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a Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China;
b Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China;
c Graduate School, The Chinese Academy of Sciences, Beijing, China;
d Oregon National Primate Research Center, Portland, Oregon, USA;
e Institute of Zoology, Chinese Academy of Sciences, Beijing, China;
f Yunnan Key Laboratory for Animal Reproductive Biology, Kunming, Yunnan, China
Key Words. Embryonic stem cells • Rhesus monkey feeders • Stem cell markers • Self-renewal • Wnt signaling
Correspondence: Weizhi Ji, Ph.D., Kunming Primate Research Center and Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan, 650223, China. Telephone: 86-871-5139413; Fax: 86-871-5139413; e-mail: wji{at}mail.kiz.ac.cn; and Qi Zhou, Ph.D., Institute of Zoology, Chinese Academy of Sciences, Beijing 100086, China. Telephone: 86-10-62650042; e-mail: qzhou{at}ioz.ac.cn
In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor ß1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.
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