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.

Fig. 1.

Analyses of expression profiles from cloned and fertilization-derived ES cell lines (NT-ESC and F-ESC, respectively). Mean signal intensities (MSI) of five NT-ESC lines were plotted against the corresponding MSI of five F-ESC lines. Dotted lines indicate 1.5-fold regulation.

Fig. 2.

Hierarchical clustering of individual ESC line expression profiles. (a) Heat map of clustering results (blue, no or very low expression; white, low expression; red, high expression). (b) Sample tree obtained from hierarchical clustering. ES cell line expression profiles cluster by genetic background (colored octagons) rather than by type of donor blastocyst (NT, cloned; F, fertilized; numbers next to nodes display multiscale bootstrap resampling probability based on 10,000 replications).

Fig. 3.

Analysis of variability in gene expression among cloned and fertilization-derived ES cell lines. (a) Comparison of gene expression levels in NT-ES (open bars) and F-ES (filled bars) cell lines. Columns display mean signal intensities; error bars display standard deviation. (b) Comparison of standard deviation levels across all probes in the data set. As a measure of gene expression variability, standard deviation levels were calculated for the log2 probe signal values for each group. Probes in each group (NT-ES and F-ES cell lines) were ordered by their standard deviation levels, and then standard deviation levels were compared at different percentiles. Data sets display closely matched standard deviation levels for different percentiles, indicating highly similar variability in gene expression between the two groups.

Fig. 4.

Retention of epigenetic memory in blastocysts and loss of epigenetic memory in NT-ES cell lines. The outcome of postimplantation development depends strictly on the origin of the donor nucleus. Fertilized blastocysts develop into normal mice with high efficiency, whereas blastocysts derived after nuclear transplantation from donor cells, such as ES cells, fibroblasts, immune cells, or neurons, develop into abnormal mice with an efficiency that depends on the differentiation status of the respective donor nucleus (15, 16). For example, cloned blastocysts derived from ES cell donor nuclei develop to birth with high efficiency, whereas those derived from fibroblasts, immune cells or neurons develop to birth with low or very low efficiency. In contrast to postimplantation development, the process of deriving embryonic stem cells entails rigorous selection for in vitro proliferation and results in the loss of the epigenetic memory of the donor nucleus.