{"title":"培养角膜上皮干细胞序列传代过程中异质性和分子变化的单细胞分析。","authors":"Usanee Reinprayoon, Supaporn Khramchantuk, Natthida Kittimawikrom, Praewphan Ingrungruanglert, Sarinya Phodang, Thanakorn Jaemthaworn, Sira Sriswasdi, Nipan Israsena","doi":"10.1093/stmcls/sxaf041","DOIUrl":null,"url":null,"abstract":"<p><p>The maintenance of corneal epithelial homeostasis relies on limbal stem cells (LSCs) located at the limbus. Although short-term cultured LSC transplantation effectively treats LSC deficiency, prolonged culture leads to stemness loss and abortive colony formation, and the mechanisms remain elusive. In this study, we employed single-cell transcriptomics to investigate LSC population dynamics and changes in gene expression during extended serial culture. Transcriptomic data from 22,708 cells revealed 19 clusters, identifying three distinct limbal progenitor populations (Progenitors 1-3) with unique transcriptional profiles and cell division kinetics. All progenitor subgroups expressed stemness-related genes such as ANLN, AURKB, and HMGB2 and were detected at all stages of the cell cycle. Notably, Progenitor3 exhibited the highest levels of genes associated with stemness and the G2/M checkpoint, including ANLN, PLK1, AURKA, HMGB2, and TOP2A, and had the largest proportion of cells in G2/M. Progenitor2 was marked by histone H1 expression, while Progenitor1 displayed distinctive cell cycle kinetics. Despite stable proportions of the three progenitor populations throughout prolonged passaging, mitochondrial gene downregulation and ribosomal gene upregulation were observed. Treatment with the small molecule RepSox partially preserved LSC maintenance in long-term culture by inhibiting the epithelial-mesenchymal transition program and modulating energy and metabolic pathways. These findings provide insights for optimizing in vitro LSC expansion for cell-based therapies.</p>","PeriodicalId":231,"journal":{"name":"STEM CELLS","volume":" ","pages":""},"PeriodicalIF":3.6000,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Single-cell analysis of heterogeneity and molecular changes in cultured corneal epithelial stem cells during serial passage.\",\"authors\":\"Usanee Reinprayoon, Supaporn Khramchantuk, Natthida Kittimawikrom, Praewphan Ingrungruanglert, Sarinya Phodang, Thanakorn Jaemthaworn, Sira Sriswasdi, Nipan Israsena\",\"doi\":\"10.1093/stmcls/sxaf041\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The maintenance of corneal epithelial homeostasis relies on limbal stem cells (LSCs) located at the limbus. Although short-term cultured LSC transplantation effectively treats LSC deficiency, prolonged culture leads to stemness loss and abortive colony formation, and the mechanisms remain elusive. In this study, we employed single-cell transcriptomics to investigate LSC population dynamics and changes in gene expression during extended serial culture. Transcriptomic data from 22,708 cells revealed 19 clusters, identifying three distinct limbal progenitor populations (Progenitors 1-3) with unique transcriptional profiles and cell division kinetics. All progenitor subgroups expressed stemness-related genes such as ANLN, AURKB, and HMGB2 and were detected at all stages of the cell cycle. Notably, Progenitor3 exhibited the highest levels of genes associated with stemness and the G2/M checkpoint, including ANLN, PLK1, AURKA, HMGB2, and TOP2A, and had the largest proportion of cells in G2/M. Progenitor2 was marked by histone H1 expression, while Progenitor1 displayed distinctive cell cycle kinetics. Despite stable proportions of the three progenitor populations throughout prolonged passaging, mitochondrial gene downregulation and ribosomal gene upregulation were observed. Treatment with the small molecule RepSox partially preserved LSC maintenance in long-term culture by inhibiting the epithelial-mesenchymal transition program and modulating energy and metabolic pathways. These findings provide insights for optimizing in vitro LSC expansion for cell-based therapies.</p>\",\"PeriodicalId\":231,\"journal\":{\"name\":\"STEM CELLS\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.6000,\"publicationDate\":\"2025-06-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"STEM CELLS\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1093/stmcls/sxaf041\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"STEM CELLS","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/stmcls/sxaf041","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Single-cell analysis of heterogeneity and molecular changes in cultured corneal epithelial stem cells during serial passage.
The maintenance of corneal epithelial homeostasis relies on limbal stem cells (LSCs) located at the limbus. Although short-term cultured LSC transplantation effectively treats LSC deficiency, prolonged culture leads to stemness loss and abortive colony formation, and the mechanisms remain elusive. In this study, we employed single-cell transcriptomics to investigate LSC population dynamics and changes in gene expression during extended serial culture. Transcriptomic data from 22,708 cells revealed 19 clusters, identifying three distinct limbal progenitor populations (Progenitors 1-3) with unique transcriptional profiles and cell division kinetics. All progenitor subgroups expressed stemness-related genes such as ANLN, AURKB, and HMGB2 and were detected at all stages of the cell cycle. Notably, Progenitor3 exhibited the highest levels of genes associated with stemness and the G2/M checkpoint, including ANLN, PLK1, AURKA, HMGB2, and TOP2A, and had the largest proportion of cells in G2/M. Progenitor2 was marked by histone H1 expression, while Progenitor1 displayed distinctive cell cycle kinetics. Despite stable proportions of the three progenitor populations throughout prolonged passaging, mitochondrial gene downregulation and ribosomal gene upregulation were observed. Treatment with the small molecule RepSox partially preserved LSC maintenance in long-term culture by inhibiting the epithelial-mesenchymal transition program and modulating energy and metabolic pathways. These findings provide insights for optimizing in vitro LSC expansion for cell-based therapies.
期刊介绍:
STEM CELLS, a peer reviewed journal published monthly, provides a forum for prompt publication of original investigative papers and concise reviews. STEM CELLS is read and written by clinical and basic scientists whose expertise encompasses the rapidly expanding fields of stem and progenitor cell biology.
STEM CELLS covers:
Cancer Stem Cells,
Embryonic Stem Cells/Induced Pluripotent Stem (iPS) Cells,
Regenerative Medicine,
Stem Cell Technology: Epigenetics, Genomics, Proteomics, and Metabonomics,
Tissue-Specific Stem Cells,
Translational and Clinical Research.