Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan.
J Cell Biochem. 2013 Mar;114(3):584-8. doi: 10.1002/jcb.24395.
Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.
无饲养层培养的人诱导多能干细胞（hiPS）对于其临床应用是必要的，以避免外来蛋白的不良影响。hiPS 细胞在无饲养层条件下，用激活素（A）、CHIR99021（C）、碱性成纤维细胞生长因子（F）和白血病抑制因子（L）组合培养。培养 12 代或细胞形态从多能性改变时终止培养。通过碱性磷酸酶（ALP）染色和 Oct3/4、Nanog、SSEA4 和 TRA-1-60 抗体的免疫染色分析多能性。在培养物中添加激活素抑制剂 SB431542（SB），观察细胞形态。经过 12 代培养的 A、AC 和 ACL 培养的 hiPS 细胞 ALP 染色阳性。A、AC 和 ACL 培养的 hiPS 细胞 Oct3/4 阳性。A 和 AC 培养的 hiPS 细胞 Nanog 阳性，但 ACL 培养的细胞 Nanog 信号较弱。A 和 AC 培养的 hiPS 细胞 SSEA4 阳性，但 ACL 培养的细胞几乎为阴性。A、AC 和 ACL 培养的 hiPS 细胞 TRA-1-60 阳性。当用 A + SB 培养时，hiPS 细胞在 6 代时失去未分化形态，用 AC + SB 培养时在 5 代时失去未分化形态，用 ACL 培养时在 9 代时失去未分化形态。我们得出结论，无饲养层培养 hiPS 细胞需要 A 或 AC 来维持多能性。
