Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus

Abstract

Reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells, demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methyltransferase-1, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.

Figure 1

Oct4 and Nanog are not expressed in neural stem (NS) cells but are expressed in derivative NT ES cells. Northern analysis of Oct4, Nanog, and Sox2 expression in wild-type ES cells, NS cells, and NT-derived ES cells from the three independent neural stem cell lines, NS5, NSV6.5, and Cor1-5. Nanog shows two bands. The upper band corresponds to the expected size for the Nanog transcript. The nature of the lower band is unknown. Abbreviations: ES, embryonic stem; NSC, neural stem cell; NT, nuclear transfer; WT, wild-type.

Figure 2

Bisulfite sequencing of the Oct4 and Nanog promoters in the three NS cell lines and NT-derived ES cells from the Cor1-5 NS cell line. Wild-type ES cells and whole adult brain are shown as controls. Differentially methylated CpG sites are depicted below the bar graphs as closed circles along the promoters of Oct4 and Nanog. Bars represent the percentage of sequence reads that showed methylation for each site. Number of reads for each line is shown in brackets. Four independent cell lines were used in the Cor1-5 analysis. Abbreviations: ES, embryonic stem; NT, nuclear transfer; WT, wild-type.

Figure 3

Bisulfite sequencing of hypomethylated (chip/c) versus wild-type fibroblasts. Abbreviation: WT, wild-type.

Figure 4

NT-derived ES cells are pluripotent. NT-derived ES cells from both neural stem (NS) cell (A–C) and hypomethylated tail tip fibroblast (D–F) donors are pluripotent. Representative NT ES cell lines were targeted with a ubiquitously expressed green fluorescent protein (GFP) (R26-GFP). Resulting ES cells were injected into wild-type blastocysts to produce chimeras. Chimerism was analyzed in adults by coat color contribution (A) and anti-GFP antibody staining in E14.5 embryos. The NS cells were derived from outbred mice with white coat color and were injected into wild-type 129/B6 F2 blastocysts, which produce tan, brown, and black coat color adults. Therefore, any white coat color must derive from the NT ES cells. Darkly labeled cells represent positive GFP staining. Contribution to all three germ layers was seen including ectoderm ([A], melanocytes; [D], neural tissue), mesoderm ([B, E], cartilage), and endoderm ([C], intestine; [F], liver). Abbreviations: ES, embryonic stem; NSC, neural stem cell; NT, nuclear transfer; TT, tail tip.