Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene Running head: Development of embryos cloned from stem cells

We assessed the developmental ability of embryos cloned from porcine neural stem cells, amniotic fluid-derived stem cells, fetal fibroblast cells, adult fibroblast and mammary gland epithelial cells. The five cell lines were transfected with enhanced green fluorescence protein gene respectively using lipofection. Neural stem and amniotic fluid-derived stem cells were induced to differentiate in vitro, respectively. Stem cells and their differentiated cells were harvested for analysis of the markers using reverse transcription polymerase chain reaction. The five cell lines were used for neuclear transfer, respectively. The two-cell stage cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that both neural stem and Page 1 of 32 Reproduction Advance Publication first posted on 4 March 2009 as Manuscript REP-08-0469 Copyright © 2009 by the Society for Reproduction and Fertility. 2 amniotic fluid-derived stem cells expressed Oct4, CD90 and Sox2, and they were both induced to differentiate into astrocyte (GFAP + ), oligodendrocyte (GalC + ), neuron (NF + , NSE + and MAP2 + ), adipocyte (LPL + and PPARγ-D + ), osteoblast (osteonectin + and osteocalcin + ), myocyte (myf-6 + and myoD + ) and endothelium (CD31 + , CD34 + , CD144 + and eNOS + ), respectively. Seven cloned fetuses (28 days and 32 days) derived from stem cells were obtained. The in vitro developmental ability (morula–blastocyst rate were 28.26%-30.07%) and in vivo developmental ability (pregnancy rate were 1.67%-2.17%) of the embryos cloned from stem cells were higher (P < 0.05) than that of the embryos cloned from somatic cells (morula–blastocyst rate were 16.27%-19.28%, and pregnancy rate were 0.00%), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.