Sadie Smith

Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning

Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%–5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category “RNA processing” was over‐represented among the genes that were down‐regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos. Mol. Reprod. Dev. 76: 38–47, 2009.

Nuclear reprogramming by somatic cell nuclear transfer - the cattle story.

Somatic cell nuclear transfer (cloning) returns a differentiated cell to a totipotent status; a process termed nuclear reprogramming. Nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. To understand the deficiencies of nuclear reprogramming, our research has focused on both candidate genes (imprinted and X-linked genes) and global gene expression patterns in cloned bovine embryos/offspring as compared to those generated by conventional reproduction. We found aberrant expression patterns of H19 and Igf2r as well as X-linked genes in term cloned calves. The expression profiles of cloned blastocysts, however, closely resembled those of the naturally fertilized embryos but were considerably different from those of their nuclear donor cells. Our findings suggest that cloned embryos have undergone significant nuclear reprogramming by the blastocyst stage. However, it is possible that during re-differentiation in later development gene expression aberrancies occur. Additionally, small initial nuclear reprogramming errors may be manifested during subsequent development.

Reply to “On the cloning of animals from terminally differentiated cells”

blastocyst-stage embryos that developed from hematopoietic stem cell (HSC) donors (4%) compared with granulocyte donors (35%) and on the failure to obtain cloned mice from HSC donor nuclei. A trivial explanation for this finding is that the nuclei of HSCs, in contrast to those of granulocytes, are more easily damaged by nuclear transfer, by FACS or by freezethaw procedures used with all donor cells. Cleavage of reconstructed oocytes into blastocysts is particularly sensitive to experimental parameters, such as the cell-cycle stage and physical condition of the transferred nucleus, and is therefore not an acceptable measure of cloning efficiency8,9. However, once an embryo has reached the blastocyst stage, it has a relatively consistent probability of developing into a mouse or giving rise to ES cells. Therefore, measuring the potential of a cloned blastocyst to generate a viable animal or ES cells provides more defined readouts for cloning efficiency than just development through cleavage stages. Sung et al. did not transfer any cloned blastocysts derived from HSCs into female mice to compare the efficiency of postimplantation development with that of granulocyte donors. The main conclusion of the paper is thus based on the two cloned newborns derived from granulocyte nuclear donors (2 newborn pups from 394 cleaved embryos, corresponding to a 0.5% efficiency or a 1.1% efficiency when calculated from 182 embryos transplanted into the uterus) versus none derived from HSC nuclear donors (Table 1). It is curious that one of the co-authors recently cloned newborn pups from HSC donor nuclei with an efficiency of approximately 0.5% (2 newborn pups obtained from 411 transferred cloned embryos10; Table 1). Thus, given this insignificant difference in cloning efficiencies between HSCs and granulocytes and the failure to unequivocally demonstrate the identity of the donor cells, one wonders whether the evidence presented in this paper justifies the sweeping conclusion “that the cloning efficiency increases directly with the state of differentiation”, thus “revers[ing] the current popular hypothesis”. Konrad Hochedlinger1 & Rudolf Jaenisch2 1Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 2Whitehead Institute and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. e-mail: jaenisch@wi.mit.edu