Isolation, culture, characterization, and adipogenic differentiation of heifer endometrial mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) have been isolated from various tissues of different species. The mammalian endometrium has morphological and functional modifications throughout the estrous cycle undergoing periodic proliferation and degeneration. The aim of this study was to isolate, culture, and characterize and to determine adipogenic differentiation of endometrial mesenchymal stem cells (En-MSCs) in heifers. Uteri of healthy heifers were collected from Shiraz Slaughterhouse, Iran and transferred to Stem Cell Laboratory of Stem Cell and Transgenic Technology Research Center to isolate En-MSCs from endometrial tissue samples. The tissue samples were exposed to collagenase type IA to reach the primary culture of En-MSCs. Isolated En-MSCs were sub-cultured up to passage 4. A specified number of En-MSCs were seeded into 12- and 24-well culture plates, and the number of cells was counted to evaluate the growth behavior of isolated cells and the population doubling time (PDT). RT-PCR for CD45 marker (hematopoietic stem cells) and CD73 marker (MSCs) and differentiation to adipocytes were performed for MSCs confirmation of En-MSCs of heifer. After cell culture, spindle-shape En-MSCs were visible adherent to the culture flasks. The cell count and the growth curves using 12- and 24-well culture plates showed that the PDT of En-MSCs was 37, 159.5, 52.9, and 136.3 h after seeding 2.2 × 104 and 20 × 104 (12-well) and 5 × 104 and 10 × 104 (24-well) En-MSCs, respectively. Using RT-PCR, heifer En-MSCs were negative for CD45 marker and positive for CD73 marker. Moreover, after culture of En-MSCs in differentiation medium, the cells differentiated toward adipocytes as verified by positive staining with Oil Red O staining. The morphology, growth kinetic, and differentiation of bovine En-MSCs demonstrated that these cells may be a good choice in bovine cell therapy and if necessary, they can be easily and safely administered in bovine tissue regeneration.