Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

Nesfatin-1/NUCB2, which is secreted from the brain, is known to control appetite and energy metabolism. Recent studies have been shown that nesfatin-1/NUCB2 was expressed not only in the brain, but it was also expressed in the gastric organs and adipose tissue. However, little is known about the expression of nesfatin-1/NUCB2 in the male reproductive system. Therefore, we examined whether the nesfatin-1/NUCB2 and its binding site exists in the male reproductive organs. Nesfatin-1/NUCB2 mRNA and protein were detected in the mouse testis and epididymis by PCR and Western blot analysis. As a result of the immunohistochemistry staining, the nesfatin-1 protein was localized at the interstitial cells and Leydig cells in the testis. Nesfatin-1 binding sites were also displayed at boundary cells in the tunica albuginea. Furthermore, in order to examine if the expression of nesfatin-1/NUCB2 mRNA in the testis and epididymis were affected by gonadotropin, its mRNA expression was analyzed after PMSG administration into mice. NUCB2 mRNA expression levels were increased in both of the testis and epididymis after PMSG administration. These results demonstrated for the first time that nesfatin-1 and its binding site were expressed in the mouse testis and epididymis. In addition, nesfatin-1/NUCB2 mRNA expression was controlled by gonadotropin, suggesting a possible role of nesfatin-1 in the male reproductive organs as a local regulator. Due to this, further study is needed to elucidate the functions of nesfatin-1 on the male reproductive system.