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Pluripotent Stem Cells from Cloned Human Embryos: Success at Long Last

2013; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês

10.1016/j.stem.2013.05.022

1934-5909

Alan Trounson, Natalie DeWitt,

Renal and related cancers

Recently in Cell, Mitalipov and colleagues report an advance that has eluded scientists for over a decade—the successful derivation of embryonic stem cell lines using somatic cell nuclear transfer, or SCNT (Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Cell. 2013; 153 (this issue): 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Recently in Cell, Mitalipov and colleagues report an advance that has eluded scientists for over a decade—the successful derivation of embryonic stem cell lines using somatic cell nuclear transfer, or SCNT (Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Cell. 2013; 153 (this issue): 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). After the isolation and propagation of human embryonic stem cells (ESCs) was first reported in 1998, many stem cell and reproductive biologists set their sights on being the first to isolate ESCs from SCNT-generated (or “cloned”) human embryos. SCNT is a technique where the nucleus of a somatic cell is inserted into a recipient oocyte. Then cleavage of the oocyte containing the donor nucleus can be induced to initiate embryogenesis, which is supported by reprogramming factors in the oocyte. At the time, SCNT was the only potential source of pluripotent cells genetically matched to an individual for histocompatible cell transplants. Thus generating SCNT-ESCs became something of a holy grail, and the public was keenly interested. Now, 14 years later, Mitalipov and colleagues report that they have been able to efficiently derive ESC lines from human embryos generated by SCNT (Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Cell. 2013; 153 (this issue): 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Cell. 2013; 153 (this issue): 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar report production of human SCNT-ESCs with remarkable efficiency. They produced a total of six SCNT-ESC lines, starting with a total of 28 oocytes derived from three egg donors. Fetal dermal fibroblasts or cultured skin fibroblasts were used as the nuclear donor cells. They attributed their success to overcoming challenges that they believed had thwarted previous attempts—namely the oocytes’ premature exit from meiosis and suboptimal oocyte activation procedures. It is important to note that this group is made up of some of the most experienced nuclear transfer scientists in the world who have been responsible for many of the major advances in primate reproductive cell biology over the last two decades, including the production of SCNT-ESCs in the monkey (Byrne et al., 2007Byrne J.A. Pedersen D.A. Clepper L.L. Nelson M. Sanger W.G. Gokhale S. Wolf D.P. Mitalipov S.M. Nature. 2007; 450: 497-502Crossref PubMed Scopus (476) Google Scholar). Mitalipov and colleagues spent considerable effort optimizing their protocol, using monkey oocytes in many of their experiments to minimize their use of human oocytes. One step they cite as especially critical to their success was to more effectively activate oocytes by electrical stimulation. Another barrier to success they cite is the oocytes’ premature exit from meiosis upon enucleation. They report that treating the oocytes with caffeine, a protein phosphatase inhibitor, prevented premature exit from meiosis. Another optimization step they reported is the use of 10 nM Trichostatin A (TSA), an inhibitor of histone deacetylase, to enhance embryogenesis, as they had previously used in SCNT with monkey oocytes. They also observed that performing SCNT with oocytes that might normally be discarded was effective in their hands. Specifically, 12 hours after activation of oocytes with 6-methylaminopurine (6-DMAP), one would expect two pronuclei to form. However, the SCNT oocytes that went on to form ESC lines with normal chromosome numbers had zero, one, or two pronuclei, or were already in cleavage. This suggests that the activation of the oocytes is not closely synchronized, and that discarding oocytes with zero or one pronuclei or cleaved cells could alter the efficiency of SCNT. Overall, the study reported a relatively small number of SCNTs, which precludes any firm statistical conclusions; therefore, further studies will be necessary to confirm the importance of factors identified for success. In addition, concerns have been raised about the duplication of figures in the Tachibana paper that will require explanation and independent genetic verification of the results. The cloning of an animal by transplantation of differentiated nuclei into eggs was pioneered by John Gurdon who used it in amphibian cell reprogramming (Gurdon, 1962Gurdon J.B. J. Embryol. Exp. Morphol. 1962; 10: 622-640PubMed Google Scholar). Then with the cloning of “Dolly the sheep” (Campbell et al., 1996Campbell K.H. McWhir J. Ritchie W.A. Wilmut I. Nature. 1996; 380: 64-66Crossref PubMed Scopus (1468) Google Scholar) came a veritable menagerie of cloned mammals. SCNT-ESCs were isolated and cultured from many of the cloned species, including monkeys (Byrne et al., 2007Byrne J.A. Pedersen D.A. Clepper L.L. Nelson M. Sanger W.G. Gokhale S. Wolf D.P. Mitalipov S.M. Nature. 2007; 450: 497-502Crossref PubMed Scopus (476) Google Scholar), suggesting it should be possible to do the same for humans. In 2004, the Korean veterinarian Woo-Suk Hwang claimed to have isolated ESCs from cloned human embryos. However, key data were later found to be fabricated and the papers were retracted. His group did manage to produce one parthenogenetic ESC line from human blastocysts as a result of activation of a recipient oocyte rather than nuclear transfer (Kim et al., 2007Kim K. Ng K. Rugg-Gunn P.J. Shieh J.H. Kirak O. Jaenisch R. Wakayama T. Moore M.A. Pedersen R.A. Daley G.Q. Cell Stem Cell. 2007; 1: 346-352Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). When Shinya Yamanaka reported the reprogramming of human adult cells by the introduction of only four transcription factors (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14964) Google Scholar), reprogramming technology spectacularly leap-frogged over SCNT. This development lessened the urgency of establishing human SCNT-ESCs, and attention instead became focused on improving factor-mediated reprogramming technology and evaluating the safety of induced pluripotent cells (iPSCs) and their equivalency to ESCs. In 2012, the Nobel Prize in Physiology and Medicine was awarded to Shinya Yamanaka and John Gurdon for their achievements. Nonetheless, a number of groups made progress toward the goal of producing SCNT-ESCs (reviewed by Grieshammer et al., 2011Grieshammer U. Shepard K.A. Nigh E.A. Trounson A. Nat. Biotechnol. 2011; 29: 701-705Crossref PubMed Scopus (8) Google Scholar). Notably, despite having relatively few oocytes available, French et al., 2008French A.J. Adams C.A. Anderson L.S. Kitchen J.R. Hughes M.R. Wood S.H. Stem Cells. 2008; 26: 485-493Crossref PubMed Scopus (197) Google Scholar reported that 23% of SCNTs they attempted resulted in human blastocyst stage embryos that had the genomic contribution from the adult cell donor. Noggle et al., 2011Noggle S. Fung H.L. Gore A. Martinez H. Satriani K.C. Prosser R. Oum K. Paull D. Druckenmiller S. Freeby M. et al.Nature. 2011; 478: 70-75Crossref PubMed Scopus (161) Google Scholar observed that enucleation of the oocyte prior to nuclear transfer resulted in embryos not developing more than a few cleavage divisions. If they left the oocyte nucleus in place and transferred a somatic cell nucleus, triploid blastocysts developed, from which they derived triploid pluripotent ESC lines. Although representing a partial success in SCNT technology, these triploid lines are not useful therapeutically. The current study signifies several “firsts.” It will now be possible to evaluate how different reprogramming approaches affect developmental capacity, genetic stability, and graft survival by directly comparing SCNT-ESCs and iPSCs derived from the same donor. New reprogramming factors identified in oocytes may be used to improve the efficiency and speed of iPSC reprogramming. SCNT may also provide a way of enabling cell therapies for patients with mitochondrial diseases because healthy mitochondria will derive from the oocyte donor, which isn’t the case for iPSC technology (see Figure 1). Perhaps most importantly, the study supports the feasibility of using SCNT for efficiently generating pluripotent cell lines for cell replacement therapy. It is also much more rapid than iPSC derivation, which could be crucial for patients urgently needing autologous cell transplants. The main constraint presently to widespread application of SCNT is likely to be the availability of mature, developmentally competent human oocytes, which will again raise commentary on the financial and ethical issues of donation of human oocytes for research. This is in large part due to the difficulty in procuring sufficient numbers of mature human oocytes to carry out what have typically been low-efficiency experiments. Recently, however, it was reported that mouse ESCs and iPSCs could produce viable oocytes and live, healthy young (Hayashi et al., 2012Hayashi K. Ogushi S. Kurimoto K. Shimamoto S. Ohta H. Saitou M. Science. 2012; 338: 971-975Crossref PubMed Scopus (524) Google Scholar). If applied to human cells, this advance may eventually lead to successful production of mature human oocytes in vitro for such research purposes. This may relieve some of the ethical and financial barriers presently involved in oocyte donation for SCNT. The generation of SCNT-ESC lines in this study shows it is feasible to generate cellular derivatives that may be more robust, genetically stable, and “adult-like” due to the absence of somatic cell memory and without the introduction of genetic elements and oncogenes used to derive iPSCs. Time will be needed to test whether this is the case, and whether SCNT-ESC-derived cells, tissues, and organs fair better in differentiation to mature, functional cells and in graft survival. What is certain is that this breakthrough will rekindle interest in the potential of human SCNT for basic research and cell therapies. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear TransferTachibana et al.CellMay 15, 2013In BriefFor the first time, diploid human embryonic stem cells are derived from somatic cell nuclear transfer. Full-Text PDF Open ArchivePluripotent Stem Cells from Cloned Human Embryos: Success at Long LastTrounson et al.Cell Stem CellAugust 01, 2013In Brief(Cell Stem Cell 12, 636–638; June 6, 2013) Full-Text PDF Open Archive