Abstract
We have earlier reported pluripotent, very small embryonic-like stem cells (VSELs) and slightly bigger endometrial stem cells (EnSCs) in adult mouse uterus and their regulation by gonadotropin and steroid hormones. VSELs can differentiate into cells of all three lineages in vitro; however, they neither expand readily in vitro nor compliment a developing embryo. In the present study, a robust protocol is described to enrich uterine stem/progenitor cells along with their characterization and variation across estrus cycle. After enzymatic digestion of adult mouse uterus, single-cell suspension obtained was spun at 1000 rpm (250 g) to pellet majority of cells. Stem cells remain buoyant at this speed and were pelleted by spinning supernatant at 3000 rpm (1000 g). Spherical, darkly stained VSELs (2–6 μm) with high nucleo-cytoplasmic ratio and EnSCs (> 6 μm) expressed OCT-4, NANOG, SSEA-1, SCA-1, and c-KIT. OCT-4-positive cells co-expressed SSEA-1, ERα, ERβ, PR, and FSHR. Transcripts specific for pluripotent state (Oct-4, Oct-4a, Sox-2, Nanog), primordial germ cells (Stella, Fragilis), and receptors for pituitary and steroid hormones (ERα, ERβ, PR, FSHR 1 and 3) were studied by RT-PCR in 3000 rpm pellet. Cell pellet collected at 3000 rpm showed 10-fold enrichment of VSELs (2–6 μm, viable cells with surface phenotype of LIN-CD45-SCA-1+) by flow cytometry and upregulation of pluripotent transcripts by qRT-PCR compared with 1000 rpm pellet. VSELs were maximal during estrus and metestrus phases of estrus cycle. To conclude, VSELs/EnSCs can be enriched from adult uterus using the strategy described here, vary in numbers across estrus cycle, and are vulnerable to endocrine disruption as they express steroid receptors.
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Acknowledgments
The help from Ms Gayatri and Ms Sushma for flow cytometry studies and Ms Reshma and Ms Shobha for confocal studies is acknowledged. (NIRRH/MS/RA/849/12-2019)
Funding
The study was supported by core funds provided by the Indian Council of Medical Research, Government of India, New Delhi. PS acknowledges the DST-INSPIRE fellowship (IF170144).
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Singh, P., Bhartiya, D. Pluripotent Stem (VSELs) and Progenitor (EnSCs) Cells Exist in Adult Mouse Uterus and Show Cyclic Changes Across Estrus Cycle. Reprod. Sci. 28, 278–290 (2021). https://doi.org/10.1007/s43032-020-00250-2
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DOI: https://doi.org/10.1007/s43032-020-00250-2