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Producing primate embryonic stem cells by somatic cell nuclear transfer

A Corrigendum to this article was published on 10 December 2014

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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Figure 1: Chromatograms of the rhesus macaque mitochondrial D-loop hypervariable region 2 informative domain 1 (RhDHV2 ID1).
Figure 2: Morphological and immunocytochemical analysis of CRES cells and their differentiated derivatives.
Figure 3: Cytogenetic analysis of CRES cells.

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Acknowledgements

The authors acknowledge the Division of Animal Resources and the Endocrine Services Cores at the Oregon National Primate Research Center for assistance and technical services. We thank M. Sparman, C. Ramsey and V. Dighe of the Assisted Reproductive Technology Core for their embryological and logistical assistance; J. Fanton and D. Jacobs for laparoscopic oocyte retrievals; B. Ferguson for performing the SNP analysis; C. Penedo for microsatellite analysis; and R. Stouffer, M. Grompe and R. Reijo Pera for reviewing this manuscript. Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. This study was supported by funds from ONPRC and NIH grants to S. Mitalipov, R. Stouffer and D. Dorsa.

Author Contributions S.M.M. and J.A.B. designed experiments, conducted SCNT and ES cell derivation. L.L.C. performed DNA/RNA isolations and stemness gene expression. J.A.B. analysed the microarray data and performed the mitochondrial DNA analysis. D.A.P. assisted with ES cell derivation and performed ES cell culture, characterization and differentiation. W.G.S. and M.N. performed the cytogenetic analysis. S.G. analysed teratomas. S.M.M., J.A.B. and D.P.W. analysed the data and wrote the paper.

Microarray data, including CEL and CHP files, and Supplementary Data files containing microarray analyses (Supplementary Data 3–7) have been deposited in the Gene Expression Omnibus (GEO) database with accession number GSE7748 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7748).

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Correspondence to S. M. Mitalipov.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-9 with Legends, Supplementary Tables 1-5 and the MIAME Checklist. (PDF 1085 kb)

Supplementary Video

The file contains Supplementary Video 1 showing contracting cardiomyocytes derived from differentiated CRES cell lines. (MOV 10738 kb)

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Byrne, J., Pedersen, D., Clepper, L. et al. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450, 497–502 (2007). https://doi.org/10.1038/nature06357

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