Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells

¹ Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China; Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China; Graduate School, The Chinese Academy of Sciences, Beijing 100039, China
² Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China; Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China; Yunnan Key Laboratory for Animal Reproductive Biology, Kunming Yunnan 650223, China
³ Institute of Zoology, Chinese Academy of Sciences, Beijing 100086, China
⁴ Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China; Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China
⁵ Oregon National Primate Research Center, Portland, OR97006, USA

^* To whom correspondence should be addressed. E-mail: wji{at}mail.kiz.ac.cn.

	Abstract

In the present study, 5 homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rES). Monkey ear skin fibroblasts (MESF), oviductal fibroblasts (MOF), follicular granlosa fibroblast-like cells (MFG), granulosa epithelium-like cells (MFGE) and clonally derived fibroblasts from MESF (CMESF) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rES growth. MESF, MOF, MFG and CMESF, but not MFGE, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rES cells. 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 1, bone morphogenetic protein 4, and WNT3A while WNT2, WNT4 and WNT5A were down regulated, compared with MFGE. Additionally all of the feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signally pathway but not WNT1, WNT8B or Dkk2. rES cells grown on homologous feeders maintained normal karyotypes and displayed the characteristics of ES cells including morphology, alkaline phosphatase, Oct4, the cell surface markers SSEA-3, SSEA-4, 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 rES cells.

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