Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium

Cell Regeneration - 2021
Zhongyang Zhao1, Yujia Wang1, Yingchuan Wu1, Dandan Li1, Ting Zhang2, Yu Ma2, Xiaoming Teng3, Wei Zuo2
1East Hospital, School of Medicine, Tongji University, Shanghai, China
2Super Organ R&D Center, Regend Therapeutics, Shanghai, China
3Shanghai First Maternity and Infant Hospital, Tongji University, Shanghai, China

Tóm tắt

AbstractInformation about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.

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Tài liệu tham khảo

Avraham R, Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol. 2011;12(2):104–17.

Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell. 2004;118(5):635–48.

Cantz T, Manns MP, Ott M. Stem cells in liver regeneration and therapy. Cell Tissue Res. 2008;331(1):271–82.

Cao J, Spielmann M, Qiu X, Huang X, Ibrahim DM, Hill AJ, et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019;566(7745):496–502.

Chumduri C, Gurumurthy RK, Berger H, Dietrich O, Kumar N, Koster S, et al. Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia. Nat Cell Biol. 2021;23(2):184–97.

Chumduri C, Gurumurthy RK, Berger H, Koster S, Meyer TF. Transition of Wnt signaling microenvironment delineates the squamo-columnar junction and emergence of squamous metaplasia of the cervix; 2018.

Clevers H, Loh KM, Nusse R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science. 2014;346(6205):1248012.

Cliffe LJ, Humphreys NE, Lane TE, Potten CS, Booth C, Grencis RK. Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion. Science. 2005;308(5727):1463–5.

Darwish AM, Zahran KM. Trichloroacetic acid application versus spray monopolar diathermy for treating benign cervical lesions: a randomized controlled clinical trial. J Lower Genit Tract Dis. 2013;17(3):248–54.

Farin HF, Van Es JH, Clevers H. Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology. 2012;143(6):1518–29 e7.

Finak G, McDavid A, Yajima M, Deng J, Gersuk V, Shalek AK, et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015;16:278.

Geirsson G, Woodworth F, Patten S Jr, Bonfiglio T. Epithelial repair and regeneration in the uterine cervix. I. An analysis of the cells. Acta Cytol. 1977;21(3):371–8.

Haegebarth A, Clevers H. Wnt signaling, lgr5, and stem cells in the intestine and skin. Am J Pathol. 2009;174(3):715–21.

Hwang LY, Ma Y, Benningfield SM, Clayton L, Hanson EN, Jay J, et al. Factors that influence the rate of epithelial maturation in the cervix in healthy young women. J Adolesc Health. 2009;44(2):103–10.

Kang HM, Huang S, Reidy K, Han SH, Chinga F, Susztak K. Sox9-positive progenitor cells play a key role in renal tubule epithelial regeneration in mice. Cell Rep. 2016;14(4):861–71.

Kumar PA, Hu Y, Yamamoto Y, Hoe NB, Wei TS, Mu D, et al. Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell. 2011;147(3):525–38.

Kusaba T, Lalli M, Kramann R, Kobayashi A, Humphreys BD. Differentiated kidney epithelial cells repair injured proximal tubule. Proc Natl Acad Sci U S A. 2014;111(4):1527–32.

Li Y, Zhang QY, Sun BF, Ma Y, Zhang Y, Wang M, et al. Single-cell transcriptome profiling of the vaginal wall in women with severe anterior vaginal prolapse. Nat Commun. 2021;12(1):87.

Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The molecular signatures database (MSigDB) hallmark gene set collection. Cell Systems. 2015;1(6):417–25.

Ma Q, Ma Y, Dai X, Ren T, Fu Y, Liu W, et al. Regeneration of functional alveoli by adult human SOX9+ airway basal cell transplantation. Protein Cell. 2018;9(3):267–82.

Malviya VK, Deppe G, Pluszczynski R, Boike G. Trichloroacetic acid in the treatment of human papillomavirus infection of the cervix without associated dysplasia. Obstet Gynecol. 1987;70(1):72–4.

Martens JE, Arends J, Van Der Linden PJ, De Boer BA, Helmerhorst TJ. Cytokeratin 17 and p63 are markers of the HPV target cell, the cervical stem cell. Anticancer Res. 2004;24(2B):771–6.

Martens JE, Smedts FM, Ploeger D, Helmerhorst TJ, Ramaekers FC, Arends JW, et al. Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal. Int J Gynecol Pathol. 2009;28(4):381–8.

Maru Y, Kawata A, Taguchi A, Ishii Y, Baba S, Mori M, et al. Establishment and Molecular Phenotyping of Organoids from the Squamocolumnar Junction Region of the Uterine Cervix. Cancers. 2020;12(3):694.

Menéndez Velázquez JF, González Sánchez JL, Rodríguez de Santiago JD, Muñoz Reyes R, Bailón Uriza R. The treatment of cervical human papillomavirus (HPV) infection with trichloroacetic acid. Ginecol Obstet Mex. 1993;61:48–51.

Meyer-Blazejewska EA, Call MK, Yamanaka O, Liu H, Schlötzer-Schrehardt U, Kruse FE, et al. From hair to cornea: toward the therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency. Stem Cells. 2011;29(1):57–66.

Noah TK, Shroyer NF. Notch in the intestine: regulation of homeostasis and pathogenesis. Annu Rev Physiol. 2013;75:263–88.

Parent R, Marion M-J, Furio L, Trépo C, Petit M-A. Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology. 2004;126(4):1147–56.

Phadnis S, Atilade A, Bowring J, Kyrgiou M, Young M, Evans H, et al. Regeneration of cervix after excisional treatment for cervical intraepithelial neoplasia: a study of collagen distribution. BJOG Int J Obstet Gynaecol. 2011;118(13):1585–91.

Qiu X, Mao Q, Tang Y, Wang L, Chawla R, Pliner HA, et al. Reversed graph embedding resolves complex single-cell trajectories. Nat Methods. 2017;14(10):979–82.

Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147–55.

Reich O, Regauer S, McCluggage WG, Bergeron C, Redman C. Defining the cervical transformation zone and Squamocolumnar junction: can we reach a common Colposcopic and histologic definition? Int J Gynecol Pathol. 2017;36(6):517–22.

Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011;141(5):1762–72.

Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet. 2007;370(9590):890–907.

Singer A. Dynamic anatomy of the cervical epithelium. Adv Exp Med Biol. 1977;89:77–89.

Slack JM. Stem cells in epithelial tissues. Science. 2000;287(5457):1431–3.

Smedts F, Ramaekers F, Leube RE, Keijser K, Link M, Vooijs P. Expression of keratins 1, 6, 15, 16, and 20 in normal cervical epithelium, squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma. Am J Pathol. 1993;142(2):403.

Song T, Seong SJ, Kim B-G. Regeneration process after cervical conization for cervical intraepithelial neoplasia. Obstet Gynecol. 2016;128(6):1258–64.

Suh K-S, Silverberg SG. Tubal metaplasia of the uterine cervix. Int J Gynecol Pathol. 1990;9(2):122–8.

Tian H, Biehs B, Warming S, Leong KG, Rangell L, Klein OD, et al. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature. 2011;478(7368):255–9.

Trapnell C, Cacchiarelli D, Grimsby J, Pokharel P, Li S, Morse M, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol. 2014;32(4):381–6.

Tsuda L, Nagaraj R, Zipursky SL, Banerjee U. An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling. Cell. 2002;110(5):625–37.

Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science. 2004;303(5656):359–63.

van der Marel J, van Baars R, Alonso I, del Pino M, van de Sandt M, Lindeman J, et al. Oncogenic human papillomavirus–infected immature Metaplastic cells and cervical Neoplasia. Am J Surg Pathol. 2014;38(4):470–9.

VanDussen KL, Carulli AJ, Keeley TM, Patel SR, Puthoff BJ, Magness ST, et al. Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development (Cambridge, England). 2012;139(3):488–97.

Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature. 2018;563(7731):347–53.

Voojis P, van Aspert-van Erp AJ, Bulten J, Bibbo M, Wilbur D. Benign proliferative reactions, intraepithelial neoplasia, and invasive cancer of the uterine cervix. In: Comprehensive Cytopathology. 3rd ed. Philadelphia: Saunders; 2008. p. 131–213.

Wang X, Yamamoto Y, Wilson LH, Zhang T, Howitt BE, Farrow MA, et al. Cloning and variation of ground state intestinal stem cells. Nature. 2015;522(7555):173–8.

Weinreb I. Hyalinizing clear cell carcinoma of salivary gland: a review and update. Head Neck Pathol. 2013;7(Suppl 1):S20–9.

Wen L, Tang F. Single-cell sequencing in stem cell biology. Genome Biol. 2016;17(1):71.

Wiik J, Sengpiel V, Kyrgiou M, Nilsson S, Mitra A, Tanbo T, et al. Cervical microbiota in women with cervical intra-epithelial neoplasia, prior to and after local excisional treatment, a Norwegian cohort study. BMC Womens Health. 2019;19(1):30.

Wu B, An C, Li Y, Yin Z, Gong L, Li Z, et al. Reconstructing lineage hierarchies of mouse uterus epithelial development using single-cell analysis. Stem Cell Rep. 2017;9(1):381–96.

Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics. 2012;16(5):284–7.

Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun. 2019;10(1):1523.

Zuo W, Zhang T, Wu DZA, Guan SP, Liew A-A, Yamamoto Y, et al. p63+ Krt5+ distal airway stem cells are essential for lung regeneration. Nature. 2015;517(7536):616–20.

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