Isolation, Characterization, and In-Vitro Establishment of Mammary Epithelial Stem Cells From Beetal Goats

Background: The mammary gland has a remarkable ability to renew and repair itself, making mammary epithelial stem cells (MESCs) critical targets for understanding lactation biology and developing therapeutic interventions for intramammary infections. Objective: The objective of this study was to isolate, culture, and characterize goat mammary epithelial stem cells (GMESCs) from Beetal goats to optimize growth conditions and establish a foundation for future biomedical research. Methods: Mammary tissues were harvested from Beetal goats and subjected to enzymatic digestion to isolate GMESCs. To identify the optimal culture environment, the cells were screened across multiple growth and differentiation media containing varying growth factors. Optimization was performed based on proliferation index and population doubling time. Purification of isolated cells was performed using the EasySep™ PE positive selection kit targeting the CD24 luminal marker. Characterization of stem cells, milk secretion, and proliferation markers was performed using phase-contrast microscopy, colony-forming unit (CFU) assays, qualitative real-time-polymerase chain reaction (qRT-PCR), and immune cytochemistry. Results: The optimal basal growth medium was Dulbecco modified eagle medium (DMEM)/F12 fortified with 10% fetal bovine serum (FBS), three essential growth factors (epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and insulin), and 1% antibiotics. Under these conditions, the GMESCs exhibited a characteristic cobblestone morphology and successfully differentiated into functional units, including domes, alveoli-like structures, and interconnecting tubules. Although CFU assays revealed relatively low colony-forming efficiency in these epithelial cells, the identity of GMESCs was validated by the presence of specific markers, including endogenous stem cell markers and milk-secreting proteins. Purified CD24+ luminal cells maintained stable morphological characteristics after 10 days of culture. Conclusion: This study successfully established a robust protocol for the isolation and expansion of GMESCs from Beetal goats. By defining the specific markers and media requirements of these cells, this study provides a valuable model for mammary tissue repair and broader applications in veterinary regenerative medicine and sustainable production.