Anti-Nestin monoclonal antibody, clone 3D14C10

Human teratoma is a germ cell tumor that contains normal tissues (e.g., hair, skin or cartilage) differentiated from embryonal germ layers. Because of the feature of this tumor, we hypothesized that human teratomas contain multipotent stem cells that can develop into various non-cancerous normal tissues. In this study, we cultured neurospheres originally derived from a human infantile teratoma tissue, and the sphere cells were found to possess the characteristics of neural stem cells. Tumor tissues were obtained from an infantile immature teratoma at the time of surgical resection. In the primary cell culture, colonies were formed in two weeks and were individually cultured in serum-free conditioned neural stem cell medium (NSC medium). Colonies changed into spheres and grew in smooth round forms, or attached to the bottom of the dishes and extended processes and filaments around. Sphere cells were dissociated into single cells, and new spheres (secondary spheres) were formed in NSC medium. Cell differentiation was induced by culturing cells in serum-containing medium (differentiation medium), as cells spread and attached to the bottom of dishes and changed form. The expression of Nestin, Sox2, CXCR4, and (stem cell markers), beta 3-tubulin (a neural marker) GFAP (a glial marker) CNPase, SOX10 (oligodendrocyte markers) and NF-L in cells was analyzed by immunofluorescence and a Q-PCR. Nestin, SOX2, CXCR4 were abundant in both primary and secondary spheres. Neural and glial markers (beta 3-tubulin and GFAP, respectively) were increased in cells cultured in differentiation medium while stem cell markers were diminished. The oligodendrocyte markers SOX10 and CNPase were also found in both spheres and differentiated cells. In conclusion, spheres with the characteristics of neural stem cells were obtained from the primary culture of a human infantile teratoma. These spheres are considered to have the potential to undergo a natural course of neural development in humans.

Adipose-derived mesenchymal stem cells (ADSC) are adult pluripotent cells and important resources for cell-based therapies of animals. There are presently different kinds of somatic cells used as donor cells for clone successfully. However, studies on somatic cell nuclear transplantation (SCNT) using ADSC as donor cells from Mongolian sheep have not been reported up to now. This study tested optimal methods of isolating, purifying, and proliferating Mongolian sheep ADSC, and determine their multiple differentiation potentiality. Adipose tissue was removed from approximately 2-year-old sheep and ADSC were harvested by pancreatic enzyme decomposition and adherent culture method. The growth curves of the Passages 1, 5, and 10 cultures were plotted and the exponential growth was determined as a population doubling time of 34.1 h. The expression of OCT4, SOX2, and NANOG genes were increased at Passage 3 (P3) as seen by reverse transcription polymerase chain reaction (RT-PCR) analysis. ADSC from Passage 3 were induced to undergo neurogenesis and form cardiomyocytes and pancreatic islet-like cells under inductive environments in vitro. The differentiation properties of cardiomyocytes and islet-like cells were confirmed by histological staining with toluidine blue, periodic acid-Schiff, and dithizone. The expression of specific genes in these cells were also detected by RT-PCR. Our study results confirm that isolated cells were indeed ADSC and may provide valuable materials for somatic cell clone and transgenic research.