Producing primate embryonic stem cells by somatic cell nuclear transfer.

Oregon National Primate Research Center, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, Oregon 97006, USA.
Nature (Impact Factor: 42.35). 12/2007; 450(7169):497-502. DOI: 10.1038/nature06357
Source: PubMed

ABSTRACT Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

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    ABSTRACT: Parthenogenesis is the process by which an oocyte develops into an embryo without fertilization. Parthenogenetic activation can be performed at various stages of meiosis, yielding embryos with a distinct genetic pattern of homozygousity and heterozygousity. The heterozygousity pattern specific to parthenogenetic embryonic stem (pES) cells derived from such embryos, can be predicted using genome-wide single nucleotide polymorphism (SNP) analysis to determine whether extrusion of the first or second polar body is prohibited. The heterozygous pES cells carrying the full complement of major histocompatibility complex (MHC) antigen matched to the oocyte donor, could therefore provide a potential source of MHC matched cells or tissue for cell replacement therapy. In this review, we summarized the mechanism of deriving heterozygous MHC-matched pES cells using a mouse and human models.
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    ABSTRACT: Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog tran-scripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were meth-ylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic en-richment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the pa-rameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.