Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells.

The aim of this study was to determine the developmental ability of cloned embryos derived from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, differentiated cells from NS and AFS cells, fetal fibroblast (FF) cells, adult fibroblast (AF) cells and mammary gland epithelial (MGE) cells. NS, AFS and FF cells were isolated from embryonic day 30 porcine fetus, AF and MGE cells were isolated from adult pig. NS and AFS cells were induced to differentiate into different cell types, respectively. Stem cells and their differentiated cells were harvested for analysis of the markers using reverse transcription PCR. NS and AFS cells, their differentiated cells, FF, AF and MGE cells were used for nuclear transfer, respectively. A total of 100 two-cell stage cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that the neurospheres were positive for the undifferentiated neural cell marker, Nestin and NS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, Desmin, CD-90, Nanog and Sox2. AFS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Nanog, Sox2, Oct4, Desmin and CD-90. Both NS and AFS cells were differentiated 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+). Four cloned fetuses (28 and 32 days) derived from NS and AFS cells were obtained. The developmental potential of the cloned embryos derived from stem cells (NS and AFS cells) were higher (P < 0.05) than that of the cloned embryos derived from somatic cells (the differentiated cells from NS and AFS cells, FF cells, AF cells and MGE cells), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.