Keith's MAGIC: Cloning and the Cell Cycle

Abstract

Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

Introduction

Keith worked on nuclear transfer (NT) in the group lead by Professor Sir Ian Wilmut at the Roslin Institute, in Scotland. Along with Bill Ritchie and Jim McWhir, as well as colleagues from PPL Therapeutics, also in Roslin, his studies on cell cycle coordination in NT and the resulting nuclear–cytoplasmic interactions culminated in the birth of undoubtedly the most famous sheep in the world, namely “Dolly,” cloned from a mammary gland cell from a 6-year-old ewe. This was an extension of John Gurdon's pioneering work in the amphibian Xenopus laevis between the late 1950s and 1970s, for which he was awarded the Nobel Prize in Physiology or Medicine in 2012. Gurdon had demonstrated the totipotency of nuclei from differentiated cells at larval stages to generate fertile frogs following nuclear transplantation, but the cloning from adult cells to create fully mature animals was not accomplished during this era (Gurdon, 2013). And so Dolly's announcement in 1997, as the first animal to be cloned from an adult cell, truly astounded the world and resulted in phenomenal media attention and public debate. Much of this focused on the specter of cloning humans, with the prospect of science fiction finally becoming reality. However, Keith's legacy is the impetus that this breakthrough from the Roslin team gave to other researchers to study the epigenetic basis behind reprogramming and cellular plasticity, contributing toward valuable agricultural and medical applications for the future.

The Road to Dolly

After fertilization, embryo development proceeds by successive cell division and progressive differentiation to form all cell types of the mature animal. Biologists had long wondered what controlled this cellular specialization and questioned whether genetic material was lost or inactivated during this process and if differentiation was reversible. In the early 1900s, delayed nucleation experiments in species such as sea urchin and salamanders clearly demonstrated that the nuclei of early cleavage-stage embryos were equipotent (for review, see Di Berardino, 2001). However, the nuclear equivalence of more differentiated cells at later developmental stages was left unresolved. The concept of NT was essentially described by Hans Spemann in 1938 as a “somewhat fantastical thought experiment” (Spemann, 1938). He proposed to test the nuclear equivalence of increasingly differentiated cell types in the context of the whole organism and disprove the theory that cell differentiation entails the loss or irreversible inactivation of genetic information required for development.

NT essentially involves the transfer of a donor nucleus into the cytoplasm of an oocyte whose own nuclear material has been removed or destroyed (termed the recipient cytoplast). Although, he had no way technically at the time to perform NT, Spemann anticipated that every cell had the potential to initiate normal development. The technology to conduct the experiment in amphibians was developed quite independently in the early 1950s (Briggs and King, 1952). In both Rana pipiens and Xenopus laevis, it was evident that NT success decreased as nuclei were obtained from more specialized cells from increasingly advanced developmental stages (for review, see Di Berardino, 2001). Despite this ever-increasing restriction, the nuclei from differentiated larval cells, as obtained from the intestinal epithelia of feeding-stage tadpoles, could in a few cases elicit completely normal development (Gurdon, 1962) and even generate sexually mature adults (Gurdon and Uehlinger, 1966), revealing their inherent nuclear totipotency. While nuclei transferred from fully differentiated adult cells (such as skin keratinocytes) supported development to juvenile swimming tadpole stages (Laskey and Gurdon, 1970), it is important to note that no development to mature adults was reported with adult donor cells (Gurdon et al., 1975; Gurdon, 2013). This therefore, left open the question as to whether a differentiated adult nucleus could be fully reprogrammed. It took nearly another 40 years after these early results in Xenopus for this to be achieved first in mammals.

NT experiments were considerably more difficult to perform in mammals than amphibians. Oocytes were harder to obtain in large numbers and, being much smaller, were more difficult to manipulate. Furthermore, media and culture systems to adequately mature oocytes and grow embryos in vitro to a suitable stage for embryo transfer had to be devised. Numerous technical refinements and species-specific modifications now enable NT in a wide range of mammalian species.

The first studies in mice in the 1980s incorrectly concluded that developmental potential following NT in mammals was considerably more restricted than in amphibians, with totipotency of transferred nuclei being rapidly lost after the one-cell stage (McGrath and Solter, 1984). However, these early experiments were disadvantaged by using zygotes as recipient cytoplasts. During zygote enucleation, critical reprogramming factors were removed together with the intact pronuclei, compromising development potential. Strategies that more selectively removed chromatin from pronuclei while leaving sufficient nuclear reprogramming factors behind finally overcame this technical problem (Greda et al., 2006). Alternatively, chromatin removal shortly after fertilization at telophase II (Schurmann et al., 2006) or from mitotic zygotes (Egli et al., 2007) was successful.

Like the mouse, initial research in livestock focused on using undifferentiated embryonic blastomeres from cleavage-stage embryos as donor cells. Importantly, switching from zygotes to cytoplasts obtained from metaphase II (MII) stage oocytes, similar to the unfertilized eggs used in amphibia, led to cloning success in sheep (Willadsen, 1986), cattle (Prather et al., 1987), and pigs (Prather et al., 1989). The early belief that nuclear totipotency was restricted to these undifferentiated cell types was reinforced by the failure of cloned pregnancies (in cattle) following the use of adult cumulus cells for NT (Collas and Barnes, 1994). In hindsight, this was probably due to other limitations in the methodology and number of NT attempts, rather than any developmental restriction on the part of the somatic nuclei.

In identifying a suitable cell type for NT in mammals, there was encouragement with the use of pluripotent cells as nuclear donors. Cells from the inner cell mass (ICM) of blastocyst-stage embryos in both sheep (Smith and Wilmut, 1989) and cattle (Keefer et al., 1994) were shown to result in full-term development following NT. The ICM is the founder pluripotent tissue from which embryonic stem cells (ESCs) had been isolated in mouse a decade earlier (Evans and Kaufman, 1981; Martin, 1981). Because of their ability to be genetically manipulated in vitro and colonize the germ line in chimeras, ESCs have been an extremely powerful tool to introduce targeted genetic modifications into the genome of mice (Bradley et al., 2012). Thus, by the early 1990s, many large animal embryology laboratories invested considerable effort attempting to isolate ESCs in livestock species with the prime objective of using them to introduce desired genetic modifications into the germ line of farm animals. Rather than the chimera route, which would be very time-consuming in livestock species due to their long generation intervals, it was envisioned that NT was the more direct approach. By extrapolation from the proven nuclear totipotency of ICM cells, this was also considered feasible.

Despite many attempts starting in the late 1980s, the isolation of bona fide livestock ESCs still has not been demonstrated. Furthermore, the use of bovine ES-like cells for NT showed only limited success with pregnancies failing by around day 55 of gestation, largely due to an absence of cotyledon formation (which subsequently proved to be an all too common cloning deficit) (Stice et al., 1996). So, by the mid-1990s the prospect of isolating authentic ESCs in livestock and the ability to generate NT animals from them appeared to be remote prospects. Therefore, it was such a surprise to those of us in the field at the time that the Roslin researchers reported success in sheep with cells more differentiated than ESCs (Campbell et al., 1996b).

Because of the failure to isolate pluripotent ESCs, the Roslin team initially used differentiated epithelial cell cultures derived from the embryonic discs of 9-day-old sheep embryos. At first, actively growing embryonic cells (where the cell cycle stage was unknown) were fused to preactivated cytoplasts (see below). These NT experiments did in fact produce lambs at term from early (passages 1–3) (Campbell et al., 1995a) but not later-passage cells (passages 6–13) (Campbell et al., 1995b; Campbell et al., 1996b). It was only in subsequent studies, where the cells were deprived of serum for 5 days and reported to be quiescent, that cells from higher passages yielded lambs following fusion with cytoplasts either before, after, or simultaneous with the activation stimulus, all at similar overall efficiencies (Campbell et al., 1996b; Campbell et al., 1996c). The two surviving lambs were named Megan and Morag. To many in the field, these sheep were more significant than Dolly because they demonstrated for the first time the nuclear totipotency of differentiated mammalian cells. These differentiated embryonic cells were described as “TNT” cells; that is, “totipotent for nuclear transfer.” They also signified, at least initially, the importance of quiescence in NT.

The Roslin team extended their observations the following year with three new populations of differentiated sheep cell lines derived from a day-9 embryo, a day-26 fetus, and, most significantly, in the case of Dolly, the mammary gland of an adult ewe (Wilmut et al., 1997). This breakthrough finally disproved the suggestion that “cloning of mammals by simple NT is biologically impossible” (McGrath and Solter, 1984). Consequently, it opened new directions in research and provided practical opportunities for agriculture and medicine (Wilmut, 1998; Gurdon and Colman, 1999). The use of differentiated cells for cloning has been termed somatic cell nuclear transfer (SCNT) to distinguish it from NT with embryonic cells.

Although it was clear that Dolly originated from an adult cell, there was some uncertainty as to the degree of cellular differentiation in the successful donor used for NT. Was she in fact derived from a rare mammary stem cell in the cultured population? While nuclear reprogramming and cloning efficiency are much greater with early embryonic blastomeres, it still has not been conclusively determined whether differentiation status affects nuclear totipotency within somatic cell lineages (Oback and Wells, 2007)—that is, whether a putative mammary gland stem cell would actually be easier to reprogram. The definitive proof that terminally differentiated cells could be fully reprogrammed directly following NT was demonstrated in the mouse with natural killer T lymphocytes (Inoue et al., 2005).

Beyond Dolly

The significance of performing NT with in vitro–cultured cell lines cannot be overstated. Because there is access to large numbers of cells, it made cloning more practical to multiply genetically valuable farm animals compared to early embryos and more amenable to the generation of genetically modified livestock following NT, as first demonstrated with “Polly” (Schnieke et al., 1997). Transfecting cultured cells and selecting those that had undergone the desired genetic alteration to generate farm animals by NT was significantly more efficient and cost-effective compared to the available alternatives at the time, most notably, pronuclear injection of DNA. Cultured cells were amenable to both random integration of DNA constructs (Schnieke et al., 1997) and (with some difficulty) gene targeting, to either knockout or knockin genes of interest (McCreath et al., 2000; Dai et al., 2002). The combination of cell-mediated genetic modification and NT has been especially useful for advancing potential biomedical applications (including biopharming, organs for xenotransplantation, and livestock as biomedical models of human diseases), as well as those for agriculture (Niemann and Kues, 2007). There are limitations, however, with primary somatic cell lines, principally in their shortened in vitro life span and possibly lower frequency of homologous recombination compared to the preferred immortal ESCs (equivalent to what is available in the mouse).

G0 Cloning

Keith's idea to deprive cultured cells of serum and force them to exit the cell division cycle was initially perceived as the key to the first successful births of cloned animals derived from differentiated somatic cells. Serum starvation is a convenient way to synchronize cells into quiescence, where they can be easily maintained for days in culture in a uniform state for experimentation. Keith hypothesized that quiescent somatic cells were more amenable to NT and provided a greater opportunity for reprogramming given their altered chromatin state and reduced transcriptional profile (Campbell et al., 1996b; Wilmut et al., 1997).

Proliferating cells move through a continuous cell cycle, which has four distinct stages: Gap 1 (G1), DNA Synthesis (S), Gap 2 (G2), and Mitosis (M). Transition from one stage to the other is governed by cyclins and their respective cyclin-dependent kinases (CDKs). Cells can exit this cell cycle and rest in a stage called quiescence (G0). In G0, they are neither dividing nor preparing to divide, but can re-enter the cell cycle and proliferate upon certain stimuli (Zetterberg and Larsson, 1985). Quiescence as a distinct cellular stage has been well established. Experimentally, serum starvation, growth to confluency, and inhibition of adhesion can all induce quiescence. Strikingly, cells induced into quiescence by all these three methods exhibit distinct, although overlapping, expression profiles (Coller et al., 2006). G0 cells not only downregulate genes involved in cell proliferation, but also actively inhibit senescence, apoptosis, and differentiation (Coller et al., 2006; Coller, 2011).

Definitive molecular markers to characterize the quiescent state and distinguish between G0 and G1 cells in the diploid population is challenging (Cheung and Rando, 2013). Having exited from the cell cycle, negative markers for the G0 state include: The downregulation of cyclins, proliferating cell nuclear antigen, and Ki-67 antigen (Pellicciari et al., 1995); the absence of 5′-bromo-2′-deoxyuridine (BrdU) incorporation; and lower cellular RNA and protein content, as may be determined by dual-parameter flow cytometry, albeit with somewhat arbitrary gating thresholds (Boquest et al., 1999). Because quiescent cells are poised to re-enter the cell cycle at short notice, they cannot deviate far from the proliferating state, and so universal positive markers for G0 are equivocal. At present, the best positive indicators for entry into G0 include: the relative accumulation of CDK inhibitors (Cheung and Rando, 2013); stable E2F–p130 complex (Smith et al., 1996); and the upregulation of statin, growth-arrest-specific genes (Pellicciari et al., 1995), as well as the transcriptional repressor HES1, allowing quiescence to be reversible by preventing premature cell death or differentiation (Sang et al., 2008). In most NT experiments, more could have been done to better characterise the cell cycle stage of donors used for NT.

Quiescence Is Not Absolutely Necessary

With the dramatic increase in research activity in NT following the original breakthroughs (Campbell et al., 1996b; Wilmut et al., 1997), many groups demonstrated shortly afterward that with enough painstaking effort somatic cells at other stages of the cell cycle could also sometimes be reprogrammed correctly to generate cloned offspring. The requirement for quiescence was first questioned when randomly picked donor cells from non-serum-starved, proliferating cell cultures also resulted in viable offspring following NT with nonactivated MII cytoplasts in cattle (Cibelli et al., 1998; Vignon et al., 1998; Zakhartchenko et al., 1999) and mice (Wakayama and Yanagimachi, 1999). However, none of these early studies determined the exact stage of the cell cycle at the time of NT that resulted in those few reconstructed embryos developing into viable offspring.

Subsequent studies that selected somatic cells at specific stages of the cell cycle have proven that quiescence is not essential for cloning success. Several groups have produced cloned calves from selected G1 donors (Kasinathan et al., 2001; Gibbons et al., 2002; Wells et al., 2003; Urakawa et al., 2004; Goto et al., 2013). In addition, mitotically arrested mouse ESCs (Wakayama et al., 1999; Ono et al., 2001b) as well as somatic donor cells have also resulted in viable cloned mice (Ono et al., 2001a), pigs (Lai et al., 2002), and calves (Tani et al., 2001; Heyman et al., 2002) after NT with MII cytoplasts. In summary, various studies have demonstrated that somatic donor cells in G0, G1, and M phases can all result in viable offspring upon transfer into nonactivated MII cytoplasts, whereas G2- and S-phase donors cannot. Although cell cycle stages other than G0 do work, donors at specific stages may be reprogrammed more effectively.

The Importance of Cell Cycle Coordination

Keith understood clearly the importance of the cell cycle stage of the donor nucleus in relation to that of the recipient cytoplast at the time of NT to, first, prevent DNA damage and maintain normal ploidy and, second, maximize the opportunity for reprogramming to improve development. He summarized this insight in several excellent reviews (Campbell et al., 1996a; Campbell, 1999; Campbell and Alberio, 2003).

Recipient cytoplasts differ in the activity of cyclin B and CDK 1 complexes (M-CDK; formerly maturation-promoting factor or MPF). MII-arrested cytoplasts maintain high M-CDK activity, whereas activation enables degradation of cyclin B, destroying M-CDK activity. Therefore, two types of cytoplast may be used for NT—nonactivated (high M-CDK) or activated (low M-CDK).

When an interphase donor nucleus is fused with a nonactivated cytoplast, the high M-CDK induces immediate nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) of the donor chromatin. The chromatin of S-phase nuclei exposed to cytoplasts high in M-CDK has a typically pulverized appearance, with fragmentation of partially replicated DNA. By contrast, the chromatin of G0/G1 (2C) or G2 (4C) nuclei condenses in a high M-CDK environment, forming elongated chromosomes with either single- or double-stranded chromatids, respectively. Following a suitable activation stimulus, the nuclear envelope reforms around the donor chromatin, which then undergoes DNA synthesis regardless of its previous cell cycle stage. Thus, donor nuclei in G0/G1 initiate DNA synthesis, which is compatible with normal development. In contrast, nuclei in S phase re-replicate DNA, so that by the end of the first embryonic cell cycle, the DNA content in the two daughter cells is incorrect, leading to abnormal early embryonic development. The outcome with G2 nuclei ultimately depends on whether a diploid pseudo-polar body forms to remove the extra set of chromatin. With somatic donors in cattle, it appears this does not occur, and G2-derived blastocysts have not resulted in viable offspring, probably due to ploidy errors (Tani et al., 2001). However, this does not appear to be a limitation with G2 mouse embryonic blastomeres (Cheong et al., 1993).

Cells in metaphase (4C) could be considered maximally compatible with metaphase-arrested cytoplasts. To maintain normal ploidy, the reconstructed NT embryo must extrude a diploid pseudo-polar body and form a diploid pronucleus. Alternatively, a modified NT procedure incorporating cytoskeletal inhibitors to suppress polar extrusion body to generate two diploid pronuclei available for serial transplantation into zygotic cytoplasts has also resulted in live offspring from mitotic four-cell mouse embryos (Kwon and Kono, 1996).

By contrast, when interphase nuclei are transferred to cytoplasts after the decline of M-CDK, following a sufficient interval after activation, NEBD does not occur (so therefore neither does PCC), and it is the donor nucleus that controls DNA replication in accordance with its stage in the cell cycle at the time of NT. Thus, nuclei in G0/G1 or S phases initiate or continue replication, respectively, while those in G2 are not induced to enter another round of DNA synthesis. Such preactivated cytoplasts have been termed “Universal Recipients” (Campbell et al., 1994) and are capable of coordinating the development of donor cells at any stage of the cell cycle, apart from perhaps those in M phase (Campbell and Alberio, 2003). The use of universal recipients has been especially important for cloning preimplantation embryos, where most undifferentiated blastomeres are in S phase at any one time (80–90%) and are therefore most compatible with transfer to cytoplasts low in M-CDK. Although exemplified in sheep (Campbell et al., 1996b), it is generally regarded that somatic nuclei in a low M-CDK environment are not reprogrammed efficiently and nonactivated MII cytoplasts are the preferred recipients typically resulting in better development (Tani et al., 2001; Du et al., 2002).

Intimately associated with correctly coordinating the cell cycles of donors and recipients is the order of events in the NT procedure, particularly the temporal relationship between fusion and activation. Keith described the three possible combinations as related to the TNT cells in a poster presented at the 1996 meeting of the International Embryo Transfer Society (a copy of which was pinned above this author's desk for at least a decade afterward, given the significance of the results subsequently published in Nature; Campbell et al., 1996b). Keith described these combinations with intriguing acronyms in the poster, but denoted them more plainly in the associated abstract as recipient cytoplasts 1, 2, and 3 (Campbell et al., 1996c):

Method 1 represented “GOAT,” signifying G0 Activated at the time of Transfer (otherwise, Activation and Fusion).

Method 2 represented “MAGIC” and stood for Metaphase Arrested G1/G0 accepting Cytoplast (otherwise, Postactivated).

Method 3 represented “UNIVERSAL” and was the standard preactivated oocyte cytoplast capable of maintaining ploidy at any donor interphase stage (otherwise, Preactivated).

We and others found Keith's MAGIC method especially useful for increasing the success of somatic cell NT in cattle (Wells et al., 1999; Tani et al., 2001). The prolonged exposure of donor chromatin at the G0/G1 stage to the oocyte factors present in the MII cytoplast for 4–6 h before activation proved beneficial for reprogramming and restoring the totipotency of differentiated somatic cells.

Is Quiescence Beneficial for NT Reprogramming?

The question remains, however, whether any of the cell cycle stages compatible with maintaining ploidy are superior for epigenetic reprogramming and overall cloning efficiency, in terms of live offspring from embryos transferred to recipients. Three independent studies have directly compared cloning efficiency in cattle with somatic donor cells in either the G1 or G0 stages of the cell cycle, and all indicated that G1 donors were superior: 5% versus 0% (Kasinathan et al., 2001), 11% versus 2% (Gibbons et al., 2002), 13% versus 3% (Goto et al., 2013). These studies are, however, all in conflict with our own data with conventional bovine somatic cells, perhaps emphasizing the variable results dependent upon other methodological differences between laboratories.

In our experiments, we directly compared bovine cloning efficiencies of presumptive G0- and G1-phase donor cells from six independent cell lines in nonactivated MII cytoplasts, keeping all other experimental conditions the same (Wells et al., 2003; Tucker et al., 2004; Kallingappa et al., 2013). G0 cells were obtained following serum deprivation by culture in medium containing 0.5% fetal calf serum (Campbell et al., 1996b) for 9–11 days with granulosa cells from ovarian follicles and 5–7 days with both fetal and adult fibroblasts. Following serum starvation, the proportion of BrdU-positive follicular and fibroblast cells after labeling for 24 h was significantly reduced from 44% to 11% and 81% to 2%, respectively.

G1 cells were obtained by individually picking mitotic cells in medium containing 10% fetal calf serum and fusing individual cells after manual separation to cytoplasts within 1–3 h of cell division. This timing was prior to entry into S phase based on lack of BrdU incorporation in control cells. The cells had not entered quiescence or senescence as BrdU labeling for 24 h produced almost complete progression into S phase (98%) and cell numbers had doubled. Collectively, these results suggest that donors were in the early G1 phase of the cell cycle at the time of NT.

There was no effect of G0 versus G1 donor cell cycle stage on the rate of in vitro blastocyst development on day 7 following NT. However, we found that for all three cell types (granulosa, as well as fetal and adult fibroblasts) the overall output of viable cloned calves at weaning was significantly higher with serum-starved G0 donor cells than with G1 cells (16% vs. 6%) (Wells et al., 2003; Tucker et al., 2004).

To investigate the epigenetic basis for the improved reprogrammability of quiescent compared to proliferating cells, we examined the chromatin composition of bovine G0 and G1 adult fibroblasts and the blastocysts derived from them. Both DNA and lysine (K) residues on histone H3, specifically H3K4me3, H3K9me3, and H3K27me3, were significantly hypomethylated in G0 fibroblasts compared to early G1 cells (Kallingappa et al., 2013). Furthermore, G0 cells significantly downregulated the abundance of most chromatin-related proteins. Following NT into nonactivated cytoplasts, G0-derived blastocysts remained hypomethylated at H3K9me3, but not at H4K4me3 and H3K27me3, compared to blastocysts derived from G1 cells, in both the ICM and trophectoderm (Kallingappa et al., 2013). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. From these studies, we conclude that quiescence induces long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlate with increased donor cell reprogrammability and significantly improves the production of cloned calves (Wells et al., 2003; Tucker et al., 2004; Kallingappa et al., 2013). In support of our findings in cattle, naturally quiescent lymphocytes in mice have also been shown to contain hypomethylated histones compared to proliferating B cells, which correlated with three-fold better NT reprogramming efficiency in vitro (Baxter et al., 2004).

Keith's Legacy

Keith's work was certainly the catalyst for an exponential increase in publications on NT and epigenetic reprogramming, as well as leading to the formation of this Journal dedicated to the subject. Crucially, he realized that cell cycle coordination between the donor nucleus and the enucleated cytoplast could enable NT from differentiated cells and increase cloning efficiency. Keith's choice to use quiescent cells was of fundamental importance to overturn the prevailing thinking in cell biology. To date, the molecular signature and regulation of cell quiescence still remains to be fully elucidated. Several independent lines of evidence suggest that quiescence is a hallmark of plasticity in adult stem cells (Cheung and Rando, 2013). A key feature of the undifferentiated stem cell state and potential for life-long self-renewal (“stemness”) is the interaction with the immediate environment, forming the so-called stem cell niche (Spradling et al., 2001). The niche has been shown to slow down cell growth without inducing terminal differentiation, thereby providing a mechanism for placing stem cells on “stand-by,” locked into a quiescent, yet responsive, state (Heissig et al., 2002). Therefore, quiescence imposed by the environment appears to be a crucial condition to actively maintain stemness. A better understanding of this phenomenon will follow from deciphering the epigenetic and transcriptional regulation underlying stem cell quiescence and plasticity.

New technologies do not always live up to initial expectations. NT with cultured somatic cells has fulfilled much of the promise for the generation of valuable genetically modified farm animals (Niemann and Kues, 2007). However, cell-mediated transgenesis incorporating NT will likely become unnecessary for some of the new genome editing technologies (Tan et al., 2012). Somatic cell NT has fallen short of early hopes that it would be useful in multiplying valuable breeding stock. As a consequence of faulty reprogramming, the high frequency of development anomalies in somatic cell clones limits the practical application and consumer acceptance of the current technology in food-producing animals. Without greatly improved reprogramming strategies, this situation is unlikely to change. The identification and exogenous application of reprogramming factors present in oocytes (Pfeiffer et al., 2011; Awe and Byrne, 2013) and cytoplasmic extracts (Rathbone et al., 2010) could aid in releasing the epigenetic constraints of differentiated cells to increase the success of NT in generating viable animals.

There has been much discussion concerning the potential use of somatic cell NT in humans for the derivation of ESCs (termed ntESCs), for so-called “therapeutic cloning” and autologous cell-based therapy (Gurdon and Colman, 1999; Wilmut, 1998). Although exemplified in the mouse (Rideout et al., 2002; Tabar et al., 2008), genuine human ntESCs have only recently been reported (Tachibana et al., 2013). As was foreseen after Dolly (Wilmut, 1998), progress in reprogramming has led to methods to dedifferentiate somatic cells to pluripotency directly, without having to use NT to produce an embryo. Since 2006, the need for therapeutic cloning has been largely superseded by the advent of cellular reprogramming using specific transcription factors to generate induced pluripotent stem cells (iPSCs) directly from cultured somatic cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). This is an appealing alternative to ntESCs to provide autologous cells for cell-based therapies (Hanna et al., 2007; Wernig et al., 2008) and for which Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine, jointly with John Gurdon, in 2012.

It remains to be determined whether iPSCs or ntESCs will prove more useful for drug screening, disease modeling, and regenerative medicine in humans. Both cell types have their current deficiencies. At least for approaches that avoid integration of viruses or transgenes (Bayart and Cohen-Haguenauer, 2013), iPSCs are appealing because of their comparative ease of generation and are without the ethical burden associated with ntESCs, in that they do not require oocytes, cloning, or the destruction of embryos. However, it appears that current iPSCs are less well reprogrammed than ESCs cells and retain an epigenetic memory of their donor cell origin (Cahan and Daley, 2013). So, is there still a need for oocyte-mediated reprogramming for therapeutic cloning? Even if not, the discovery of oocyte factors may provide auxiliary molecules to promote more complete reprogramming in iPSCs. I am certain that Keith would have recommended continued investigation of both approaches. He would have also been fascinated with the recent use of caffeine to increase M-CDK in human oocytes to improve reprogramming following NT with quiescent somatic cell donors (Tachibana et al., 2013). Confirming Keith's own observations in sheep (Lee and Campbell, 2008; Choi and Campbell, 2010), caffeine was a contributing factor to improved blastocyst development, allowing the subsequent isolation of personalized human ntESCs for potential cell therapy (Tachibana et al., 2013). Dolly paved the way for these remarkable discoveries and clinical opportunities and this is something that Keith Campbell will always be remembered for.

Footnotes

Acknowledgments

The critical reading of this paper by Dr Björn Oback is most appreciated.

Author Disclosure Statement

The author declares that no conflicting financial interests exist.

References

Awe

J.P.

, Byrne

J.A.

2013. Identifying candidate oocyte reprogramming factors using cross-species global transcriptional analysis. Cell. Reprogram., 15:126–133.

Baxter

, Sauer

, Peters

et al. 2004. Histone hypomethylation is an indicator of epigenetic plasticity in quiescent lymphocytes. EMBO J., 23:4462–4472.

Bayart

, Cohen-Haguenauer

2013. Technological overview of iPS induction from human adult somatic cells. Curr. Gene Ther., 13:73–92.

Boquest

A.C.

, Day

B.N.

, Prather

R.S.

1999. Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biol. Reprod., 60:1013–1019.

Bradley

, Anastassiadis

, Ayadi

et al. 2012. The mammalian gene function resource: The International Knockout Mouse Consortium. Mamm. Genome, 23:580–586.

Briggs

, King

T.J.

1952. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc. Natl. Acad. Sci. USA, 38:455–463.

Cahan

, Daley

G.Q.

2013. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat. Rev. Mol. Cell Biol., 14:357–368.

Campbell

K.H.

1999. Nuclear equivalence, nuclear transfer, and the cell cycle. Cloning, 1:3–15.

Campbell

K.H.

, Alberio

2003. Reprogramming the genome: role of the cell cycle. Reprod. Suppl., 61:477–494.

10.

Campbell

K.H.

, Loi

, Cappai

, Wilmut

1994. Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol. Reprod., 50:1385–1393.

11.

Campbell

, McWhir

, Ritchie

, Wilmut

1995a. Production of live lambs following nuclear transfer of cultured embryonic disc cells. Theriogenology, 43:181.

12.

Campbell

K.H.S.

, McWhir

, Ritchie

W.A.

, Wilmut

1995b. Development of ovine nuclear transfer embryos during and after isolation of an established cell line from embryonic disc cells. J. Reprod. Fertil.Abstract Series 1531.

13.

Campbell

K.H.

, Loi

, Otaegui

P.J.

, Wilmut

1996a. Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev. Reprod., 1:40–46.

14.

Campbell

K.H.

, McWhir

, Ritchie

W.A.

, Wilmut

1996b. Sheep cloned by nuclear transfer from a cultured cell line. Nature, 380:64–66.

15.

Campbell

K.H.S.

, McWhir

, Ritchie

W.A.

, Wilmut

1996c. Live lambs by nuclear transfer from an established cell line. Theriogenology, 45:287.

16.

Cheong

H.T.

, Takahashi

, Kanagawa

1993. Birth of mice after transplantation of early cell-cycle-stage embryonic nuclei into enucleated oocytes. Biol. Reprod., 48:958–963.

17.

Cheung

T.H.

, Rando

T.A.

2013. Molecular regulation of stem cell quiescence. Nat. Rev. Mol. Cell Biol., 14:329–340.

18.

Choi

, Campbell

K.H.

2010. Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos. Reprod. Fertil. Dev., 22:1000–1014.

19.

Cibelli

J.B.

, Stice

S.L.

, Golueke

P.J.

, Kane

J.J.

, Jerry

, Blackwell

, Ponce de Leon

F.A.

, Robl

J.M.

1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280:1256–1258.

20.

Collas

, Barnes

F.L.

1994. Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei. Mol. Reprod. Dev., 38:264–267.

21.

Coller

H.A.

, Sang

, Roberts

J.M.

2006. A new description of cellular quiescence. PLoS Biol., 4:329–349.

22.

Coller

H.A.

2011. Cell biology. The essence of quiescence. Science, 334:1074–1075.

23.

Dai

, Vaught

T.D.

, Boone

et al. 2002. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat. Biotechnol., 20:251–255.

24.

Di Berardino

M.A.

2001. Animal cloning—the route to new genomics in agriculture and medicine. Differentiation, 68:67–83.

25.

, Sung

L.Y.

, Tian

X.C.

, Yang

2002. Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle. Mol. Reprod. Dev., 63:183–191.

26.

Egli

, Rosains

, Birkhoff

, Eggan

2007. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature, 447:679–685.

27.

Evans

M.J.

, Kaufman

M.H.

1981. Establishment in culture of pluripotential cells from mouse embryos. Nature, 292:154–156.

28.

Gibbons

, Arat

, Rzucidlo

, Miyoshi

, Waltenburg

, Respess

, Venable

, Stice

2002. Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol. Reprod., 66:895–900.

29.

Goto

, Hirayama

, Takeda

, Tukamoto

, Sakata

, Kaeriyama

, Geshi

2013. Effect of synchronization of donor cells in early G1-phase using shake-off method on developmental potential of somatic cell nuclear transfer embryos in cattle. Anim. Sci. J. 10.1111/asj.12047.

30.

Greda

, Karasiewicz

, Modlinski

J.A.

2006. Mouse zygotes as recipients in embryo cloning. Reproduction, 132:741–748.

31.

Gurdon

J.B.

1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol., 10:622–640.

32.

Gurdon

J.B.

2013. The egg and the nucleus: A battle for supremacy. Development, 140:2449–2456.

33.

Gurdon

J.B.

, Colman

1999. The future of cloning. Nature, 402:743–746.

34.

Gurdon

J.B.

, Uehlinger

1966. “Fertile” intestine nuclei. Nature, 210:1240–1241.

35.

Gurdon

J.B.

, Laskey

R.A.

, Reeves

O.R.

1975. The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. J. Embryol. Exp. Morphol., 34:93–112.

36.

Hanna

, Wernig

, Markoulaki

et al. 2007. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318:1920–1923.

37.

Heissig

, Hattori

, Dias

et al. 2002. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell, 109:625–637.

38.

Heyman

, Zhou

, Lebourhis

, Chavatte-Palmer

, Renard

J.P.

, Vignon

2002. Novel approaches and hurdles to somatic cloning in cattle. Cloning Stem Cells, 4:47–55.

39.

Inoue

, Wakao

, Ogonuki

et al. 2005. Generation of cloned mice by direct nuclear transfer from natural killer T cells. Curr. Biol., 15:1114–1118.

40.

Kallingappa

P.K.

, Turner

, Green

, Oliver

, Eichenlaub

, Chibnall

, Wells

, Oback

2013. Quiescence induces long-term epigenetic changes in bovine fibroblasts that improve their reprogramming into cloned animals. Reprod. Fertil. Dev., 25:171.

41.

Kasinathan

, Knott

J.G.

, Wang

, Jerry

D.J.

, Robl

J.M.

2001. Production of calves from G1 fibroblasts. Nat. Biotechnol., 19:1176–1178.

42.

Keefer

C.L.

, Stice

S.L.

, Matthews

D.L.

1994. Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves. Biol. Reprod., 50:935–939.

43.

Kwon

O.Y.

, Kono

1996. Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos. Proc. Natl. Acad. Sci. USA, 93:13010–13013.

44.

Lai

, Park

K.W.

, Cheong

H.T.

et al. 2002. Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol. Reprod. Dev., 62:300–306.

45.

Laskey

R.A.

, Gurdon

J.B.

1970. Genetic content of adult somatic cells tested by nuclear transplantation from cultured cells. Nature, 228:1332–1334.

46.

Lee

J.H.

, Campbell

K.H.

2008. Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer. Cloning Stem Cells, 10:381–390.

47.

Martin

G.R.

1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA, 78:7634–7638.

48.

McCreath

K.J.

, Howcroft

, Campbell

K.H.

, Colman

, Schnieke

A.E.

, Kind

A.J.

2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature, 405:1066–1069.

49.

McGrath

, Solter

1984. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science, 226:1317–1319.

50.

Niemann

, Kues

W.A.

2007. Transgenic farm animals: An update. Reprod. Fertil. Dev., 19:762–770.

51.

Oback

, Wells

D.N.

2007. Donor cell differentiation, reprogramming, and cloning efficiency: Elusive or illusive correlation? Mol. Reprod. Dev., 74:646–654.

52.

Ono

, Shimozawa

, Ito

, Kono

2001a. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol. Reprod., 64:44–50.

53.

Ono

, Shimozawa

, Muguruma

, Kimoto

, Hioki

, Tachibana

, Shinkai

, Ito

, Kono

2001b. Production of cloned mice from embryonic stem cells arrested at metaphase. Reproduction, 122:731–736.

54.

Pellicciari

, Mangiarotti

, Bottone

M.G.

, Danova

, Wang

1995. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content. Cytometry, 21:329–337.

55.

Pfeiffer

M.J.

, Siatkowski

, Paudel

, Balbach

S.T.

, Baeumer

, Crosetto

, Drexler

H.C.

, Fuellen

, Boiani

2011. Proteomic analysis of mouse oocytes reveals 28 candidate factors of the “reprogrammome.” J. Proteome Res., 10:2140–2153.

56.

Prather

R.S.

, Barnes

F.L.

, Sims

M.M.

, Robl

J.M.

, Eyestone

W.H.

, First

N.L.

1987. Nuclear transplantation in the bovine embryo: Assessment of donor nuclei and recipient oocyte. Biol. Reprod., 37:859–866.

57.

Prather

R.S.

, Sims

M.M.

, First

N.L.

1989. Nuclear transplantation in early pig embryos. Biol. Reprod., 41:414–418.

58.

Rathbone

A.J.

, Fisher

P.A.

, Lee

J.H.

, Craigon

, Campbell

K.H.

2010. Reprogramming of ovine somatic cells with Xenopus laevis oocyte extract prior to SCNT improves live birth rate. Cell. Reprogram., 12:609–616.

59.

Rideout

W.M.

3rd , Hochedlinger

, Kyba

, Daley

G.Q.

, Jaenisch

2002. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell, 109:17–27.

60.

Sang

, Coller

H.A.

, Roberts

J.M.

2008. Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science, 321:1095–1100.

61.

Schnieke

A.E.

, Kind

A.J.

, Ritchie

W.A.

, Mycock

, Scott

A.R.

, Ritchie

, Wilmut

, Colman

, Campbell

K.H.

1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science, 278:2130–2133.

62.

Schurmann

, Wells

D.N.

, Oback

2006. Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring. Reproduction, 132:839–848.

63.

Smith

E.J.

, Leone

, DeGregori

, Jakoi

, Nevins

J.R.

1996. The accumulation of an E2F-p130 transcriptional repressor distinguishes a G0 cell state from a G1 cell state. Mol. Cell. Biol., 16:6965–6976.

64.

Smith

L.C.

, Wilmut

1989. Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. Biol. Reprod., 40:1027–1035.

65.

Spemann

1938. Embryonic Development and Induction. Yale University Press: New Haven, CT.

66.

Spradling

, Drummond-Barbosa

, Kai

2001. Stem cells find their niche. Nature, 414:98–104.

67.

Stice

S.L.

, Strelchenko

N.S.

, Keefer

C.L.

, Matthews

1996. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol. Reprod., 54:100–110.

68.

Tabar

, Tomishima

, Panagiotakos

et al. 2008. Therapeutic cloning in individual parkinsonian mice. Nat. Med., 14:379–381.

69.

Tachibana

, Amato

, Sparman

et al. 2013. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 153:1228–1238.

70.

Takahashi

, Yamanaka

2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.

71.

Takahashi

, Tanabe

, Ohnuki

, Narita

, Ichisaka

, Tomoda

, Yamanaka

2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.

72.

Tan

W.S.

, Carlson

D.F.

, Walton

M.W.

, Fahrenkrug

S.C.

, Hackett

P.B.

2012. Precision editing of large animal genomes. Adv. Genet., 80:37–97.

73.

Tani

, Kato

, Tsunoda

2001. Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming. Biol. Reprod., 64:324–330.

74.

Tucker

, Eichenlaub

, Miller

, Oliver

, Forsyth

, Berg

, Cockrem

, Oback

, Wells

2004. Increased cattle cloning efficiency with serum-starved compared to G1 somatic donor cells. The 37th Annual Meeting of the Society for the Study of Reproduction. Biol. Reprod. Special IssueAbstract 577.

75.

Urakawa

, Ideta

, Sawada

, Aoyagi

2004. Examination of a modified cell cycle synchronization method and bovine nuclear transfer using synchronized early G1 phase fibroblast cells. Theriogenology, 62:714–728.

76.

Vignon

, Chesne

, Le Bourhis

, Flechon

J.E.

, Heyman

, Renard

J.P.

1998. Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. C. R. Acad. Sci. III, 321:735–745.

77.

Wakayama

, Yanagimachi

1999. Cloning of male mice from adult tail-tip cells. Nat. Genet., 22:127–128.

78.

Wakayama

, Rodriguez

, Perry

A.C.

, Yanagimachi

, Mombaerts

1999. Mice cloned from embryonic stem cells. Proc. Natl. Acad. Sci. USA, 96:14984–14989.

79.

Wells

D.N.

, Misica

P.M.

, Tervit

H.R.

1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod., 60:996–1005.

80.

Wells

D.N.

, Laible

, Tucker

F.C.

et al. 2003. Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology, 59:45–59.

81.

Wernig

, Zhao

J.P.

, Pruszak

, Hedlund

, Fu

, Soldner

, Broccoli

, Constantine-Paton

, Isacson

, Jaenisch

2008. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc. Natl. Acad. Sci. USA, 105:5856–5861.

82.

Willadsen

S.M.

1986. Nuclear transplantation in sheep embryos. Nature, 320:63–65.

83.

Wilmut

1998. Cloning for medicine. Sci. Am., 279:30–35.

84.

Wilmut

, Schnieke

A.E.

, McWhir

, Kind

A.J.

, Campbell

K.H.

1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810–813.

85.

Zakhartchenko

, Durcova-Hills

, Stojkovic

, Schernthaner

, Prelle

, Steinborn

, Muller

, Brem

, Wolf

1999. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J. Reprod. Fertil., 115:325–331.

86.

Zetterberg

, Larsson

1985. Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA, 82:5365–5369.