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Therapeutic Cloning. Critical Reprogramming Factors in Human MII Oocytes are Physically Associated with the Chromosomes?
Posted on: May 16, 2023

Cytoplasmic factors present in mature, metaphase II (MII) arrested oocytes have a unique ability to reset the identity of transplanted somatic cell nuclei to the embryonic state. Since the initial discovery in amphibians, somatic cell nuclear transfer (SCNT) success in a range of different mammalian species has demonstrated that such reprogramming activity in enucleated or spindle-free oocytes (cytoplasts) is universal. However, despite numerous applications of SCNT for animal cloning, the nature of reprogramming oocyte factors and their mechanism of action remain largely unknown.

In humans, SCNT was envisioned as a means of generating personalized embryonic stem cells from patients' somatic cells, which could be used to study disease mechanisms and ultimately for cell-based therapies. However, the derivation of human nuclear transfer embryonic stem cells (NT-ESCs) has not been achieved despite numerous attempts during the past decade.

The roadblock responsible for failure is early embryonic arrest of human SCNT embryos precluding derivation of stable NT-ESCs. Typically, SCNT embryos fail to progress beyond the eight-cell stage, presumably due to an inability to activate critical embryonic genes from the somatic donor cell nucleus. In a few cases, when SCNT embryos did reach the blastocyst stage, either stable ESCs were not recovered or derivation was not attempted. Though the underlying cause of early developmental arrest remains unclear, most of these studies involving human oocytes applied SCNT protocols developed for nonprimate species.

Previously, scientists demonstrated that SCNT procedures, when adapted to primates, succeeded in reprogramming rhesus macaque adult skin fibroblasts into NT-ESCs. Therefore, they reasoned that, similar to other mammals, human MII oocytes must contain reprogramming activity. Several recent observations are relevant. Removal of human oocytes' nuclear genetic material (chromosomes) negatively impacts the cytoplast's subsequent ability to induce reprogramming. However, when a somatic cell nucleus is transplanted into an intact oocyte containing its own chromosomes, the resulting polyploid embryos are able to develop to blastocysts and support ESC derivation.

One possible explanation for these observations is that critical reprogramming factors in human MII oocytes are physically associated with the chromosomes or spindle apparatus and are depleted or critically diminished upon enucleation.

Alternatively, it is possible that one or more of the steps in SCNT – namely, oocyte enucleation, donor cell nucleus introduction, or cytoplast activation – negatively impact cytoplast quality, rendering it incapable of inducing sufficient reprogramming.

In considering distinct biological features of human oocytes that could be involved, scientists focused on the recent observation that meiotic arrest in human MII oocytes is unstable and can be easily perturbed by mechanical manipulations. Earlier, they have suggested that retention of meiosis specific activities in the cytoplast is critical for nuclear remodeling after transplantation of an interphase-stage somatic cell nucleus. This remodeling is positively correlated with onward development of SCNT embryos after activation.

Therefore, scientists systematically evaluated modifications in oocyte enucleation and donor cell introduction that might work to retain meiosis factors in human cytoplasts. They also determined that routine cytoplast activation treatments were insufficient to support subsequent human SCNT embryo development. They initially used rhesus macaque oocytes to evaluate factors affecting successful SCNT reprogramming in a primate system. Subsequently, they refined SCNT approaches with high-quality human oocytes donated by healthy volunteers and demonstrated that methodological alterations significantly improve blastocyst formation from human SCNT embryos. Moreover, they derived several human NT-ESC lines from these embryos and validated that their nuclear DNA is an exclusive match to parental somatic cells, whereas mitochondrial DNA originated almost exclusively from oocytes. They also conducted extensive pluripotency assays on human NT-ESCs to verify reprogramming.

Source: Tachibana et al., Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2023),
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