On the cloning of animals from terminally differentiated cells
To the Editor: In a recent Nature Genetics Letter entitled 'Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer' Sung et al.1 draw two major conclusions that are not convincingly supported by the data presented. The authors claim to provide the first unequivocal evidence for the generation of cloned animals from terminally differentiated cells and to prove that differentiated cells are more efficient donor cells for nuclear transfer than adult stem cells. The question of whether the nuclei of differentiated cells remain equivalent to the nuclei of embryonic cells was raised over 50 years ago and led to the seminal cloning experiments in amphibians and later to the generation of Dolly from an adult mammary gland cell2. However, due to the heterogeneity of the donor cell populations and because of a lack of genetic markers, it was impossible to unambiguously determine the identities and differentiation states of the donor cells that gave rise to cloned mammals. In fact, the low efficiency of cloning usually, only 1% 3% of cloned embryos develop into live animals did not exclude the possibility that only rare adult stem cells rather than differentiated cells could serve as donors for cloned animals. A line of solid genetic experiments laid this idea to rest. The generation of cloned mice from terminally differentiated lymphocytes3 and from postmitotic olfactory neurons4,5 provided unequivocal evidence for the nuclear totipotency of terminally differentiated cells. In these initial studies, cloned mice were produced using a two step approach that involved the derivation of embryonic stem (ES) cells from cloned blastocysts followed by the generation of entirely ES cell derived mice by injecting ES cells into tetraploid host blastocysts. Importantly, ES cells derived by nuclear transfer from donor neurons supported the development of cloned mice in a second round of nuclear transfer, hence unambiguously proving the genomic totipotency of postmitotic terminally differentiated cells4. This finding was later corroborated by the generation of cloned mice from terminally differentiated natural killer T cells by direct nuclear transfer6. Sung et al. argue that the cloning of mice from lymphocytes and neurons did not resolve the question of 'nuclear equivalence' because of the two step cloning procedure. The authors used a single surface marker (Gr 1) and cellular granularity (side scatter) as parameters to isolate a population of terminally differentiated granulocytes that was alleged to be of 99.4% purity, based on morphological criteria. Two viable clones were produced from sorted granulocytes and were claimed to be the first true examples of animals derived from terminally differentiated cells without the recourse of ES cell derivation. These conclusions are not convincing. Sorting of Gr 1 positive cells from bone marrow will enrich not only for mature but also for immature granulocytes as well as for monocytes7. Parameters such as granularity and morphology are poor markers to determine the differentiation state of a cell, and expression profiling of a bulk population is of little value to prove the postmitotic state of two randomly picked donor cells. Thus, in the absence of a genetic marker for terminal differentiation, as was used in the previous experiments3 6, the use of a heterogeneous donor cell population7 and statistical calculations do not exclude the possibility that less differentiated cells or contaminating bone marrow cells served as donors for the two surviving clones. The claim that these experiments demonstrate for the first time the development of clones from terminally differentiated donor cells is, therefore, not only based on poor and ambiguous evidence but is also redundant given previous findings. The second claim of the paper is the generalized statement that differentiated cells are more efficient donor cells than adult stem cells. This claim is based on the lower number of
Nature Genetics