Somatic cell nuclear transfer

In genetics and developmental biology , somatic cell nuclear transfer ( SCNT ) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte (egg cell) and implanting a donor nucleus from a somatic (body) cell. It is used in both therapeutic and reproductive cloning . Dolly the Sheep became famous for being the first successful case of the reproductive cloning of a mammal. [1] ” Therapeutic cloning ” refers to the potential use of SCNT in regenerative medicine; This approach has been championed as an answer to the many issues concerning embryonic stem cells (ESC) and the destruction of viable embryos for medical use, however questions remain on how homologous to the two cell types truly are.

Process

The process of somatic cell nuclear transplant involves two different cells. The first being a female gamete, known as the ovum (egg / oocyte). In human SCNT (Somatic Cell Nuclear Transfer), these eggs are obtained through consenting donors, utilizing ovarian stimulation. The second being a somatic cell, referring to the cells of the human body. Skin cells, fat cells, and liver cells are only a few examples. The nucleus of the donor is removed and discarded, leaving it ‘deprogrammed.’ What is left is a somatic cell and an unnucleated egg cell. These are then fused by inserting the somatic cell into the ’empty’ ovum. [2] After being inserted into the egg, the somatic cell nucleus is reprogrammedby its host egg cell. The ovum, now containing the somatic cell nucleus, is stimulated with a shock and will begin to divide. The egg is now viable and capable of producing an adult organism containing all the necessary genetic information from just one parent. This cell will have an early blastocyst (early stage embryo with about 100 cells) with an identical genome to the original organism (ie a clone). [3] Stem cells can be obtained by the destruction of this clone embryo for use in therapeutic cloning or in the cloning of the clone embryo is implanted into a host mother for further development and brought to term.

Applications

Stem cell research

Somatic cell nuclear transplantation has become a focus of study in stem cell research . The aim of carrying this procedure is to obtain pluripotent cells from a cloned embryo. These cells genetically matched the donor organism from which they came.This gives them the ability to create specific patient pluripotent cells, which could then be used in therapies or disease research. [4]

Embryonic stem cells are undifferentiated cells of an embryo. These cells are considered to be pluripotent potential because they have the ability to give rise to all tissues found in an adult organism. This ability can be transferred to other cells, which can be transplanted to replace damaged or destroyed cells. Controversy surrounds human ESC work due to the destruction of viable human embryos. SCNT is one such method.

Human Embryonic Stem cell colony on a mouse embryonic fibroblast feeder layer.

A potential use of stem cells is genetically matched to a patient who would have been associated with a particular patient. By doing so, an in vitro model could be created, potentially discovering its pathophysiology, and discovering therapies. [5] For example, if a person with Parkinson ‘s disease , the stem cells resulting from SCNT would have contributed to Parkinson’ s disease. The disease specific stem cells could then be studied in a better understanding of the condition. [6]

Another application of SCNT stem cell research is the specific patient stem cell lines to generate tissue or even organs for transplant into the specific patient. [7] The resulting cells would be genetically identical to the somatic cell donor, thus avoiding any complications from immune system rejection . [6] [8]

Only a handful of the labs in the world are currently using SCNT techniques in human stem cell research. In the United States , scientists at the Harvard Stem Cell Institute , the University of California San Francisco , the Oregon Health & Science University , [9] Stemagen (La Jolla, CA) and possibly Advanced Cell Technology are currently researching a technique to use somatic cell nuclear transfer to produce embryonic stem cells . [10] In the United Kingdom , the Human Fertilization and Embryology Authority has granted permission to research groups.Roslin Instituteand the Newcastle Center for Life . [11] SCNT may also be occurring in China. [12]

In 2005, Professor Hwang Woo-suk published a report to the South Korean research team led by SCNT, [13] but supported claims with fabricated data. [14] Recent evidence has been made that stem from a parthenote . [15] [16]

But there are many successes with cloning animals, questions about the mechanisms of reprogramming in the ovum. Despite many attempts, success in creating human embryonic stem cells. There is a problem in the human cell’s ability to form a blastocyst; the cells fail to progress the eight cell stage of development. This is thought to be a result of the somatic cell nucleus being unable to turn on embryonic genes crucial for proper development. These experiments were developed in non-primate animals with little success. A research group from the Oregon Health & Science UniversitySCNT procedures developed for primates successfully reprogrammed skin cells into stem cells. The key to their success was utilizing oocytes in metaphase II (MII) of the cell cycle. Egg cells in MII contain special factors in the cytoplasm that have a special ability in reprogramming implanted somatic cell nuclei into cells with pluripotent states. When the ovum’s nucleus is removed, the cell loses its genetic information. This has been blamed for why they are hampered in their reprogramming ability. It is theorized the critical embryonic genes are closely linked to oocyte chromosomes, enucleation negatively affects these factors. Another possibility is removing the egg nucleus or inserting the somatic nucleus causes damage to the cytoplast, affecting reprogramming ability. SCNT stem cells. In May 2013, the Oregon group reported the successful derivation of human embryonic stem cell lines derived through SCNT, using fetal and infant donor cells. Using MII oocytes from volunteers and their SCNT procedure, human clone embryos were successfully produced. These embryos were poorly produced, lacking a substantial inner cell mass and poorly constructedtrophectoderm . The imperfect embryos prevented the acquisition of human ESC. The addition of caffeine during the removal of the ovum nucleus and injection of the somatic nucleus improved blastocyst formation and ESC isolation. The ESCs were found to be capable of producing teratomas, expressed pluripotent transcription factors, and expressed a normal 46XX karyotype, indicating that these SCNTs were in fact ESC-like. [9] This was the first instance of successfully using SCNT to reprogram human somatic cells. This study used fetal and infant somatic cells to produce their ESC.

In April 2014, an international research team expanded on this break through. There remained the question of whether or not it was possible to become accustomed to somatic cells. Epigenetic and age related changes are thought to be possible in the future. They have been able to grow cells stemming from SCNT using adult cells from two donors, aged 35 and 75.Indicating age does not impede a cells ability to be reprogrammed [17] [18]

Late April 2014, the New York Stem Cell Foundation was successful in creating SCNT stem cells derived from adult somatic cells. One of these lines of stem cells was derived from the donor cells of a diabetic type. The group was then able to successfully culture these stem cells and induce differentiation. When injected into mice, cells of all three of the germ layers The most significant of these cells have been able to secrete the hormone. [19] These insulin producing cells could be used for replacement therapy in diabetics, demonstrating real SCNT stem cell therapeutic potential.

The impetus for SCNT-based stem cell research has been reduced by the development and improvement of alternative methods of generating stem cells. Plasmid cells in pluripotent stem cells were developed in humans in 2007. The following year, this method achieved a key goal of SCNT-based stem cell research: the derivation of pluripotent stem cells that have all genes linked to various diseases . [20]Some scientists working on SCNT-based stem cell research have recently moved to the new methods of induced pluripotent stem cells. Recent studies have been in question how similar are embryonic stem cells. Epigenetic memory in iPS affects the cell lineage it can differentiate into. For instance, an iPS cell derived from a blood cell will be more efficient at differentiating into blood cells, while it will be less efficient at creating a neuron. [21] These are the types of cells that can be used in a variety of ways. SCNT stem cells do not pose such a problem and continue to remain relevant in stem cell studies.

Reproductive cloning

Main article: Cloning
BTX ECM 2001 Electrofusion generator used for SCNT and Cloning applications

This technique is currently the basis for cloning animals (such as the famous Dolly the sheep ), [22] and has been theoretically proposed as a possible way to clone humans. Using SCNT in reproductive cloning has proven difficult with limited success. High fetal and neonatal death make the process very inefficient. Resulting cloned offspring are also plagued with development and imprinting disorders in non-human species. For these reasons, along with moral and ethical objections, [23]Most researchers believe that it will be possible to use the current cloning technique to produce a clone that will develop to term. It remains a possibility, although critical adjustments will be required to overcome current limitations during early embryonic development in human SCNT. [24] [25]

There is also the potential for treating diseases associated with mutations in mitochondrial DNA. Recent studies show SCNT of the nucleus of a body cell afflicted with one of these diseases in a healthy oocyte prevents the inheritance of the mitochondrial disease. This treatment does not involve cloning but would produce a child with three genetic parents. A mother providing a sperm cell, a mother providing a mother and a daughter providing a sperm cell. [7]

Interspecies nuclear transfer

Interspecies nuclear transfer (iSCNT) is a means of somatic cell nuclear transfer used to facilitate the recovery of endangered species, or even to restore species after extinction. The technique is similar to SCNT cloning which typically is between domestic animals and rodents, or where there is a ready supply of oocytes and surrogate animals. However, the cloning of highly endangered or extinct species requires the use of an alternative method of cloning. Interspecies nuclear transfer utilizes a host of two different organisms that are closely related species and within the same genus. In 2000, Robert Lanza was able to produce a cloned fetus of a gaur , Bos gaurus, combining it with a domestic cow, Bos taurus . [26]

Interspecies nuclear transfer provides evidence of the universality of the triggering mechanism of cell nucleus reprogramming. For example, Gupta et al., [27] explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Moreover, NCSU23 medium, which was designed for in vitro culture of embryo pigs, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Furthermore, ovine oocyte cytoplast can be used for remodeling and reprogramming of human somatic cells back to the embryonic stage. [28]

Limitations

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SCNT can be inefficient. Stresses placed on both the egg cell and the introduced nucleus in early research, resulting in a low percentage of successfully reprogrammed cells. For example, in 1996, 267 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood. [22] As the procedure was not automated, but had to be performed under a microscope , SCNT was very resource intensive. The biochemistry involved in reprogramming the differentiatedsomatic cell nucleus and activating the egg is also far from understood. HOWEVER, by 2014, Researchers Were reporting success rates of 70-80% with cloning pigs [29] and in 2016 Korean company, Sooam Biotech Was Reported to be cloned embryos Producing 500 a day. [30]

In SCNT, not all of the donor’s genetic information is transferred, as the donor’s mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures that originally belonged to the egg. As a result, clones such as these are born from SCNT are not perfect copies of the donor of the nucleus. This fact may also hamper the potential benefits of SCNT derived tissues / organs for therapy, as it may be an immunoresponse to the non-self mtDNA after transplant.

Controversy

Further information: stem cell controversy
Human Blastocyst , showing the inner cell mass (top, right).

Proposals to use nucleus transfer techniques in human stem cell research raised a set of concerns beyond the moral status of any created embryo. These are some of the subjects that are not specifically addressed to human embryonic stem cells, or to SCNT research. [31] [32] [33]

One concern is that blastula creation in SCNT-based human stem cell research will lead to the reproductive cloning of humans. Both processes use the same first step: the creation of a nuclear embryo, most likely via SCNT. Those who hold this concern often advocate for the regulation of SCNT to preclude implantation of any derived products for the purpose of human reproduction, [34] or its prohibition. [31]

A second important concern is the appropriate source of the eggs that are needed. SCNT requires human eggs , which can only be obtained from women. The most common source of these diseases is that they are produced during the course of IVF treatment. This is a minimally invasive procedure, but it does carry some health risks, such as ovarian hyperstimulation syndrome .

One vision for successful stem cell therapies is to create custom stem cell lines for patients. Each cell line would consist of a collection of identical stem cells each carrying the patient’s DNA, thus reducing or eliminating any of the following causes of transplantation. For example, to treat a man with Parkinson’s disease, a cell nucleus from one of his cells would be transplanted by SCNT into an egg cell from an egg donor, creating a unique lineage of stem cells almost identical to the patient’s own cells. (There would be differences, for example, the mitochondrial DNA would be the same as that of the egg donor.In comparison, his own cells would carry the mitochondrial DNA of his mother.)

Potentially millions of patients could benefit from stem cell therapy, and each patient would require a large number of patients. Such large numbers of donated eggs would be more likely to be delivered by assisted reproduction technology . Therefore, healthy young women would be able to be used in the creation of custom stem cells that could be purchased by the medical industry and sold to patients. It is so far unclear where all these eggs would come from.

Stem cell experts consider it unlikely that such large numbers of human donations would occur in the future because of the long-term health effects of treating large numbers of healthy young women with heavy doses of hormones in order to induce hyperovulation (ovulating several eggs at once). Although such treatments have been performed for a long time, the long-term effects have not been studied or declared safe. Longer-term treatments with much lower doses of hormones. Whether hormone treatments to induce hyperovulation could have similar effects is unknown. There are also ethical issues surrounding paying for eggs. In general, marketing body parts is considered unethical and is banned in most countries.

To address the problem of creating a human egg market, some stem cell researchers are investigating the possibility of creating artificial eggs. If successful, human egg donations would not be needed to create custom stem cell lines. However, this technology may be a long way off.

Policies regarding human SCNT

SCNT involving human cells is currently legal for research purposes in the United Kingdom , having been incorporated into the Human Fertilization and Embryology Act 1990 . [35] [2] Permission must be obtained from the Human Fertilization and Embryology Authority in order to perform or attempt SCNT.

In the United States, the practice remains legal, as it has not been addressed by federal law. [36] However, in 2002, a moratorium on United States federal funding for SCNT prohibits funding the practice for the purposes of research. Thus, though legal, SCNT can not be federally funded. [37] American scholars have recently argued that the product of SCNT is a clone embryo, rather than a human embryo, these are morally wrong and should be revised. [38]

In 2003, the United Nations adopted a proposal submitted by Costa Rica , calling on member states to “prohibit all forms of human cloning in as much as they are incompatible with human dignity and the protection of human life.” [39]This sentence may include SCNT, depending on interpretation.

The Council of Europe’s Convention on Human Rights and Biomedicine and its Additional Protocol to the Convention for the Protection of Human Rights and the Dignity of the Human being with regard to the Application of Biology and Medicine, on the Prohibition of Cloning Human Behavior SCNT of human beings. Of the Council’s 45 member states, the Convention has-been signed by 31 and 18. Ratified by The Additional Protocol has-been signed by 29 member nations and Ratified by 14. [40]

See also

  • Induced stem cells
  • Stem cell research
  • Stem cell controversy
  • embryogenesis
  • In vitro fertilization
  • Cloning
  • New Jersey legislation S1909 / A2840
  • Rejuvenation

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