Reprogramming refers to the process of erasing the existing differentiation memory of somatic cells and returning them to a state similar to embryonic cells, and regaining totipotency or pluripotency. The development of reprogramming technology overcomes the defects of scarce sources of embryonic stem cells and large individual differences, and avoids a series of ethical controversies caused by the destruction of embryos. The other directions have broader application prospects and become a hot spot in the field of stem cells. There are currently three main methods of somatic cell reprogramming: somatic cell nuclear transfer (SCNT), induced pluripotent stem cells (iPSCs) and cell fusion technology. Among them, the cell fusion technology produced in the 1960s and 1970s used chemical stimulation or electric shock to fuse somatic cells and embryonic stem cells to obtain pluripotency. However, because the cells produced after fusion are tetraploid, it is often difficult to apply to clinical research, and this technique has not been promoted.
- 1. Somatic cell nuclear transfer technology
The mammalian sperm and egg are combined in the fallopian tube to form a fertilized egg. The fertilized egg then undergoes multiple cleavages and cell differentiation to eventually develop into a new individual with thousands of cell types. In the early embryonic development, the cell development includes intra-embryo ability of the entire biological individual including extraembryonic tissue, which is defined as totipotency. In addition to normal fertilization and development, artificial techniques of somatic cell nuclear transplantation, which are well-known for cloning, can also achieve cell totipotency. In the 1960s, the Nobel Prize winner in Physiology or Medicine John Gurdon obtained the normal development of tadpoles and sexually mature adult cloned animal Xenopus laevis through SCNT technology, which proved for the first time that differentiated donor cells can be reprogrammed in enucleated fertilized eggs to obtain totipotency to develop into cloned individuals. In 1997, British scientist Wilmut fused serum-starved adult goat mammary epithelial cells with enucleated oocytes to obtain the cloned animal star “Dolly” sheep, which proved for the first time that cloning technology could be applied to mammals. In 2018, the Institute of Neuroscience of the Chinese Academy of Sciences used nuclear transfer of cynomolgus monkey fetal skin fibroblasts to successfully obtain two healthy cloned monkey individuals. This is the first time that SCNT technology has been used to truly clone primates. However, there are still some obstacles in the practical application of SCNT technology, mainly manifested in the low cloning efficiency of almost all species, abnormal development of extra-embryonic tissues of cloned embryos, and abnormalities such as immunodeficiency or even early death of cloned animals after birth. Due to insufficient understanding of the obstacles in the SCNT reprogramming process, the cloning efficiency of mammals has not been effectively improved. In recent years, the invention and promotion of sequencing technology have made it possible to study the changes in the embryo transcriptome and epigenetic group during SCNT reprogramming, and gradually improve the nuclear transfer embryo development mechanism.
Figure 1. Cellular and molecular events during somatic cell nuclear transfer embryo development
1.1. The development mechanism of SCNT embryo
The core of SCNT technology mainly includes three parts, removal the genetic material of the recipient oocyte or fertilized egg, injection the donor cell nucleus or fusion of the donor cell with the enucleated recipient cell, and the activation of the recombinant embryo. The nuclear membrane of the donor cell nucleus ruptures rapidly after entering the enucleated oocyte, forming a concentrated chromosome. This process is called premature chromosome condensation (PCC), which is caused by the M-phase promoting factor in the cytoplasm of the oocyte. (M-phase-promoting factors, MPFs). The PCC process is necessary for the reprogramming of SCNT embryos, and SCNT embryos that do not undergo the PCC process will have developmental disorders. MPFs are the most important cytoplasmic influence factor in oocytes, and their activity peaks during the MI and MII phases of meiosis in oocytes. In the selection of SCNT receptor cells, the use of MII stage oocytes is better than the prokaryotic stage zygotes, because the high level of MPFs in the MII stage oocytes can effectively mediate the PCC of the donor nucleus, while the prokaryotic stage zygotes MPFs activity has begun to decline rapidly due to fertilization or embryo activation. As the fertilization proceeds normally, the phospholipase CZ1 (PLCZ1) carried by the sperm triggers calcium ion oscillations in the cytoplasm of the oocyte, accompanied by MPFs inactivation, which activates the oocyte and initiates subsequent development. Since PLCZ1 is not present in donor cells, the most common method for activating mouse SCNT recombinant embryos is artificial strontium chloride treatment, which adds strontium chloride to the embryo culture medium to simulate fertilization signals.
In the normal fertilized zygote, the two nuclei from the sperm and oocyte are called the male and female pronucleus, respectively. One unique feature of the pronucleus is its large size. In the early stage of mammalian zygote formation, the transcription of oocytes and sperm is silent, and then the zygote genome gradually begins to be transcribed in a process called zygotic genome activation (ZGA). This process is accompanied by the rapid degradation of maternal RNA, and Zygote RNA is synthesized in large quantities. In SCNT embryos, the donor cell genome forms a nuclear membrane and then forms a pseudo-pronucleus in the G1 phase. According to the random distribution of PCC chromosomes, SCNT embryos usually form one or two pseudo-pronuclei. Like the pronucleus of normal embryos, the pseudo-pronucleus of SCNT embryos is much larger than the donor cell nucleus. Although the DNA replication and ZGA dynamic changes of SCNT embryos are similar to those of normal embryos, the time of DNA replication for each SCNT embryo is inconsistent, and many genes in SCNT embryos cannot be effectively activated during ZGA. Compared with normal fertilized embryos or in-vitro-fertilized (IVF) embryos, SCNT embryos have some defects in the transcriptome, epigenetic group, and chromatin structure, which may be the main reason for limited success rate of mammalian cloned embryos for a long time.
To be continued in Part Two…