Navarro-Alvarez Nalú, Soto-Gutierrez Alejandro, Yuasa Takesui, Yamatsuji Tomoki, Shirakawa Yasuhiro, Nagasaka Takeshi, Sun Sheng D, Javed Muhammad Shahid, Tanaka Noriaki, Kobayashi Naoya
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Cell Transplant. 2008;17(1-2):27-33.
Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.
人类多能胚胎干细胞（hESCs）在人类发育生物学研究、开发用于治疗疾病的细胞疗法、毒理学和药物发现等方面具有巨大潜力。传统上，未分化的hESCs是在小鼠胚胎成纤维细胞（MEFs）上培养的，这阻碍了hESCs的临床应用。在此，我们研究了日本hESC系（KhES-1）在无饲养层培养中的长期稳定性。将KhES-1细胞与MEF条件培养基（CM）和不同剂量的碱性成纤维细胞生长因子（bFGF）在表面涂有基质胶的六孔板中培养。KhES-1细胞维持培养至少40代。在这种培养系统中，细胞维持稳定的增殖速率，并稳定表达Oct-4、Nanog和碱性磷酸酶。此外，未与饲养层直接接触而维持培养的KhES-1细胞形成了表达三个胚层标志物的胚胎体。在此我们证明，日本人类胚胎干细胞KhES-1可以通过无饲养层方法长期培养，同时在体外保持多能性。
