Identification Of Stem Cell Subpopulation And Investigation Of The Stem Cell Maintenance Mechanism In Mammalian Esophageal Squamous Epithelia

Esophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumors which ranks the sixth in cancer morbidities and the fourth in mortalities of China.The strong heterogeneity of ESCC was prone to drug resistance,recurrence and metastasis,which eventually leads to treatment failure.The basic reason of cancer heterogeneity was cancer stem cells(CSCs).There are so far no widely accepted markers of CSCs of ESCC.CSCs have many similar properties with normal stem cells and could transform from normal stem cells.However,the research on esophageal stem cells remains controversial.Currently,there are two conflicting theoretical models:heterogenous or homogenous model.Therefore,the identification of esophageal stem cells and the investigation of the stem cell maintenance mechanism can lay an important theoretical foundation for the identification and isolation of CSCs.In the study,we used the BrdU labeling experiment in animals to examine the proliferation pattern of esophageal basal cells.The results showed that the esophageal basal cells were heterogeneous and there were two cell subpopulations.Short-quiescent stem-like cells(SCs)were a small part of esophageal basal cells,out of cell cycle and in a lower metabolic state.Actively proliferating progenitor cells(PCs)accounted for the majority of esophageal basal cells and were in cell cycle.Both subpopulations expressed Keratinl4,p63,Sox2,Bmi1,Oct4,Integrin?6,Integrin?4,and did not express CD34.Next,using whole genome bisulfite sequencing technology,we found that there were differences in methylation profiles between SCs and PCs.The CHG and CHH methylation levels of SCs were significantly different from PCs.Cluster analysis showed that PCs had a high similarity with suprabasal differentiated cells(DCs)and a low similarity with SCs.DMR-related genes were significantly enriched in biological processes such as cell differentiation and regulation of cell shape and biological pathways such as Wnt signaling pathway,pluripotent stem cell regulatory signaling pathway,and cAMP signaling pathway.Furthermore,using organoid culture system in vitro,we proved the heterogeneity in the basal layer of organoids and there were SCs.Further functional assay showed that inhibition of Wnt signals could significantly increase the proportion of SCs,which indicated that SCs were maintained by a low Wnt signal.In conclusion,this study defined the heterogeneity of esophageal basal cells and revealed the existence of short-quiescent stem-like cells(SCs).And our results demonstrated the critical role of Wnt signaling pathway in esophageal stem cell fate determination.To a certain extent,it provided new and strong evidences for the research of esophageal heterogenous model and offered theoretical supports for finding the key molecules and regulatory mechanisms of Wnt signaling pathway.