epithelial stem cells.Nature. 2006; 439: 993-997Crossref PubMed Scopus (1258) Google Scholar). Therefore, we hypothesized that a similar assay could be applied to the esophagus. To test this, we removed the esophagus from mice and enzymatically dissociated the mucosa into single cells followed by suspension in Matrigel (Figures 1A–1C). We found that growth media supplemented with exogenous stem cell factors was required to generate 3D organoids (Figure 1D and Table S1). The organoids were morphologically similar to normal esophageal tissue after 9 days in culture, with small basal-like cells in contact with the extracellular matrix, large flat suprabasal-like cells in the interior, and hardened keratinized material in the center (Figures 1E and 1F). We then compared the cellular composition of the organoids to primary tissue using markers that are specific for the basal and more differentiated suprabasal cell layers (Figure 1G). The organoid outer cell layer was CK14+, p63+, and contained proliferating cells (incorporated EdU during a 2-hr incubation), similar to esophageal basal cells found in primary tissue. The organoid interior consisted of differentiated cells as shown by CK13+ immunostaining, as well as abundant keratinization. Next, we determined if organoids were generated from single esophageal epithelial cells. Initially, we combined a single cell suspension of primary esophageal cells from GFP+ and GFP− mice. After organoids formed, they were always completely GFP+ or GFP−, indicating that they did not form by aggregation (Figures S1A and S1B). We then sorted primary esophageal cells with fluorescence-activated cell sorting (FACS) at the clonal level, and single cells were suspended in Matrigel to initiate organoid formation. After 9 days, organoids grew from a single cell with a similar morphology to those plated in a single cell suspension (Figure S1C). We then assessed the role of Wnt and BMP signaling on esophageal organoid generation and self-renewal because these pathways govern esophageal development as well as the intestinal stem cell niche (Barker, 2014Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration.Nat. Rev. Mol. Cell Biol. 2014; 15: 19-33Crossref PubMed Scopus (793) Google Scholar, Jacobs et al., 2012Jacobs I.J. Ku W.Y. Que J. Genetic and cellular mechanisms regulating anterior foregut and esophageal development.Dev. Biol. 2012; 369: 54-64Crossref PubMed Scopus (63) Google Scholar). Primary esophageal cells were suspended in Matrigel followed by the addition of complete stem cell medium, stem cell medium lacking Wnt agonists (Wnt3a/R-Spondin 2), or stem cell medium lacking the BMP inhibitor noggin. Organoids were generated with a similar efficiency under each condition, with a small decrease in organoid formation in the absence of noggin (Figure S1D). However, upon dissociation of organoids to single cells and replating, we found that exogenous noggin and Wnt agonists were required for self-renewal. On the other hand, organoids maintained in the presence of stem cell medium showed no decrease in self-renewal potential (passaged at least five times). We then asked whether the ability to generate organoids was restricted to basal epithelial cells, because these cells are thought to contain stem/progenitor cells (Messier and Leblond, 1960Messier B. Leblond C.P. Cell proliferation and migration as revealed by radioautography after injection of thymidine-H3 into male rats and mice.Am. J. Anat. 1960; 106: 247-285Crossref PubMed Scopus (463) Google Scholar). Previous studies reported that Sox2 is constitutively expressed in all basal epithelial cells that give rise to Sox2-negative differentiated cells, which we confirmed by immunostaining (Figure 2A; Arnold et al., 2011Arnold K. Sarkar A. Yram M.A. Polo J.M. Bronson R. Sengupta S. Seandel M. Geijsen N. Hochedlinger K. Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice.Cell Stem Cell. 2011; 9: 317-329Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, Liu et al., 2013Liu K. Jiang M. Lu Y. Chen H. Sun J. Wu S. Ku W.Y. Nakagawa H. Kita Y. Natsugoe S. et al.Sox2 cooperates with inflammation-mediated Stat3 activation in the malignant transformation of foregut basal progenitor cells.Cell Stem Cell. 2013; 12: 304-315Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). To separate primary live basal epithelial cells from other cells (i.e., hematopoietic, mesenchymal, or suprabasal), we used a genetic mouse model that had EGFP knocked in to one of the endogenous Sox2 alleles (Figure 2B). After isolating cells from the esophagus and separating the Sox2+ and Sox2− cells by FACS, we found that only Sox2+ cells generated organoids (Figures 2C, 2D, and S2A). We also found that the Sox2+ population could be distinguished from the Sox2− population with the cell-surface marker EpCam, but EpCam expression is dim compared to that of GFP (Figure S2B). We confirmed that the Sox2+ cell population was specific to basal cells, because all of the primary GFP+ cells expressed the β1 integrin (Itgb1, CD29) and p75 (Figure 2E), which were previously used to separate esophageal basal cells (Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar, Liu et al., 2013Liu K. Jiang M. Lu Y. Chen H. Sun J. Wu S. Ku W.Y. Nakagawa H. Kita Y. Natsugoe S. et al.Sox2 cooperates with inflammation-mediated Stat3 activation in the malignant transformation of foregut basal progenitor cells.Cell Stem Cell. 2013; 12: 304-315Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Furthermore, we quantified the percent of EdU uptake in esophageal basal cells by both immunostaining and flow cytometry, and found no significant difference in the percent of EdU+ cells when comparing the two methods (Figure S2C). Together, these data show that the sorted Sox2 GFP+ population does not contain differentiated postmitotic suprabasal cells. We next confirmed that Sox2+ basal cells gave rise to differentiated cells using lineage tracing in the in vitro organoid assay. To label the Sox2+ cells and their progeny, we used a tamoxifen inducible Sox2CreERT2 knockin mouse crossed with a mouse that contains a floxed stop signal to prevent EYFP expression (Figure 3A). Esophageal cells isolated from the Sox2CreERT2/EYFP mice were suspended in Matrigel to generate organoids followed by a 12 hr tamoxifen pulse to activate EYFP expression. After 9 days in culture, we found a majority of organoids with EYFP expression in all cells of the organoid, indicating that Sox2+ cells generated the organoids (Figure 3B). However, treatment with 1 μM tamoxifen was not 100% efficient at labeling all cells (Figures S3A and S3B). We then generated Sox2CreERT2/floxed mice to genetically remove Sox2 upon tamoxifen administration (Figure 3C). We confirmed the loss (∼80%) of Sox2 expression in organoids after the addition of tamoxifen using quantitative PCR analysis (Figure 3D). Previous studies showed that ablation of Sox2 expressing cells disrupted the esophageal basal epithelium (Arnold et al., 2011Arnold K. Sarkar A. Yram M.A. Polo J.M. Bronson R. Sengupta S. Seandel M. Geijsen N. Hochedlinger K. Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice.Cell Stem Cell. 2011; 9: 317-329Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Here, we found that genetically reducing Sox2 expression resulted in an ∼20% decrease in the total number of organoids formed in vitro (Figure 3E). We also found a significant decrease in the size of the organoids after Sox2 deletion (Figure S3C). Upon organoid dissociation and passaging, we found that Sox2 is required to generate new organoids (Figure 3F). Together, these data indicate that Sox2 plays an important role in the generation and self-renewal of organoids, and Sox2+ basal cells contain a stem cell population. After establishing an in vitro assay to test for stemness and focusing on the basal cell compartment, we asked if stem cell heterogeneity could be observed among the basal cell population. Cell-surface proteins have been used to separate epithelial subpopulations to test for differences in stem cell potential (Croagh et al., 2007Croagh D. Phillips W.A. Redvers R. Thomas R.J. Kaur P. Identification of candidate murine esophageal stem cells using a combination of cell kinetic studies and cell surface markers.Stem Cells. 2007; 25: 313-318Crossref PubMed Scopus (73) Google Scholar, Kaur et al., 2004Kaur P. Li A. Redvers R. Bertoncello I. Keratinocyte stem cell assays: an evolving science.J. Investig. Dermatol. Symp. Proc. 2004; 9: 238-247Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). If all proliferating esophageal basal cells represent a single functionally equivalent cell population (Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar), then we predict that separate cell populations would show similar stem cell characteristics. Using flow cytometry analysis, we found that esophageal basal cells (Sox2+) express a range of low, medium, and high levels of α6 integrin (Itga6, CD49f) and β4 integrin (Itgb4, CD104), which form the integrin pair for laminin (Figure 4A). We also found heterogeneous expression of Itgb4 on basal cells by immunostaining (Figure 4B). We used FACS to separate primary esophageal basal cells based on their Itga6/Itgb4 expression. Limiting dilution analysis showed that the colony-forming unit frequency of cells expressing high levels of Itga6/Itgb4 was enriched 2.6-fold above the bulk population of basal cells (Figure 4C). In addition, the organoid-forming unit frequency of Itga6/Itgb4High cells was significantly higher than the Itga6/Itgb4Low and bulk cell populations (Figure 4D). These data show that Itga6/Itgb4High expressing basal cells enrich for cells that have stem cell features (i.e., increased colony-forming frequency and 3D organoid generation), similar to previous reports that correlated integrin expression with epithelial stemness (Adams and Watt, 1990Adams J.C. Watt F.M. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface.Cell. 1990; 63: 425-435Abstract Full Text PDF PubMed Scopus (400) Google Scholar, Croagh et al., 2007Croagh D. Phillips W.A. Redvers R. Thomas R.J. Kaur P. Identification of candidate murine esophageal stem cells using a combination of cell kinetic studies and cell surface markers.Stem Cells. 2007; 25: 313-318Crossref PubMed Scopus (73) Google Scholar, Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, Mascré et al., 2012Mascré G. Dekoninck S. Drogat B. Youssef K.K. Broheé S. Sotiropoulou P.A. Simons B.D. Blanpain C. Distinct contribution of stem and progenitor cells to epidermal maintenance.Nature. 2012; 489: 257-262Crossref PubMed Scopus (406) Google Scholar). Previous work suggested that CD34+ cells were candidate esophageal stem cells, but subsequent studies showed that epithelial cells do not express CD34 (Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar, Kalabis et al., 2008Kalabis J. Oyama K. Okawa T. Nakagawa H. Michaylira C.Z. Stairs D.B. Figueiredo J.L. Mahmood U. Diehl J.A. Herlyn M. Rustgi A.K. A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.J. Clin. Invest. 2008; 118: 3860-3869PubMed Google Scholar). In our hands, CD34 appears to label an EpCam− stromal cell population of mesenchymal origin (Figure 4E). Next, we asked whether the Itga6/Itgb4High-expressing cells could be further enriched for stem cell activity. While screening the esophageal cells for mesenchymal cell-surface marker expression, we noticed that a subpopulation of Itga6/Itgb4High epithelial cells were CD73+. We hypothesized that CD73 may be a stem cell marker for esophageal basal epithelial cells. We found CD73+ basal cells by immunostaining, consistent with the predicted localization of an esophageal stem cell (Figure 5A). Using flow cytometry, the Itga6/Itgb4High population was separated into two populations, Itga6/Itgb4HighCD73+ and Itga6/Itgb4HighCD73−, and compared to the bulk population for the ability to generate organoids in vitro (Figure 5B). We found that the CD73+ cell population had a significantly higher organoid-forming unit frequency compared to both the Itga6/Itgb4HighCD73− and the bulk cell populations (Figure 5C). Our data are consistent with a hierarchical model, in which the Itga6/Itgb4HighCD73+ population represents cells with the greatest stem cell potential followed by a continuum of increased differentiation (Figure 5D). To test this model further, we performed quantitative PCR analysis. We sorted primary esophageal basal cells based on their Itga6/Itgb4 and CD73 expression and assessed gene expression of cytokeratin 14 (Krt14), cytokeratin 13 (Krt13), cytokeratin 4 (Krt4), and involucrin (Ivl; Figure 5E). Each sorted cell population expressed the same levels of Krt14 (basal cell marker), confirming that each population represents basal cells equally. On the other hand, we observed increased expression of the differentiating cell markers Krt13 and Krt4 as cells progress from the Itga6/Itgb4HighCD73+ population, to the Itga6/Itgb4HighCD73− population, to the Itga6/Itgb4Low population. Ivl, a marker of differentiation in suprabasal cells, was below the threshold of detection in each population. Next, we determined whether esophageal organoids recapitulate the cell-surface heterogeneity observed on cells from primary tissue. Cells were isolated from mice and placed in Matrigel to generate organoids. After 9 days, organoids were collected and dissociated to single cells for subsequent analysis by flow cytometry (Figure S4A). Similar to primary tissue, we observed a range of Itga6 and Itgb4 expression. We found high CD73 expression on dissociated organoids in both the Itga6/Itgb4High and Itga6/Itgb4Low populations. However, the mean fluorescence intensity of CD73 was higher in the Itga6/Itgb4High population compared to the Itga6/Itgb4Low population, which is consistent with primary cells. These data show that organoids retain a similar cell-surface phenotype compared to cells isolated directly from primary tissue, with differences in overall expression levels once cells are cultured in vitro. Organoids generated from primary esophageal epithelial cells can be passaged repeatedly in the presence of stem cell medium, indicating self-renewal potential (Figure S1D). We then asked if self-renewal potential was inherently lower in the Itga6/Itgb4Low population, which represents a more differentiated cell population compared to the Itga6/Itgb4High population. We sorted primary esophageal cells from the Itga6/Itgb4High and Itga6/Itgb4Low populations and generated organoids in stem cell medium. Organoids were dissociated to single cells and suspended in Matrigel to form new organoids. We found that organoids derived from both the Itga6/Itgb4High and Itga6/Itgb4Low populations were capable of self-renewal at a similar level, because they could be passaged repeatedly (Figure S4B). Even though fewer organoids were initially generated from the Itga6/Itgb4Low population (Figure 4D), these data suggest that extrinsic factors (i.e., stem cell medium) are sufficient to maintain self-renewal potential once organoids are formed. Therefore, the total number of cell divisions (self-renewal potential) does not appear to be intrinsically defined by the current differentiation status of a given basal cell, similar to the regulation observed in proliferating intestinal epithelial cells (Ritsma et al., 2014Ritsma L. Ellenbroek S.I. Zomer A. Snippert H.J. de Sauvage F.J. Simons B.D. Clevers H. van Rheenen J. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.Nature. 2014; 507: 362-365Crossref PubMed Scopus (349) Google Scholar). Although a quiescent long-term label-retaining epithelial cell may not be present in the basal layer (Doupé et al., 2012Doupé D.P. Alcolea M.P. Roshan A. Zhang G. Klein A.M. Simons B.D. Jones P.H. A single progenitor population switches behavior to maintain and repair esophageal epithelium.Science. 2012; 337: 1091-1093Crossref PubMed Scopus (217) Google Scholar), we predict that an activated esophageal stem cell would undergo cell division less frequently than a transit-amplifying cell, whereas a fully differentiated cell of the suprabasal layer would not divide (Croagh et al., 2007Croagh D. Phillips W.A. Redvers R. Thomas R.J. Kaur P. Identification of candidate murine esophageal stem cells using a combination of cell kinetic studies and cell surface markers.Stem Cells. 2007; 25: 313-318Crossref PubMed Scopus (73) Google Scholar, Potten and Loeffler, 1990Potten C.S. Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.Development. 1990; 110: 1001-1020Crossref PubMed Google Scholar). This is analogous to the proliferation kinetics observed in the intestine, where actively dividing LGR5+ cells give rise to the rapidly dividing transit-amplifying population, which eventually form the fully differentiated cell lineages of the intestine (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. Clevers H. Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (4045) Google Scholar). We examined the cell-cycle profile of the three basal epithelial cell populations that we identified (Figure 6A). Primary esophageal cells were isolated from mice and we found that the Itgb4HighCD73+ stem cell enriched population had fewer cells in G1 phase and significantly more cells in the S and G2/M phases compared to the Itgb4HighCD73- and Itgb4LowCD73− populations (Figures 6B and 6C). Next, we administered EdU to mice for 2, 12, or 24 hr and isolated cells from the esophagus. Short-term exposure to EdU (2 hr) resulted in a profile similar to the number of cells found in S phase (Figures 6D and 6E). After specifically examining EdU+ cells, we found that each of our identified cell populations were represented, indicating that each basal cell subpopulation remains actively dividing while having varying degrees of differentiation (Figure 6F). We also observed more EdU+ cells in the Itgb4HighCD73− population compared to the Itgb4HighCD73+ population after 24 hr (Figure 6E), which suggested that the CD73− population might serve as a faster dividing transit-amplifying cell population. Next, we examined the CD73− population as a whole, irrespective of Itgb4 expression levels, and found that they incorporated a higher proportion of EdU over time compared to the Itgb4HighCD73+ population, in part because the CD73− population represents the majority of basal cells (Figures S5A–S5C). We then assessed the frequency of cell division to determine if distinct basal cell subpopulations do, in fact, proliferate at different rates. The stem-like Itgb4HighCD73+ cells divided approximately 1.4 times per week (± 0.1), whereas Itgb4HighCD73− cells divided almost twice as fast at 2.3x per week (+/− 0.2), and Itgb4LowCD73− cells divided approximately 1.7 times per week (± 0.2; Figure 6G). We also determined that the entire CD73− (i.e., the putative transit-amplifying) pop" @default.
• W2024876545 created "2016-06-24" @default.
• W2024876545 creator A5000371607 @default.
• W2024876545 creator A5005294767 @default.
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• W2024876545 date "2014-10-01" @default.
• W2024876545 modified "2023-10-04" @default.
• W2024876545 title "Cellular Heterogeneity in the Mouse Esophagus Implicates the Presence of a Nonquiescent Epithelial Stem Cell Population" @default.
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