5B). Open in a separate window Figure 5 The number of mesenchymal stem cells.The expression of CD90 (A) and CD105 (B), two markers for mesenchymal stem cells in bone marrow mononuclear cells, were measured by flow cytometry. Estrogen deficiency did not switch the number of satellite cells in skeletal muscle mass By counting the number of Pax7+ satellite cells within the tibialis, we found that there was no significant difference among groups (Fig. CXCR4 and intracellular ROS levesl in bone marrow mononuclear cells (BM-MNCs).(A) The bone marrow was collected from your femur and tibia 2 months after treatments, and the number of total collected mononuclear cells from each mouse was directly counted. (B) The expression of CXCR4 was detected in freshly collected BM-MNCs by circulation cytometry. (C) The intracellular ROS level was measured as the mean fluorescence intensity in the freshly collected BM-MNCs after 30?min loading with 10-M CM-H2DCFDA. Voriconazole (Vfend) Moreover, compared with healthy mice in the C group, the expression of c-kit, a marker popularly utilized for identifying hematopoietic stem/progenitors cells, was detected to be significantly higher in the Ovx group (3.27??0.14% 2.65??0.09%, C and Ovx groups, Fig. 3). Open in a separate windows Physique 3 The number of hematopoietic stem/progenitor cells.The expression of c-kit, a marker of hematopoietic stem/progenitor cells in bone marrow mononuclear cells, was measured by flow cytometry. The results of the colony-forming assay, a method well utilized for evaluating the function of hematopoietic stem/progenitor cells 26.7??3.5, C group; Fig. 4B). Open in a separate window Physique 4 Colony-forming assay.Bone marrow mononuclear cells were isolated from mice 2 months after treatments. Colony formation was observed under microscopy at 7 days after incubation. The number of all types Rabbit polyclonal to OSBPL6 of colonies (30 cells, (A)) and mixed cell type colonies (at least two different types of cell in the colony, (B)) were counted. Estrogen deficiency increased the number of CD105+ mesenchymal stem cells in the bone marrow We also measured the expressions of CD90 and CD105, two popular markers utilized for the identifying mesenchymal stem cells. The expression of CD90 in BM-MNCs did not significantly differ among groups (Fig. 5A). However, the expression of CD105 in BM-MNCs was significantly lower in the Ovx group compared with the C group (1.78??0.25% 2.10??0.16%, C group; Fig. 5B). Open in a separate windows Physique 5 The number of mesenchymal stem cells.The expression of CD90 (A) and CD105 (B), two markers for mesenchymal stem cells in bone marrow mononuclear cells, were measured by flow cytometry. Estrogen deficiency did not switch the number of satellite cells in skeletal muscle mass By counting the number of Pax7+ satellite cells within the tibialis, we found that there was no significant difference among groups (Fig. 6). Open in a separate windows Physique 6 The number of satellite cells.Satellite cells were detected in tibialis anterior muscles by immunostaining with an anti-Pax7 antibody, and the Pax7-positive cells in randomly determined fields was counted under fluorescence microscopy. Discussion The present study was designed to examine the hypothesis that estrogen deficiency induces a decrease in the quantity and quality of tissue-specific stem cells, thereby contributing to postmenopausal secondary disorders in different tissues/organs. Using an ovariectomy model in young healthy female mice, we found that estrogen deficiency increased the number, but likely impaired the function, of hematopoietic stem/progenitor cells. Estrogen deficiency also significantly decreased the number of CD105+ mesenchymal stem cells in bone marrow, but did not switch the number of Pax7+ satellite cells in skeletal muscle tissue. Our data Voriconazole (Vfend) shows the heterogeneous effects of estrogen deficiency in different types of tissue-specific stem cells, suggesting a likely and direct relationship between the estrogen deficiency-induced impairment of stem cells and postmenopausal disorders. Although estrogens are generally known as female reproductive hormones, the functions Voriconazole (Vfend) of estrogens in non-reproductive tissues, such as brain, bone, and cardiovascular systems, are well-defined from previous studies13,14,15. The biological activities of estrogens are mediated by two estrogen receptor (ER) Voriconazole (Vfend) isoforms, namely ER and ER15. As stem cells in various tissues express ERs, we examined the role of estrogen in stem cells Estrogen deficiency heterogeneously affects tissue specific stem cells in mice. em Sci. Rep. /em 5, 12861; doi: 10.1038/srep12861 (2015). Supplementary Material Supplementary Information:Click here to view.(77K, pdf) Acknowledgments This study was supported in part by a Grant-in-Aid from your Ministry of Education, Culture, Sports, Science, and Technology, Japan. Funding from your Uehara Memorial Foundation and Mochida Memorial Foundation was also received. No additional external funding was received for this study. The founders did not participate in this study. Footnotes The authors declare no competing financial interests. Author Contributions T.L. and.
