35 IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS DERIVED FROM PORCINE EMBRYONIC GERM CELLS AND THEIR DESCENDENT NEURAL PRECURSOR CELLS

Abstract

Due to an ease of reprogramming in the process of nuclear transfer (NT), undifferentiated stem cells may support greater development of cloned embryos compared with differentiated cell types. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than somatic cells. In pigs, comparative studies of NT using stem cells and their differentiated descendant cells have not yet been reported. Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated. Porcine EG cells were isolated from cultured PGC collected from embryos on Day 23 of gestation (Shim et al. 1997 Biol. Reprod. 57, 1089–1095). Neural precursor cells, based on morphology of neural rosettes or neurospheres, were differentiated from porcine EG cells by sequential culture of the cells on gelatinized dishes in N2B27 medium with and without 10 µm retinoic acid for 7 days each. Since porcine EG cells and NP cells differentiated from the same EG cells were used in the present study, the genetic makeup of nuclear donor cells was identical, eliminating any genetic variation between undifferentiated and differentiated cells in the comparison. The efficiencies of NT using the two different cell populations were compared. The rates of fusion were not different between NT embryos from EG and NP cells. However, the rate of cleavage in NT embryos from EG cells was significantly higher (P < 0.05) than that from NP cells (141/247, 57.1% v. 105/228, 46.1%). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than that using NP cells (43/247, 17.4% v. 18/228, 7.9%). The effects on NT efficiency using either stem cells or their differentiated progeny is still controversial. The rates of blastocyst development using somatic stem cells were either reduced in mice (Sung et al. 2006 Nat. Genet. 38, 1323–1328) or remained the same in deer (Berg et al. 2007 Biol. Reprod. 77, 384–394) and cattle (Green et al. 2007 Biol. Reprod. 77, 395–406). However, the results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. This suggests that the effect on NT efficiency by using stem cells and their differentiated progeny may vary depending upon the species and cell types involved.