PerspectiveDevelopment

Is Therapeutic Cloning Dead?

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Science  21 Dec 2007:
Vol. 318, Issue 5858, pp. 1879-1880
DOI: 10.1126/science.1153229

Ever since it was first suggested by Robert Edwards and Patrick Steptoe in their 1980 book A Matter of Life (1), it seemed that the path to making immunocompatible cells to potentially treat human diseases would necessitate cloning of a human embryo. This process calls for replacing the DNA of an unfertilized egg (oocyte) with that from the patient's somatic cell, in vitro culture of the reconstructed embryo to the blastocyst stage, and subsequent isolation of pluripotent cells that could potentially differentiate into any cell type. The prediction of Edwards and Steptoe came closer to reality with the arrival of Dolly, the cloned sheep, and derivation of the first human embryonic stem cells by James Thomson's group (2, 3). This procedure of somatic cell nuclear transfer has been referred to as human therapeutic cloning (4), and although studies in mice have shown that this approach is possible (5, 6), its proof-of-principle in humans never materialized. Now, two reports in this issue—by Yu et al. on page 1917 (7) and Hanna et al. on page 1920 (8)—and a recent report by Takahashi et al. (9) suggest that human therapeutic cloning may never happen.

Takahashi et al. (9) report the generation of human induced pluripotent stem cells using four transcription factors: Oct4, Sox2, Klf4, and c-Myc. These are the same transcription factors previously reported by this group and others to produce such stem cells in mice (1014). Takahashi et al. have now shown that exogenous expression of these genes (delivered by retroviral vectors) can transform newborn and adult human fibroblasts into induced pluripotent stem cells. Two of these factors can be considered predictable in that they appear in most microarray analyses of mouse and human embryonic stem cells and are important in development. But the two other factors, c-Myc and Klf4, were unforeseen, and their presence in the “reprogramming quartet” in mice has been difficult to justify. Nevertheless, the cocktail works in human cells, and now the challenge is to determine each factor's role during the reversion of a mature cell to an earlier, unspecialized form that can then differentiate into multiple cell types.

Features of pluripotent stem cells derived by different procedures.

SCNT, somatic cell nuclear transfer; ESC, embryonic stem cells; iPS, induced pluripotent stem cells; asterisk indicates the capacity to proliferate for periods of time longer than those recorded in somatic cells of the same species while maintaining their pluripotent state.

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Yu et al. (7) also report the production of induced pluripotent stem cells, but use only two factors, Oct4 and Sox2, in combination with one more predictable transcription factor, NANOG, and a fourth largely unconsidered gene encoding Lin28, a protein thought to be involved in RNA processing (15). This combination shares only two of the factors reported by Takahashi et al., raising the question of redundancy among genes that can reprogram cells. Perhaps one master gene upstream of the six now described opens the gate to pluripotency. Yu et al. avoid expressing c-Myc, which can act as an oncogene and cause cancer, and improve gene delivery by using lentiviral vectors. It is possible that c-Myc promotes the proliferation of induced pluripotent stem cells until the stochastic and progressive process of dedifferentiation has progressed sufficiently to allow selfrenewal. c-Myc could be functionally redundant with NANOG, which maintains embryonic stem cell pluripotency. Yu et al. further show that from the eight induced pluripotent stem cell lines generated, two expressed Oct4, Sox2, and NANOG alone. It is extraordinary that just three exogenously expressed transcription factors can completely reprogram a cell.

Finally, Hanna et al. show that symptoms of sickle cell anemia can be ameliorated with induced pluripotent stem cells in a mouse model of this human disease. Adult somatic cells taken from the tail of a mouse bearing a mutated version of the human β-globin gene were converted into induced pluripotent stem cells. The mutation was then repaired and through expression of the HoxB4 transcription factor, the cells were differentiated into hematopoietic progenitor cells and transferred back into the affected animals. All three animals treated with the induced pluripotent stem cells survived up to 20 weeks, whereas untreated animals died before the seventh week of age. The treated animals also showed increases in red blood cells and hemoglobin. Although it is premature to extrapolate these experiments to humans (we have yet to obtain mature blood cells from human embryonic stem cells), Hanna et al. have shown that induced pluripotent stem cells are more than a laboratory amusement: They may not only treat, but potentially cure diseases, at least in an animal model (and for the limited time of the study).

These three reports come on the heels of the announcement by Byrne et al. that nonhuman primate embryonic stem cells can be derived from adult fibroblasts using somatic cell nuclear transfer (16). The next logical step will be to compare the cells obtained by Byrne et al. with those generated using the suite of transcription factors implicated in cell dedifferentiation. Such a comparison is necessary to determine whether the reprogramming process and function of induced pluripotentor somatic cell nuclear transfer-derived cells are equivalent or not. We may not be able to resolve these questions anytime soon, not only because the mechanism of somatic cell nuclear transfer has yet to be described in detail, but more because we can as yet only follow the transformation of a given somatic cell into an induced pluripotent stem cellwhen embryonic stem cell-like colonies first emerge, and not any earlier.

The current breakthroughs raise exciting questions. Some are technical, such as whether viral vectors can be replaced with other agents that facilitate transient expression of delivered genes. Others are mechanistic, such as whether the conversion of cells is due to epigenetic modifications of key genetic elements or to a genetic event that has yet to be identified. Another question is whether several events must occur in a given sequence, with a precise amount of each transcription factor at a particular time, for cell dedifferentiation to occur and be sustained. This also raises the issue of whether the converted cell that gives rise to an induced pluripotent cell colony is somehow predisposed to “stemness,” given that, in the context of somatic cell nuclear transfer, it is clear that pluripotent cells, such as embryonic stem cells, are easier to reprogram into viable animals than are somatic cells.

Is human therapeutic cloning no longer needed? The short answer is no, but it is likely a matter of time until all the hypothetical advantages of therapeutic cloning will be implemented with induced pluripotent stem cells. More importantly, the controversial issues (ethical and technical) specific to human therapeutic cloning may well be left behind along with the procedure itself, a refreshing change for the field, indeed.

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