1. ¹State Key Laboratory of Reproductive Biology, Chinese Academy of Sciences, Beijing 100080, China
2. ²Joint laboratory of biology of embryonic cells of mammalians (LABIOCEM), Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
3. ³Department of Reproduction and Development, Kunming Institute of Zoology & Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223, China
4. ⁴Graduate University of Chinese Academy of Sciences, Beijing 100049, China
5. ⁵INRA, UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas, F-78350, France
6. ⁶Institute of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China

Correspondence: Liu Wang, Tel/Fax: 86-10-62650042 E-mail: qzhou@ioz.ac.cn; Fanyi Zeng, Tel/Fax: 86-10-62650042 E-mail: zengfy@shsmu.edu.cn

^*These two authors contribute equally to the work.

Received 31 November 2006; Revised 13 December 2006; Accepted 13 December 2006; Published online 9 January 2007.

Abstract

Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from nuclear transfer (NT) embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC (NT-ESC) lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage R1 donor cells were able to form full term developed pups, whereas those from late passage R1 ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-R1-ESC lines that were developed from NT blastocyst of late passage R1 ESC donors. However, these NT-R1-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.