Somatic cell nuclear transfer derived stem cells

Somatic cell nuclear transfer (SCNT), colloquially known as cloning, is the process of transferring the nuclear DNA of a donor somatic cell into an enucleated oocyte, followed by embryo development (Fig. ​(Fig.2)2) (Wilmut et al., 2002). When the SCNT embryo is transferred to a surrogate recipient with the aim to achieve a live birth, the process is defined as reproductive cloning. The first success in mammals was achieved with the birth of Dolly the sheep in 1996 (Wilmut et al., 1997), cloned from a differentiated mammary epithelial cell. This successful attempt proved that it is possible to revert the differentiated status of the somatic nucleus to totipotency (reprogramming) (Wilmut et al., 2002). However, when pluripotent SCNT stem cells are harvested from the reconstructed SCNT embryo, the process is called therapeutic cloning, aiming at deriving pluripotent stem cells for future cell therapy and research purposes (Fig. ​(Fig.2).2). The advantage of therapeutic cloning over ESCs is that SCNT stem cells, like iPSCs, are genetically identical to the somatic cell they are derived from, thereby overcoming immune rejection, inherently valuable for future clinical applications. Somatic cell nuclear transfer was first attempted in amphibians due to the comparatively large size of the eggs, enabling easier micromanipulation coupled with the possibility of using considerable numbers of eggs and embryos. Tadpoles developed following transfer of nuclei from early cleavage stage embryos to enucleated eggs (Briggs and King, 1952). Subsequently, the group of Dr J. Gurdon (Gurdon et al., 1958) transplanted the nucleus of a tadpole intestinal cell into an enucleated frog egg, succeeding in the creation of viable tadpoles that were genetically identical to the one from which the intestinal cell was obtained. This was the first experiment to show that differentiated cells could be set back to an embryonic state.

Since Dolly, several attempts have been made to generate SCNT-ESCs in several mammalian species, due to their potential benefits in biomedical applications such as allo-transplantation and personalized drug selection (Matoba and Zhang, 2018). These attempts have further enabled optimization of the SCNT process, including cell cycle synchronization between donor cells and recipient oocytes, erasure of epigenetic marks by using donor cells of varying ages and from different tissues, as well as the addition of small molecules and the modification of culture conditions (Akagi et al., 2011). The first primate SCNT-ESCs were derived in the rhesus macaque from adult skin fibroblasts, partly owing to the non-invasive removal of the spindle-chromosome complex by polarized microscopy (Byrne et al., 2007). Although very successful in all species tested, pseudoblastocyst development following human SCNT was not achieved, with most SCNT embryos arresting at the stage of embryonic genome activation (Heindryckx et al., 2007). The first successfully reconstructed human SCNT pseudoblastocysts were reported by French et al. (2008), however the derivation of SCNT-ESC lines was not attempted. The key to success was minor SCNT technological adjustments and the use of in vivo matured oocytes from young donors. Subsequently, Noggle and collaborators adjusted the conventional SCNT approach by transferring the somatic nucleus into a non-enucleated recipient oocyte. The reconstructed embryos developed well, and several SCNT-ESC lines were derived, albeit triploid (Noggle et al., 2011).

The group of Dr S. Mitalipov (Tachibana et al., 2013) was the first to succeed in the production of SCNT-hESCs lines (Fig. ​(Fig.1),1), later reproduced by a handful of groups (Chung et al., 2015; Wolf et al., 2017). As S. Mitalipov highlights himself: ‘We demonstrated that cytoplasmic factors present in mature human oocytes are capable of converting the transplanted nuclear genomes from somatic cells (skin fibroblasts) to become “oocyte-like”. We then used such skin-derived oocytes to develop into blastocysts and ESCs.’ (Supplementary data).

The biggest hurdle for human SCNT applications remains the scarcity of human oocytes. A highly debated research question is whether the SCNT-ESC represent a better reprogramming method compared to iPSC (Matoba and Zhang, 2018). An issue of SCNT-ESCs and iPSCs is their propensity to retain a so-called somatic epigenetic memory, i.e. a partial epigenetic state, characteristic of the somatic cell used to derive them, which may lead to biases or limitations in their fate choice following differentiation into cells of a particular lineage, as shown in the mouse. In-depth analysis of mouse SCNT-PSCs has shown that they are molecularly closer to and functionally indistinguishable from mESCs derived from IVF-fertilized embryos, as compared to both mouse and hiPSCs (Ma et al., 2014; Mishra et al., 2018).