Somatic Cell Nuclear Transfer–Derived Embryonic Stem Cell Lines in Humans: Pros and Cons

Dediferentiation of Somatic Cells

There are several methods of dedifferentiating the somatic cell nucleus (Halley-Stott et al., 2013), but it is also evident that somatic cell nuclear transfer (SCNT) represents the most efficient artificial approach, although it has not yet fully explained why. We suggest that an important point of SCNT reprogramming efficiency is that the transferred nucleus, in the course of reprogramming, uses some oocyte components that are incorporated into the remodeled nucleus. The other reprogramming approaches—induced pluripotent stem cells (iPSCs) production, heterokaryons—are in fact “closed” systems, i.e., reprogramming is induced in a given cell with an intact nucleus. On the other hand, SCNT is an “open” system in which the nuclear membrane breakdown occurs within a very short time interval after introduction of the nucleus into the cytoplast and then the chromosomes condense. Following a subsequent artificial activation, which is usually applied approximately 1 h after nuclear transfer (NT), the pseudopronuclei start to form and, in the mouse, for example, they are typically very visible after about 6 h. Morphologically they are much larger when compared to the original transferred nucleus and contain very visible nucleoli (nucleolus precursor bodies, NPBs) that are morphologically indistinguishable from similar structures that can be found in pronuclei of normally fertilized or parthenogenetic one-cell-stage embryos. These dramatic changes cannot be detected for example in iPSCs or heterokaryons.

The Role of Oocyte Organelles in Reprogramming

The invention of the enucleolation method, in which NPBs can be microsurgically removed either from oocytes (or even from one-cell-stage embryos; Fulka Jr et al., 2003; Fulka and Fulka Jr., 2010), clearly demonstrated that if enucleolated oocytes are fertilized or parthenogenetically activated, normal-sized pronuclei are formed in one-cell stage embryos; however, these pronuclei or pseudopronuclei do not contain typical nucleoli (NPBs) and consequently embryos fail to develop (Fulka et al., 2004; Ogushi et al., 2008). In SCNT experiments, where enucleolated and subsequently matured oocytes were used for the preparation of cytoplasts, the nucleus that is transferred into the cytoplast contains its own nucleolus(i) but this nucleolus(i) cannot substitute for the original oocyte nucleolar material that is dissolved in the oocyte cytoplasm. Thus, pseudopronuclei do not contain nucleoli.

These observations clearly explain, of course to a certain extent, why early NT experiments in mammals where nuclei were transferred into enucleated zygotes were unsuccessful (McGrath and Solter, 1984). The nucleoli were removed along with the entire pronuclear material. Typically, only two-cell-stage mouse embryo nuclei supported development, but no development was observed when more advanced nuclei were used. It must be noted here that in the mouse the transformation of nucleoli in embryos begins at the late two-cell stage. Moreover, this also explains why mitotic one-cell-stage embryos can be used as cytoplasts (Noggle et al., 2011). Here the zygote nucleolar material is also dissolved in the cytoplasm. Nucleoli are released into the cytoplasm when zygotes are enucleated selectively, and this also results in successful development of reconstructed embryos (Greda et al., 2006).

The role of nucleolar material in the process of normal and SCNT embryo development is not yet understood completely. The developmentally competent oocytes and zygotes contain atypical nucleoli (NPBs) that are composed from dense fibrillar material. On the other hand, somatic and advanced embryonic cells contain nucleoli composed from fibrillar, dense fibrillar, and granular material. It has been commonly accepted that the original oocyte nucleolar material is, as the embryo develops, gradually transformed into the somatic cell nucleoli and that the oocyte nucleolus (NPB) serves as a depot or storage site of material that is, after fertilization, used by the early embryo. Some recent results, however, have forced us to reconsider the whole concept of the NPB as a passive storage site of nucleolar proteins because they indicate that this, in fact, might be very far from the primary role of this structure. Currently, it is believed that the NPB actually plays an active role in supporting early development unrelated to ribosomal RNA production and is probably involved in the regulation of certain cell cycle processes during very early embryogenesis. However, some very recent results also demonstrate that NPBs might also be essential for certain structural and chromatin modification processes that occur within a very short time interval after fertilization, when both the maternal and paternal chromatin are remodeled extensively. For further successful development, heterochromatin at pericentric satellites must be in a very close contact with NPBs (Probst and Almouzni, 2011).

All of these hints of the actual role of NPB are supported by experiments performed by Ogushi and Saitou (2010), who transferred NPBs into metaphase II oocytes originating from germinal vesicle (GV)-stage enucleolated oocytes. These oocytes were subsequently fertilized in vitro. Eventually, NPBs were transferred into early and late one-cell-stage embryos that were produced by fertilization of previously enucleolated oocytes. When NPBs were transferred into mature oocytes, the development of embryos was rescued. However, this was not the case after transfer of NPBs into zygotes without nucleoli. The involvement of NPBs in structural and chromatin modifications seems to be true also for SCNT embryos. The proper nuclear genome organization in pseudopronuclei, which is also characterized by a close association of heterochromatin with NPBs, is necessary for genome reprogramming and probably for correct gene expression during further development (Martin et al., 2006a, b). Which other of the oocyte cellular components are used in the process of transferred nucleus remodeling remains to be determined.

Taken together, the above results can explain why NT is more efficient than other reprogramming approaches, but they do not answer the question whether SCNT embryonic stem cells (ESCs) are better than iPSCs. The ESC lines can be established efficiently from SCNT embryos (Wakayama et al., 2006), even though the production of cloned offspring is still disappointingly very low (Ogura et al., 2013). It is, however, commonly accepted that the main problems in cloning is the abnormal function of the placenta. This, however, is not relevant when the aim is ESC line derivation.

Conclusion

It is our opinion that at present it is difficult to say which is the best way that will lead to an efficient approach of somatic cell reprogramming of and the production of patient-compatible stem cell lines. The production of a patient's iPSC lines may take at least few months (Takahashi and Yamanaka, 2013). The SCNT approach would be, in theory, much shorter. Thus, at present the evident advantage of SCNT ESC derivation is the speed of this approach. On the other hand, we must keep in mind that the production of SCNT ESC lines requires the supply of very high-quality human oocytes as a source of cytoplasts. This represents the first ethical problem. The second ethical problem is in the opening the gate to the production of cloned humans.