Effect of culture medium type on canine adipose-derived mesenchymal stem cells and developmental competence of interspecies cloned embryos

Abstract

Canine adipose-derived mesenchymal stem cells (ASCs) are promising as donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that different cell cultures possess different capacities to support pre-implantation development of SCNT embryos. The aim of this study is to investigate whether two culture medium (RCMEP, Dulbecco's modified Eagle's medium [DMEM]) affect gene expression of ASCs, subsequent development of interspecies SCNT (iSCNT) and gene expression of cloned embryos. The RCMEP-cultured cells contained significantly greater amounts of SOX2, NANOG, OCT4, DNMT1, and MeCP2 than DMEM-cultured cells (P < 0.05). In iSCNT, the use of DMEM medium for culturing cells resulted in similar development to the blastocyst stage than those derived from RCMEP cultured cells (4.5% and 3.2%, respectively; P > 0.05). The expression of all transcripts except for DNMT1 in cloned blastocysts from RCMEP cultured cells followed those of cloned blastocysts derived from DMEM cultured cells. The alteration of gene expression in ASCs by culture medium was not manifested in the iSCNT embryos derived from these cells. Although the culture medium can induce changes of gene expression by ASCs, such alterations in donor cells did not affect the developmental competence or gene expression patterns of iSCNT embryos.

Introduction

In efforts to improve the efficiency of somatic cell nuclear transfer (SCNT) in mammals, many studies focused on donor cells have been performed. It is critically important for development of reconstructed embryos that the cell cycle states of the donor cell and the enucleated recipient oocyte are coordinated. In SCNT studies with many species, using donor cells that were synchronized into a quiescent (G0/G1) stage improved cloned blastocyst formation and cloned offspring birth rates [1], [2], [3], [4]. Methods such as serum starvation during cell culture or roscovitine are often used to achieve cell-cycle synchronization [5], [6].

The type of nuclear donor cell, characterized by its tissue origin and extent of differentiation, is among the key factors affecting the efficiency of SCNT, but cell selection and treatment are controversial areas. In mice, greater numbers of offspring were produced through nuclear transfer with embryonic stem cells compared with somatic cells [7]. However, in vitro development of mouse embryos cloned using hematopoietic stem cells was inefficient and production of cloned pups was no better than with clones made using other somatic cells such as cumulus, Sertoli, and fibroblast cells [8]. In canine SCNT, cloned offspring have been derived using donor cells of several types, which affected the efficacy of cloning [5], [9], [10], [11].

The final factor affecting the SCNT procedure is the complete reprogramming potential of donor cells. Successful reprogramming of donor cells can be influenced by in vitro culture conditions, including passage number, serum concentration, cell density, and chemical treatment [12], [13], [14], [15]. Recent studies in mice have shown that treatment of donor cells with chemicals such as trichostatin A changed epigenetic methylation patterns and improved the quality of cloned blastocysts through induced hyperacetylation in mice [16], [17], cattle [18], and pigs [19] SCNT.

To perform this experiment, it is necessary to prepare matured oocyte and donor cell derived from dogs. However, it is difficult to obtain many high-quality recipient canine oocytes because of a limited number of in vivo mature oocytes [20] and still low in vitro maturation (IVM) rate [21], [22], [23]. Therefore, interspecies SCNT (iSCNT) is utilized to analyze gene expression patterns of donor cells and cloned embryos derived from donor cells with different culture medium. The iSCNT technique has been used widely for evaluating the developmental competence of donor cells, investigating development mechanism of the reconstructed embryos, and preserving the endangered animals [24], [25].

Thus, the purpose of the present study was to (1) compare gene expression of canine ASCs grown in two culture medium, (2) analyze in vitro development of iSCNT embryos derived from ASCs cultured in two different media, and (3) investigate expression patterns of genes related to stemness, reprogramming, and pre-implantation development in iSCNT embryos.