Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

Abstract

Abstract. The present study compared the developmental ability of miniature pig embryos cloned with fetal fibroblasts (FFs), bone marrow-derived mesenchymal stem cells (MSCs) and differentiated (osteocytes, adipocytes and chondrocytes) MSCs. MSCs were isolated from an approximately 1-month-old female miniature pig (T-type, PWG Micro-pig ® , PWG Genetics Korea). MSCs were differentiated into osteocytes, adipocytes and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, as well as FFs of miniature pig, were transferred into enucleated oocytes of domestic pigs. Data from 10 replicates involving 1567 cloned embryos were assessed in terms of developmental rates. The in vitro development rate to the blastocyst stage of embryos cloned with undifferentiated MSCs was significantly (P<0.05) higher than that of embryos cloned with differentiated MSCs and FFs. Surgical transfer of 523 two-cell stage embryos cloned with undifferentiated MSCs into five synchronized domestic pig recipients resulted in 5 cloned miniature pig offspring (1 stillborn and 4 viable) from 2 pregnant recipients. The results imply that MSCs might be multipotent and that they can be used to produce viable cloned miniature pigs that cannot be easily reproduced with differentiated somatic cells. Key words: Animal cloning, Differentiation, Mesenchymal stem cells, Miniature pig, Nuclear transfer (J. Reprod. Dev. 56: [256][257][258][259][260][261][262] 2010) he production of cloned animals by nuclear transfer (NT) has been increasingly studied as a potential approach to tissue and organ xenotransplantation with reduced immunogenicity for endstage organ failure. Several studies have suggested that miniature pigs, rather than nonhuman-primate species, are suitable as a donor animal for human xenotransplantation [1,2] because miniature pigs are superior in terms of similarities in gross anatomy and physiology to humans as well as in terms of ethical issues. The NT efficiency, however, remains relatively low due to abnormalities throughout pre-and post-implantation development regardless of the species or type of donor cell [3]. A number of factors affect NT efficiency, including the NT technical process, activation protocol, recipient oocyte age, cell cycle stage and type of donor cells, which has been implicated in the precise reprogramming of chromatin and genomic imprinting. Among these factors, incomplete selection of donor cells has also been considered a prime cause for NT inefficiency.Transfer of embryonic stem (ES) cells to produce cloned embryos has resulted in consistently higher numbers of viable offspring compared with somatic cells in mice [4,5]. Moreover, embryos cloned with porcine mesenchymal stem cells (MSCs) and their derivatives along the osteogenic lineage give rise to an increase in the rate of preimplantation development compared with adult fibroblasts [6]. Our previous study on gene expression profiles demonstrated that some ge...

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

“…However, no statistically significant difference in fusion rate, cleavage rate, and blastocyst rate was found between in vitro-cultured MSCs-embryos and Fsembryos of three investigated swine breeds. This is not consistent with that seen in previous studies (Colleoni et al, 2005;Faast et al, 2006;Jin et al, 2007;Kumar et al, 2007;Lee et al, 2010), which all reported that the MSCs-embryos have a significantly ( p £ 0.05) higher blastocyst rate than Fs-embryos.…”

“…The enhancement of full-term developmental ability observed in MSCs-embryos probably was due to an increase of total cell number at the blastocyst stage, which enhanced subsequent postimplantation development. Lee et al (2010) showed that use of MSCs as donor cells for cloning give rise to a birth rate of live cloned piglets at 0.76% ( = 4/523 = number of delivered live cloned piglets/number of transferred total cloned embryos), which is close to that (0.90% = 11/1221) observed in this study. Nevertheless, comparison of the birth rate of live cloned piglets between MSCs-embryos group and Fs-embryos group was not performed in the Lee et al (2010) study.…”

“…A similar result was also found in some reported studies Lee et al, 2010). The total cell number of preimplantation embryos was shown to be critical for the progression of normal embryonic development (Machaty et al, 1998).…”

“…It was reported that in cultured porcine embryos cloned from MSCs (MSCs-embryos), the expression pattern of developmentally important genes involved in pluripotency, epigenetic modification, imprinting, and apoptosis was more similar with that of in vivo-derived embryos, as compared with embryos cloned from Fs (Fs-embryos) . Moreover, several studies have reported that the in vitro preimplantation developmental potential of porcine MSCs-embryos was significantly higher than that of Fsembryos (Colleoni et al, 2005;Faast et al, 2006;Jin et al, 2007;Kumar et al, 2007;Lee et al, 2010).…”

“…However, because there are no porcine ESCs with proven pluripotency available now, MSCs, a type of multipotent adult stem cells, were employed as donor cells to improve the success rate of pig cloning. Although the porcine MSCs-embryos were demonstrated to have higher preimplantation developmental competence than Fs-embryos in in vitro culture (Colleoni et al, 2005;Faast et al, 2006;Jin et al, 2007;Kumar et al, 2007;Lee et al, 2010), their full-term developmental ability has not yet been compared with that of Fsembryos. In this study, we showed that porcine MSCs-embryos have higher blastocyst total cell numbers as well as full-term developmental potential, and porcine MSCs can be reprogrammed to generate live transgenic cloned pigs following stable genetic modification.…”