Kuo Hung-Chih, Pau K-Y Francis, Yeoman Richard R, Mitalipov Shoukhrat M, Okano Hideyuki, Wolf Don P
Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA.
Biol Reprod. 2003 May;68(5):1727-35. doi: 10.1095/biolreprod.102.012195. Epub 2002 Dec 11.
Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.
胚胎干细胞（ES细胞）能够自我更新、具有多能性，且能够分化为成体中所有类型的细胞。因此，它们有潜力替代退化或受损的细胞，包括中枢神经系统中的细胞。要使基于ES细胞的治疗成为临床现实，涉及非人类灵长类动物的转化研究至关重要。在此，我们报告了猴ES细胞向胚状体（EBs）、神经祖细胞（NPCs）和特定神经表型的分化情况。ES细胞在悬滴中聚集形成EBs。然后将EBs接种到无血清培养基中的黏附表面上，形成NPCs，并在含有成纤维细胞生长因子（FGF）-2的无血清培养基中扩增，之后再诱导神经分化。在每个步骤中，通过免疫细胞化学检测特定标志物的存在来对细胞进行表征。复杂/囊性EBs中的大多数细胞表达了代表所有三个胚胎胚层的抗原（甲胎蛋白、心肌肌钙蛋白I和波形蛋白）。超过70%的扩增细胞群体表达了NPCs的抗原标志物（巢蛋白和musashi1）。去除FGF-2后，约70%的NPCs分化为表达微管相关蛋白-2C（MAP2C）或神经元核抗原（NeuN）的神经元表型，约28%分化为表达胶质纤维酸性蛋白的胶质细胞类型。少量的MAP2C/NeuN阳性细胞还表达酪氨酸羟化酶（约4%）或胆碱乙酰转移酶（约13%）。这些结果表明，猴ES细胞可自发分化为所有三个胚层的细胞，能够被诱导并维持为NPCs，且可进一步分化为特定的神经谱系，包括假定的神经元和胶质细胞。
