228 Establishment of Embryonic Stem-Like Cell Lines in Rabbits

Abstract

Embryonic stem (ES) cells are pluripotent cells from the inner cell mass (ICM) of the blastocyst. They are capable of differentiating to various cell types, such as neural cells, cardiocytes, hepatic cells, and germ cells. The aim of this study was to establish rabbit ES cell lines as an animal model for human diseases. Blastocysts were collected from New Zealand White rabbits during Days 4 to 5 after breeding. After removal of the mucin coat and the zona pellucida by pronase, the embryos were directly cultured in ES cell medium on mitomycin C-treated mouse embryonic fibroblast (MEF) or STO feeder layers. In Experiment 1, the efficiencies of 2 different feeder layers, MEF and STO, in generating rabbit ES cell lines were compared. Six blastocysts were used for each STO and MEF feeder group. The primary ICM colonies were formed in 67% (4/6) of the cultures on the STO and 83% (5/6) on the MEF. Sixty percent of those primary colonies (3/5) were successfully grown into ES-like cell lines in the MEF feeder group. However, no cell lines were established on the STO feeder. In Experiment 2, whole blastocysts or ICMs isolated by immunosurgery were cultured to establish ES cell lines. A total of 21 blastocysts were recovered from 2 does. Eighteen whole blastocysts and 3 isolated ICMs were cultured on the MEF feeders. Twelve (67%) of the cultured whole blastocysts formed primary ICM colonies, of which 5 (42%) of the cultures continuously propagated and formed ES-like cell lines. In the immunosurgical group, 2 of the 3 isolated ICMs formed primary colonies but only 1 ES-like cell line was established. A total of 9 ES-like cell lines maintained morphological undifferentiation after 14 passages and expressed alkaline phosphatase activity. Seven of the 9 ES-like cells expressed Oct-4 and the stage-specific embryonic antigen-4 (SSEA-4) as detected by immunocytochemical staining. Two cell lines were further induced to differentiate into embryoid bodies in suspension culture. Another 3 cell lines were injected into SCID mice and one of them formed a teratoma. The competence of generating chimeric rabbits and the teratogenicity of the established ES-like cell lines are under evaluation. In conclusion, rabbit ES-like cells were efficiently generated and whole-blastocyst culturing on the MEF feeder appeared to be a preferred method for the isolation and maintenance of rabbit ES-like cell lines.

Successful Induction of Antisera Against Rabbit Embryos for Isolation of the ICM and Putative Embryonic Stem Cells

“…2000). Although the precise reasons are not clear yet, the similarity was also observed in rabbits in the present and other studies (Ou et al. 2007).…”

“…In addition, TE cells were thought to be inhibitory to ES cell development in humans (Thomson et al 1998;Pera et al 2000). Although the precise reasons are not clear yet, the similarity was also observed in rabbits in the present and other studies (Ou et al 2007). Morphologically, only one potential ES-like cell colony was obtained from the immunosurgically isolated ICM on day 6, but none from the whole blastocyst culture (Table 4) in this study.…”

“…More recently, human ES and embryonic germ (EG) cell lines were also established using ICM (Thomson et al 1998) and primordial germ cells (PGC; Shamblott et al 1998), respectively. Although the initial isolation and maintenance of putative rabbit ES cells have been reported (Graves and Moreadith 1993;Schoonjans et al 1996), no follow-up information is available (Fang et al 2006;Wang et al 2007;Ou et al 2007). Therefore, the understanding of ES cell biology in this species is still in its infancy compared with that in mice.…”