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ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable.

ABSTRACT: Reproductive cloning is uniformly rejected as a valid technology in humans because of the severely abnormal phenotypes seen in cloned animals. Gene expression aberrations observed in tissues of cloned animals have also raised concerns regarding the therapeutic application of "customized" embryonic stem (ES) cells derived by nuclear transplantation (NT) from a patient's somatic cells. Although previous experiments in mice have demonstrated that the developmental potential of ES cells derived from cloned blastocysts (NT-ES cells) is identical to that of ES cells derived from fertilized blastocysts, a systematic molecular characterization of NT-ES cell lines is lacking. To investigate whether transcriptional aberrations, similar to those observed in tissues of cloned mice, also occur in NT-ES cells, we have compared transcriptional profiles of 10 mouse NT- and fertilization-derived-ES cell lines. We report here that the ES cell lines derived from cloned and fertilized mouse blastocysts are indistinguishable based on their transcriptional profiles, consistent with their normal developmental potential. Our results indicate that, in contrast to embryonic and fetal development of clones, the process of NT-ES cell derivation rigorously selects for those immortal cells that have erased the "epigenetic memory" of the donor nucleus and, thus, become functionally equivalent. Our findings support the notion that ES cell lines derived from cloned or fertilized blastocysts have an identical therapeutic potential.

SUBMITTER: Brambrink T

PROVIDER: S-EPMC1348019 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable.

Brambrink Tobias T Hochedlinger Konrad K Bell George G Jaenisch Rudolf R

Proceedings of the National Academy of Sciences of the United States of America 20060117 4

Reproductive cloning is uniformly rejected as a valid technology in humans because of the severely abnormal phenotypes seen in cloned animals. Gene expression aberrations observed in tissues of cloned animals have also raised concerns regarding the therapeutic application of "customized" embryonic stem (ES) cells derived by nuclear transplantation (NT) from a patient's somatic cells. Although previous experiments in mice have demonstrated that the developmental potential of ES cells derived from ...[more]

PMID: 16418286

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Project description:The study of genomic imprinting in mammals started with analysis of parthenogenetic embryos. At the phenotypic level, embryos with two maternal genomes and no paternal genome proceed through early development unimpaired, and only begin to fail after implantation. The most recognizable early defect is reduced or non-existent trophoblast, the tissue that gives rise to the placenta. We applied the procedure for establishing Trophoblast Stem cells (TS cells) developed in the Rossant lab to parthenogenetic embryos, and were successful in making four different TS cell lines, three from MI oocyte derived blastocysts and one from MII derived blastocysts. Initial molecular characterization, including microarray analysis, indicates that these cells are indistinguishable from fertilized TS cells, with the single exception of null expression of the paternally expressed gene Snrpn. The only significant difference between parthenogenetic and fertilized TS cells was the frequency with which they could be derived. In our hands, fertilized blastocyst outgrowths produced TS cells at robust rates (10-12 colonies per blastocyst), while parthenogenetic blastocyst outgrowths produced only 4 colonies in 50 outgrowths, 100 times less frequently than fertilized embryos. This led us to hypothesize that those few TS cells that arose in parthenogenetic outgrowths were probably a result of very low frequency stochastic variation in the imprint status of a gene or genes required for either establishment or maintenance of stem cells, or both. The corollary to this hypothesis posits that the early failure of parthenogenetic embryos, and in particular parthenogenetic trophoblast, is a function of impaired stem cell function. This raises the intriguing possibility that microarray comparisons of parthenogenetic, fertilized and androgenetic blastocysts may reveal the identities of genes important for stem cell biology. To this end, my colleague Keith Latham, at the Fels Institute, Temple University, Philadelphia, made amplified cDNAs from pools of ten each of androgenetic, gynogenetic, or fertilized blastocysts. Three separate pools for each type of embryo were prepared for microarray analysis. The embryos were all produced by nuclear transfers between zygotes, a difficult technique that is Keith's special expertise. He used the Brady/Iscove protocol to generate quantitative 3' end biased cDNAs. We would like to compare the transcriptomes of these embryos using the MOE430 2.0 arrays. We expect that important insights into the biology of uniparental embryos in general, and stem cells in particular may be revealed. Keywords: other

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ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable.

Publications

ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable.

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