When was therapeutic cloning discovered

Her arrival started conversations about the implications of cloning, bringing controversies over human cloning and stem cell research into the public eye. Primates are good models for studying human disorders. Cloning identical primates would decrease the genetic variation of research animals, and therefore the number of animals need in research studies. The resulting embryos were then implanted into surrogate mothers. Out of 29 cloned embryos, two monkeys were born. One was a female named Neti, and the other was a male named Ditto.

This experiment was an exciting combination of findings from earlier work. Campbell and Wilmut had already created a clone using the nucleus of a cultured cell. Factor IX codes for a protein that helps blood clot, and it's used to treat hemophilia, a genetic disorder where blood doesn't form proper clots. To create the transgenic sheep, the scientists performed nuclear transfer using donor DNA from the cultured transgenic cells.

The result was Polly, a sheep that produced Factor IX protein in her milk. This experiment showed that sheep could be engineered to make therapeutic and other useful proteins in their milk, highlighting the potential medical and commercial uses for cloning. After the successes leading up to Dolly and Polly, other scientists wanted to see if similar techniques could be used to clone other mammalian species. Before long, several more animals had been successfully cloned.

Among them were transgenic animals, clones made from fetal and adult cells, and a male mouse; all previous clones had been female. As the list of successfully cloned animals grew, scientists began to explore cloning as a way to create animals belonging to endangered or extinct species.

A challenge to cloning endangered and extinct species is finding closely related animals to serve as egg donors and surrogates. The gaur and mouflon were chosen in part because they are close relatives of domestic cattle and sheep, respectively. In , using goast as egg donors and surrogates, another group of researchers cloned the first extinct animal, a Spanish mountain goat called the bucardo. Sadly, the one kid that survived gestation died soon after birth due to a lung defect.

Researchers took a cell from an adult monkey and fused it with an enucleated egg cell. The embryo was allowed to develop for a time, then its cells were grown in a culture dish. These cells, because they can differentiate to form any cell type, are called embryonic stem cells. This experiment showed that nuclear transfer in a primate, which researchers had tried for years without success, was possible.

It opened the door to the possibility of human therapeutic cloning: creating individual-specific stem cells that could be used to treat or study diseases.

Overcoming decades of technical challenges, Mitalipov and colleagues were the first to use somatic cell nuclear transfer to create a human embryo that could be used as a source of embryonic stem cells. Once we are able to derive nerve cells from cloned embryos, we hope not only to heal damaged spinal cords but to treat brain disorders such as Parkinsons disease, in which the death of brain cells that make a substance called dopamine leads to uncontrollable tremors and paralysis.

Alzheimers disease, stroke and epilepsy might also yield to such an approach. Besides insulin-producing pancreatic islet cells for treating diabetes, stem cells from cloned embryos could also be nudged to become heart muscle cells as therapies for congestive heart failure, arrhythmias and cardiac tissue scarred by heart attacks.

Autoimmune disorders such as multiple sclerosis and rheumatoid arthritis arise when white blood cells of the immune system, which arise from the bone marrow, attack the bodys own tissues. Preliminary studies have shown that cancer patients who also had autoimmune diseases gained relief from autoimmune symptoms after they received bone marrow transplants to replace their own marrow that had been killed by high-dose chemotherapy to treat the cancer.

Infusions of blood-forming, or hematopoietic, cloned stem cells might "reboot" the immune systems of people with autoimmune diseases. But are cloned cellsor those generated through parthenogenesisnormal? Only clinical tests of the cells will show ultimately whether such cells are safe enough for routine use in patients, but our studies of cloned animals have shown that clones are healthy. In the November 30, , issue of Science, we reported on our success to date with cloning cattle.

Of 30 cloned cattle, six died shortly after birth, but the rest have had normal results on physical exams, and tests of their immune systems show they do not differ from regular cattle. Two of the cows have even given birth to healthy calves. The cloning process also appears to reset the "aging clock" in cloned cells, so that the cells appear younger in some ways than the cells from which they were cloned.

In we reported that telomeresthe caps at the ends of chromosomesfrom cloned calves are just as long as those from control calves. Telomeres normally shorten or are damaged as an organism ages. Therapeutic cloning may provide "young" cells for an aging population. Imprinting is a type of stamp placed on many genes in mammals that changes how the genes are turned on or off depending on whether the genes are inherited from the mother or the father.

The imprinting program is generally "reset" during embryonic development. Although imprinting appears to play an important role in mice, no one yet knows how significant the phenomenon is for humans. In addition, Jaenisch and his co-workers did not study mice cloned from cells taken from the bodies of adults, such as fibroblasts or cumulus cells.

Instead they examined mice cloned from embryonic cells, which might be expected to be more variable. Studies showing that imprinting is normal in mice cloned from adult cells are currently in press and should be published in the scientific literature within several months.

Meanwhile we are continuing our therapeutic cloning experiments to generate cloned or parthenogenetically produced human embryos that will yield stem cells. Scientists have only begun to tap this important resource. WEST are vice president of research, vice president of medical and scientific development, and president and CEO, respectively, of Advanced Cell Technology, a privately held biotechnology company in Worcester, Mass. Cibelli received his D. His research led to the creation of the first cloned genetically modified calves in Conover's laboratory offers expertise in mouse embryonic stem cell derivation and characterization, as well as various differentiation analyses.

Conover is interested in understanding two developmental pathways, one that instructs stem cells to form dopaminergic neurons those lost in Parkinson's Disease and another that instructs stem cells to generate pancreatic islet cells those lost in Type I Diabetes.

A better understanding of these pathways will allow researchers to reliably obtain specific cell types for therapeutic use. Conover also has extensive experience in studying adult neural stem cells and is interested in the potential application of these cells toward the understanding and treatment of neurodegenerative diseases. With expertise in both embryonic and adult stem cells, Dr.

Conover is interested in comparing derivation, differentiation, and signaling pathways of different stem cell populations. Conover's research further extends to the study of mechanisms involved in stem cell activation and differentiation during regeneration.

In this special issue, Conover et al [ 8 ] discuss the fate of adult neural stem cells in the adult brain and the molecular mechanisms that regulate adult neurogenesis in mice. What makes a stem cell a stem cell and how stem cells differentiate into various cell types remain mysteries in biology. Theodore Rasmussen was recruited to the Center as a cellular and molecular geneticist to study the mechanisms of stem cell maintenance and differentiation.

Rasmussen's research concerning the molecular mechanisms that govern differentiation processes will further the goal of achieving highly efficient and rationally-guided differentiation.

Chromatin proteins are a diverse set of molecules that associate with DNA and regulate gene expression in a tissue-specific manner. Recent evidence suggests that chromatin proteins and chromatin remodeling activities play a substantial role in stem cell differentiation processes.

This proposition is supported by research into the mechanisms of X chromosome inactivation one of the earliest developmental changes in differentiating ES cells and nuclear transfer cloning experiments that suggest that "reprogramming" may have its basis in chromatin remodeling. The Rasmussen laboratory studies chromatin dynamics in mouse embryonic stem ES cells as a model system to understand differentiation processes on a mechanistic level.

Rasmussen is currently conducting extensive research on macroH2A1, a specialized histone variant involved in gene silencing and X inactivation. MacroH2A1 seems to be a general component of heterochromatin and is incorporated into the inactive X chromosome of female ES cells during the course of differentiation.

In addition, Dr. Rasmussen is interested in the mechanisms that target the formation of specialized chromatin to particular genomic sites. In this special issue, Dr. Rasmussen [ 9 ] provides an excellent overview on the recent advances in the understanding of the mechanisms that govern epigenetic regulation of gene expression of stem cell maintenance and differentiation dynamics.

As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events, which are exquisitely coordinated with the allocation of cell lineages, are reviewed in this article.

Understanding gene expression and regulation during natural organogenesis and tissue differentiation obviously will provide the theoretical basis to study in vitro stem cell differentiation and tissue regeneration. David Goldhamer was recruited to the Center as a developmental biologist to study fundamental mechanisms of skeletal muscle development, growth and repair, with an emphasis on regulation of cell commitment and muscle-specific gene expression.

One long-term goal of the Goldhamer laboratory is to define the genetic pathways that culminate in the activation of muscle regulatory factors in muscle precursor cells during development, an understanding of which will provide insight into how embryonic cells choose between alternative cell fates during development. A second major research area of his laboratory focuses on the biology of stem cells resident in muscle tissue.

Following injury of adult skeletal muscle, or in diseases such as Duchenne muscular dystrophy, skeletal muscle undergoes a regenerative process that in many ways resembles muscle development in the embryo.

Skeletal muscle's enormous regenerative capacity is mediated by muscle satellite cells, normally quiescent stem cells that are "activated" in response to injury or disease.

Despite their essential function, key aspects of satellite cell biology remain unresolved, including their developmental origin, potential and regulation of commitment to myogenesis. Goldhamer's recent research focuses on the use of cell marking experiments and mouse genetics to investigate these fundamental aspects of satellite cell biology. Recent evidence indicates that additional types of stem cells also exist in skeletal muscle tissue, the identification of which is essential for understanding diseases of abnormal bone formation and for developing therapies for musculoskeletal diseases.

These studies could lead to clinical applications for cancer diagnosis in humans since nuclear reprogramming signals from the host ooplasm variably reset the epigenetic profile of the nuclear donor DNA.

The derivation through SCNT of a healthy patient-specific stem line would show that cancer onset was triggered by epigenetic alterations. However, epigenetic resetting 15 following SCNT is likely to disrupt normal phenotype of the embryo-derived cell lines and the adult clone, the latter displaying an abnormally low body weight and expression level of MUP encoding genes Major Urinary Proteins as shown by Reik et al in the mouse.

The epigenetic pattern of imprinted genes that was established during gametogenesis is lost through SCNT 20 and the inactivation of early genes directing embryogenesis can explain low embryo viability and poor efficiency in the derivation of autologous ntESC lines. Blelloch et al found out, from studies on neurons, that stem cells used as the nuclear donor have a higher success rate 21 than fully differentiated cells in the derivation of autologous embryonic cells.

For instance, patient-specific cardiomyocytes produced through SCNT will not integrate into the scarred heart tissue resulting from myocardial infarction. For instance, Duchenne Muscular Dystrophy DMD is an inheritable X-linked condition characterized by reduced intramuscular dystophin levels, causing cellular necrosis and weakening Being a single-gene disorder, DMD can be treated by therapeutic cloning in combination with gene therapy to restore normal dystrophin production.

In the case where ntESC are transplanted without prior differentiation in vitro, the insertion of a transgene encoding MyoD 35 , a transcription factor responsible for commitment to the myogenic lineage, may promote muscle regeneration. The combination of gene therapy and therapeutic cloning has exciting potential for the genetic rescue of missing alleles in heritable genetic disorders such as severe combined immunodeficiency SCID , in which genetic mutations of specific genes such as RAG-1 and 2, essential for the DNA recombination allowing immunoglobulin and lymphocyte polymorphism, render the immune system completely inefficient.

Hochedlinger et al took a somatic nucleus from the tail-tip of an SCID mouse-model, created through the double-knockout of the Rag-2 gene recombination-activating gene 2 , and rescued the genetic defect through the insertion of two copies of the Rag-2 gene by homologous recombination However, mature T lymphocytes were not observed, suspected to be due to selective differentiation of the transplanted stem cells into myeloid cells bone marrow precursors instead Although more work needs to be done to elucidate the pathways leading to preferential differentiation in vivo, the combination of gene therapy for the rescue of a loss of function and therapeutic cloning to bypass graft rejection holds the potential to eventually cure other immune disorders.

Oncogenic activation following transduction constitutes a major drawback to this approach. In , the insertion of the transgene to treat X-linked SCID in the LMO2 oncogene caused the onset of leukemia in two out of seven patients recently treated Repeated graft rejection, even when derived through SCNT, remains an unsolved problem in the case of autoimmune disorders such as pernicious anemia and multiple sclerosis.

Legislative constraints and the subsequent lack of funding constitute a major impediment to the advancement of therapeutic cloning. For instance, although therapeutic cloning is not completely banned in the United States, federal funding is not permitted to be used in experiments involving the 20 cell lines in the NIH National Institute of Health registry 44 derived before August 9, Out of these cell lines approved by Bush, 12 died and the remaining is not useful for research purposes.

Researchers have to therefore rely on the scarcity of private funding, although 4 American states, including California with a yearly investment of millions, have a budget allowed specifically for stem cell research A major roadblock in the feasibility of human therapeutic cloning is the low availability of oocytes for research purposes. Currently, due to low SCNT efficiency, it is estimated that human oocytes 35 would be needed in order to derive one observe patient-specific ntESC line.

The Human Fertility and Embryo Authority, in England, allows women in fertility clinics to offer two oocytes for scientific research, provided that at least twelve oocytes are collected 25 , although the extra oocytes taken are more likely to be donated for in vitro insemination. Substantial financial gain would incite poorer women to surrender of part of a finite supply of gametes, in addition to the risks incurred through surgerical removal of the oocytes and hormonal treatments.

Ovarian hyperstimulation syndrome OHSS results, in most cases, from the administration of drugs such as gonadotropin-releasing hormone agonists 27 , to induce the simultaneous maturation of multiple follicles into oocytes.

Fertility clinics are the major source of human oocytes for research. The aged oocytes that did not fertilize during in vitro trials are not optimal for SCNT, as investigated by Hall et al, who observed overexpression of genes encoding for meiotic spindles proteins and a lower cleavage efficiency 30 of aged oocytes versus fresh ones.

After two years of debate, Harvard is the only group currently allowed to use oocytes collected for the sole purpose of research, with informed consent. Eggan and Melton intend to generate human ntESC lines from patients with diabetes, sickle cell anemia and amyotrophic lateral sclerosis ALS , a progressive neurodegenerative condition targeting motor neurons Interestingly, SCNT could provide a solution to low human oocyte availability and a promising therapeutic approach to circumvent infertility.

As reported by Nagy and Chang, artificial gametes 32 can be created by haploidization, through SCNT into an enucleated oocyte ready to undergo meiosis upon induction. Tesarik et al incorporated the nucleus of a human cumulus cell into an enucleated allogenic oocyte. However, abnormal chromosomal segregation and mitotic spindle assembly, as observed in mice, need to be resolved before haploidization through SCNT can be safely wide-spread in fertility clinics.

Once the molecular pathways of oocyte maturation are resolved, immature follicles could be collected postmortem 39 and induced to mature in vitro for research purpose. However, this option needs to be rigorously regulated, and is likely to stir an ethical controversy.

An alternative to low human oocyte availability would be to use an oocyte of a different species. Successful trials were done to generate blastocysts in vitro through the SCNT of skin fibroblast nucleus from different mammals ungulates, rodents, pigs, monkey 34 and human into an enucleated bovine oocyte.

The insertion of human nuclear genome into an animal oocyte, especially through electrofusion where both human and animal mitochondrial DNA coexist in the same ooplasm, raise objections among the detractors of therapeutic cloning, although the percentage of total residual animal DNA nuclear and mitochondrial is too low to consider the hybrid as a chimera.

Immune rejection of the ntESC in cell replacement therapy is due to mitochondrial heteroplasmy as a consequence of SCNT since the nuclear donor and ooplasmic host cells are not autologous in most cases. Mitochondrial heteroplasmy is also a major cause of SCNT embryo inviability beyond the eight-cell stage because the mitochondrial-nucleus interactions necessary for the production of most mitochondrial proteins are disrupted due to inter-species incompatibility Also, antigens such as Mta are encoded by the mitochondrial genome and trigger an autoimmune response targeting the hybrid 36 after transplantation.

Inter-species incompatibility can be circumvented to a certain degree if the donor and host species are closely related. For instance, embryonic cells derived from the injection of a human nucleus in a chimpanzee ooplasm were viable, contrarily to when SCNT was done with the ooplasm of a nonhuman primate such as the orangutan The transfer of mitochondria isolated from patient-specific biopsies 39 might circumvent the immune rejection problem due to mitochondrial heteroplasmy, and a female patient could in theory donate both the somatic nucleus and oocyte necessary for cell replacement therapy in her own body.

The latter option offers great promises since recent studies in bovine showed that autologous SCNT embryonic production was more efficient than when the nuclear donor and ooplasmic host are from allogenic origins, and epigenetic reprogramming occurs to a significantly less extend in autologous SCNT embryos The interspecies transmission of pathogens is a nonnegligible issue when injecting a human nucleus into the oocyte of another species, such as bovine or pig For instance, the porcine endogenous retrovirus PERV , although inoffensive in pigs, disrupts the transcription initiation of genes in humans, as demonstrated in vitro by Moalic et al, by integrating within the CpG islands of promoters Gene therapy approaches are being designed to reduce the infectivity of PERV due to the mannose-rich N-glycan integrated in the viral capsule Thus, through the insertion of genes such as ManIb and ManII, encoding mannosidases involved in N-glycan catabolism, the infectivity of PERV was significantly reduced but not annihilated in human cells.

Animal serum and non-proliferative mouse fibroblast used as feeder-cell layer to direct the development of the human ntESC 44 is problematic since animal contaminants can be transferred to the ntESC which in turn might trigger an immune response post-transplantation, thereby revoking the goal of therapeutic cloning.

N-glycolylneuraminic acid Neu5Gc is a mammalian sialic acid a sugar with acidic side-chains present in the membrane of all cells not found in humans, although most of us have anti-Neu5Gc antibodies.

When human ntESC are grown on animal feeder cells or in contact with animal-derived serum, the stem cells incorporate enough Neu5Gc to potentially elicit an immune response 45 in vivo, hence killing the transplanted cells. Although the murine leukemia virus is not pathogenic 44 when transmitted from mouse feeder cells to human ntESC, non-cellular matrices are being designed to counteract the problem of animal contaminants.

Neu5Gc was not reported in the cell lines cultured on this human matrix. In sum, the issue of pathogenic transmission is in the process of being solved, bringing one step further the potential for clinical application of therapeutic cloning in cell replacement therapy. NtESC are subjected to the same tumorigenicity potential as wild-type stem cells. The formation of teratomas, after in vivo transplantation, is due to co-purification of pluripotent stem cells along with the wanted differentiated cells.

The teratomas resulted from a low concentration of 0. Although the hyperglycemia associated with type I diabetes was reversed, tumorgenesis occurred 20 days post-transplantation, rendering stem cells, whether wild-type or issued from therapeutic cloning, a non-viable option for clinical applications in this instance, unless better isolation methods for the exclusive purification of differentiated stem cells are designed.

The apparition of the primitive streak directing polarized development confers to the two-week embryo a higher moral status 51 as a potential human organism, compared to the earlier embryo at the stage of a randomly-organized group of cells.

Consequently, laws prohibiting the culture of embryos for more than two-weeks, which marks the onset of gastrulation and the formation of the primitive streak, are in vigor in several countries such as the United States, based on a decision of the British Warnock Commission 29 in The ethical debate on the moral impermissibility of deliberate destruction of an embryo can be circumvented by a new technique deviced by Chung et al.

They successfully derived human ESC from a single cell without destroying the blastocyst in the process 52 , using the same manipulations normally devoted to genetic screening in preimplantation embryos. This method seems to be promising for solving the ethical concern of killing a human embryo, rendering feasible the prenatal generation of individual-specific cell lines for use in regenerative medicine later on in life.