The team at the  Roslin Institute  were seeking a way to modify the genetic constitution of sheep and cattle more effectively than the hit and miss method that was the only method and had sort of aids available at the time –  microinjection . In microinjection, DNA is injected into the pronuclei of fertilized oocytes. However, only a small proportion of the animals will integrate the injected DNA into their genome and in the rare cases that they do integrate this new genetic information, the pattern of expression of the injected piece of DNA's gene, due to the random integration, is very variable.


The team choose to combine two approaches – microinjection and  embryonic stem cells . In order to achieve this they decided to try to transfer the nucleus from one cell to another and stimulate this new cell to grow and become an animal, a process known as  nuclear transfer . The team at the Roslin Institute tried to make immortalized and undifferentiated embryonic stem cell lines in sheep, but failed. As a result, they decided to work with cultured blastocyst cells. The nuclear material of these blastocyst cells would be transferred into an unfertilized sheep egg cell, an oocyte where the nucleus had been removed. To optimize the chances of successful nuclear transfer, they put the cultured cells into a state of quiescence, which was a similar state to that of the unfertilized egg cell.


Nuclear transfer was done, using electrical stimuli both to fuse the cultured cell with the enucleated egg and to kick start embryonic development. From 244 nuclear transfers, 34 developed to a stage where they could be placed in the uteri of surrogate mothers. In the summer of 1995, five lambs were born, of which two – Megan and Morag – survived to become healthy fertile adults. These were the first mammals cloned from differentiated cells. They were born with the names 5LL2 and 5LL5 in June 1995.


The production of Megan and Morag demonstrated that viable sheep can be produced by nuclear transfer from cells which have been cultured in vitro. They signified the technical breakthrough that made  Dolly the sheep  possible. The birth of Megan and Morag, a year before Dolly, with their huge beneficial potential, made relatively few headlines. [ 4 ]  As of 2005 [update] , Megan was still alive and was the oldest cloned animal at the time. [ 5 ]


Mycocepurus smithii  is a species of  fungus-growing   ant  from  Latin America . This species is widely distributed geographically and can be found from  Mexico  in the north to  Argentina  in the south, as well as on some  Caribbean  Islands. [ 1 ] [ 2 ] [ 3 ]  It lives in a variety of forested habitats and associated open areas. [ 2 ]
Two studies published in 2009 demonstrated that some populations of the species consist exclusively of females which reproduce via  thelytokous   parthenogenesis . [ 4 ] [ 5 ]  A detailed study found evidence of sexual reproduction in some populations in the Brazilian Amazon. [ 3 ]  Accordingly,  M. smithii  consists of a mosaic of sexually and asexually reproducing populations. [ 3 ]  In asexual populations all ants in a single colony are female  clones  of the  queen . [ 3 ]  Inside the colony, the ants cultivate a garden of  fungus  grown with pieces of dead vegetable matter, dead insects, and insect droppings. [ 6 ] [ 7 ]


Ants of the genus  Mycocepurus  are distinctly recognizable for the crown-like cluster of spines on their promesonotum, the fused  mesonotum  and  pronotum  on the front of their  mesosoma  or midsection.  Mycocepurus smithii  has sharp, protruding propodeal (posterior of the alitrunk) spines unlike  M. obsoletus  whose propodeal spines are blunt. Workers also do not have developed promesonotal spines in the center of their crown, which separates  M. smithii  from  M. goeldii  and similar species. [ 1 ] [ 2 ]


Initially,  M. smithii  was thought to only reproduce asexually because no evidence of male individuals had been found. This led to  M. smithii  being recognized as the first fungus-growing ant species to reproduce via thelytokous parthenogenesis, where females, the workers and reproductive queens, are produced asexually. [ 5 ] [ 4 ]  The cytogenetic mechanism of thelytoky is either  apomixis  (mitotic parthenogenesis) or  automixis  with central fusion and low  recombination  rates. [ 3 ]  Automixis with central fusion is the  cytogenetic  mechanism that was recorded in other asexual ant species. [ 8 ]
Nests with multiple  dealated  queens are often found, suggesting that  M. smithii  is a  polygynous  species. [ 5 ] [ 6 ] [ 7 ]  This appears to be a case of secondary polygyny, and the queens may be daughters of the foundress. [ 6 ]


A detailed study of many  M. smithii  populations across their geographic distribution range (Mexico to Argentina) showed that some  M. smithii  populations in the Brazilian  Amazon  reproduce sexually. This was demonstrated using highly variable  genetic markers .  Sperm  was also found stored in the  spermathecas  of queens. Sexual reproduction was suggested as a mechanism for maintaining the  genetic diversity  seen in this species. [ 3 ]  In summary,  M. smithii  is not purely asexual, but instead consists of a "mosaic" of sexual and asexual populations.  Phylogenetic  reconstructions and the biology of the species suggest that these sexual populations gave rise to the asexual ones. [ 3 ]  The mechanism behind the shift to asexuality is still unknown. However,  antibiotic  assays and  genetic screenings  suggest that it is not an  endosymbiont  such as  Wolbachia  causing the asexuality. [ 4 ]  In fact, a comparative analysis showed that  Wolbachia  endosymbionts do not seem to cause asexuality in ants in general. [ 8 ]


The nests and colonies of  M. smithii  were studied in great detail in  Puerto Rico  and  Brazil . [ 6 ] [ 7 ] [ 5 ]  On the surface,  M. smithii  nests can be recognized by their nest mounds consisting of excavated soil and clay. A nest entrance of roughly 1.2 mm in diameter is located in the center of each nest mound. Large  M. smithii  nests, which are presumably older, can contain up to 7 or so chambers. [ 5 ] [ 6 ] [ 7 ]  Some fungus chambers are shallow whereas others can be found in great depths, as deep as 2 meters. [ 7 ]  The abandoned chambers are used to deposit waste from the fungus garden and loose soil from chamber construction. The number of nest chambers tends to increase as colonies grow older. [ 7 ]  Because  M. smithii  queens are capable of asexual reproduction, it is believed that colonies can also grow by budding  [ 7 ]  in addition to independent colony foundation. [ 5 ]  Colonies that grow by budding can result in large colony networks. [ 7 ]


Workers of  M. smithii  ants maintain narrow tunnels (diameter of 1.3 mm), which do not allow two ants to pass each other in the tunnel (head size is around 0.7 mm for workers and 0.9 mm for queens). The tunnels also have a number of slightly larger sections (about 3.6 mm diameter), which would allow passing while also facilitating information exchange. Narrow tunnels are presumably easier (energetically cheaper) to construct and may also aide in leveling the humidity or temperature of the colony or preventing predatory intrusions. [ 6 ] [ 7 ]  In general,  M. smithii  colonies are smaller than the colonies of  M. goeldii . [ 5 ] [ 7 ]


When founding a new colony, young queens either  shed their wings  prior to excavating the nest or just inside. They then excavate a tunnel to a depth of roughly 10 cm (4 in) and create a primary chamber. The dealate, or wingless, queen then carries the wings into the primary chamber and inserts them into the chamber ceiling where the surface of the wings is used as a platform for growing an incipient fungus garden. She will also forage around the nest entrance for caterpillar droppings to feed the fungus garden. The female fore wings of all so-called Paleoattini (the genera  Mycocepurus ,  Apterostigma , and  Myrmicocrypta ) have a crescent-shaped spot lacking any veins, hairs, and pigmentation, and is thought to provide an "easy to clean" platform for the fungus garden. [ 6 ]  Queens of the  socially parasitic  species  Mycocepurus castrator  do not found their colonies independently, and the clear spot is absent from their wings. [ 9 ]  This indirectly supports the idea that the wing spot has a function during the early colony founding and fungus cultivation stage of independently founding  Mycocepurus  queens. [ 9 ]
As the colony matures, workers develop and then tend to the fungus garden, feeding it dried leaves, caterpillar droppings, and other debris from the leaf-litter. [ 6 ] [ 7 ]


One trait of  M. smithii  cultivation is that, unlike higher attines, they use a wide diversity of fungal lineages for their gardens. [ 10 ] [ 11 ]
Lineages of  M. smithii  have undergone many cultivar shifts over time. This tendency to shift cultivars is hypothesized to be a mechanism for helping to offset some of the costs of asexuality. [ 12 ]
Also unlike other fungus-growing ants  M. smithii  has a  microbiome  that is distinct from the surrounding soil. [ 13 ]
A Brazilian population of  M. smithii  has a fungal cultivar with  gongylidia -like structures. This is unusual, because gongylidia are the nutrient rich food bodies produced by the fungi of  leaf-cutting ants  – and leaf-cutting ants are rather distant relatives of Mycocepurus. [ 14 ]


Nuclear transfer  is a form of  cloning .  The step involves removing the  DNA  from an  oocyte  (unfertilised egg), and injecting the  nucleus  which contains the DNA to be cloned. In rare instances, the newly constructed cell will divide normally, replicating the new DNA while remaining in a  pluripotent  state.  If the cloned cells are placed in the uterus of a  female mammal , a cloned organism develops to term in rare instances. This is how  Dolly the Sheep  and many  other species  were cloned. Cows are commonly cloned to select those that have the best milk production. On 24 January 2018, two  monkey   clones  were reported to have been created with the technique for the first time. [ 1 ] [ 2 ] [ 3 ]


Despite this, the low efficiency of the technique has prompted some researchers, notably  Ian Wilmut , creator of  Dolly  the cloned sheep, to abandon it. [ 4 ]


Nuclear transfer is a delicate process that is a major hurdle in the development of cloning technology. [ 5 ]  Materials used in this procedure are a microscope, a holding  pipette  (small vacuum) to keep the oocyte in place, and a  micropipette  (hair-thin needle) capable of extracting the nucleus of a cell using a vacuum.  For some species, such as mouse, a drill is used to pierce the outer layers of the oocyte.


Various chemical reagents are used to increase cloning efficiency.  Microtubule  inhibitors, such as  nocodazole , are used to arrest the oocyte in  M phase , during which its nuclear membrane is dissolved. Chemicals are also used to stimulate oocyte activation.  When applied the membrane is completely dissolved.


Somatic Cell Nuclear Transfer  (SCNT) is the process by which the nucleus of an oocyte (egg cell) is removed and is replaced with the nucleus of a somatic (body) cell (examples include skin, heart, or nerve cell). The two entities fuse to become one and factors in the oocyte cause the somatic nucleus to reprogram to a pluripotent state. The cell contains genetic information identical to the donated somatic cell. After stimulating this cell to begin dividing, in the proper conditions an embryo will develop. Stem cells can be extracted 5–6 days later and used for research. [ 6 ]


Genomic  reprogramming  is the key biological process behind nuclear transfer. Currently unidentified reprogramming factors present in oocytes are capable of initiating a cascade of events that can reset the mature, specialized cell back to an undifferentiated, embryonic state.  These factors are thought to be mainly proteins of the nucleus.


The  perivitelline space  is the space between the  zona pellucida  and the  cell membrane  of an  oocyte  or fertilized  ovum . [ 1 ]  In the  slow block to polyspermy , the  cortical granules  released from the ovum are deposited in the perivitelline space.  Polysaccharides  released in the granules cause the space to swell, pushing the zona pellucida farther from the oocyte. [ 1 ]  The  hydrolytic enzymes  released by the granules cause the  zona reaction , which removes the  ZP3  ligands from the zona pellucida. [ 1 ]


Clinically, the perivitelline space is relevant because it is where the  polar body  lodges after meiosis.


A  phagemid  or  phasmid  is a  DNA -based  cloning vector , which has both  bacteriophage  and  plasmid  properties. [ 1 ]  These vectors carry, in addition to the origin of plasmid replication, an  origin of replication  derived from bacteriophage. Unlike commonly used plasmids, phagemid vectors differ by having the ability to be packaged into the  capsid  of a bacteriophage, due to their having a genetic sequence that signals for packaging. Phagemids are used in a variety of biotechnology applications; for example, they can be used in a molecular biology technique called " phage display ". [ 2 ]


The term "phagemid" or "phagemids" was coined by a group of Soviet scientists, who discovered them, named them, and published the article in April 1984 in Gene magazine. [ 3 ]


A phagemid (plasmid + phage) is a  plasmid  that contains an f1  origin of replication  from an  f1 phage . [ 4 ]  It can be used as a type of  cloning   vector  in combination with  filamentous phage   M13 . A phagemid can be replicated as a plasmid, and also be packaged as single stranded  DNA  in viral particles. Phagemids contain an  origin of replication (ori)  for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. [ 4 ]  Many commonly used plasmids contain an f1 ori and are thus phagemids.


Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques, such as  transformation  and  electroporation . However, infection of a bacterial host containing a phagemid with a 'helper' phage, for example VCSM13 or M13K07, provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles.  The 'helper' phage infects the bacterial host by first attaching to the host cell's pilus and then, after attachment, transporting the phage genome into the cytoplasm of the host cell. Inside the cell, the phage genome triggers production of single stranded phagemid DNA in the cytoplasm. This phagemid DNA is then packaged into phage particles. The phage particles containing ssDNA are released from the bacterial host cell into the extracellular environment.


Filamentous phages retard bacterial growth but, contrasting with the  lambda phage  and the  T7 phage , are not generally  lytic . Helper phages are usually engineered to package less efficiently (via a defective phage origin of replication) [ 5 ]  than the phagemid so that the resultant phage particles contain predominantly phagemid DNA. F1 Filamentous phage infection requires the presence of a  pilus  so only bacterial hosts containing the  F-plasmid  or its derivatives can be used to generate phage particles.


Prior to the development of cycle sequencing, phagemids were used to generate single stranded DNA template for sequencing purposes. Today phagemids are still useful for generating templates for  site-directed mutagenesis . Detailed characterisation of the filamentous phage life cycle and structural features lead to the development of  phage display  technology, in which a range of peptides and proteins can be expressed as fusions to phage coat proteins and displayed on the viral surface. The displayed peptides and polypeptides are associated with the corresponding coding DNA within the phage particle and so this technique lends itself to the study of  protein-protein interactions  and other ligand/receptor combinations.


Polly and Molly  (born 1997), two  ewes , were the first  mammals  to have been successfully  cloned  from an adult somatic  cell  and to be  transgenic  animals at the same time. [ 1 ]  This is not to be confused with  Dolly the Sheep , the first animal to be successfully cloned from an adult somatic cell where there wasn’t modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the  Roslin Institute  in  Edinburgh ,  Scotland .


The creation of Polly and Molly built on the somatic nuclear transfer experiments that led to the cloning of Dolly the Sheep. The crucial difference was that in creating Polly and Molly, scientists used cells into which a new gene had been inserted. The gene chosen was a therapeutic protein to demonstrate the potential of such recombinant DNA technology combined with animal cloning. This could hopefully be used to produce pharmacological and therapeutic proteins to treat human diseases. The protein in question was the human blood clotting  factor IX . [ 2 ]  Another difference from Dolly the Sheep was the source  cell type  of the nucleus that was transferred. Although Polly and Molly were nuclear clones, they had different  mtDNA  that was different from the nuclear cells where they received their DNA. [ 3 ]


Prior to the production of Polly and Molly, the only demonstrated way to make a transgenic animal was by  microinjection  of DNA into the  pronuclei  of fertilized  oocytes  (eggs). However, only a small proportion of the animals will integrate the injected DNA into their genome. In the rare cases that they do integrate this new genetic information, the pattern of expression of the injected transgene's protein due to the random integration is very variable. As the aim of such research is to produce an animal that expresses a particular protein in high levels in, for example, its milk, microinjection is a very costly procedure that does not usually produce the desired animal.


In mice, there is an additional option for genetic transfer that is not available in other animals.  Embryonic stem cells  provide a means to transfer new DNA into the  germline . They also allow precise genetic modifications by  gene targeting . Modified embryonic stem cells can be selected  in vitro  before the experiment moves on further for the production of an animal. Embryonic stem cells capable of contributing to the germline of livestock species such as sheep have not been isolated.


The production of Dolly the Sheep and also  Megan and Morag , the two sheep that led to the production of Dolly, demonstrated that viable sheep can be produced by  nuclear transfer  from a variety of  somatic cell  types which have been cultured in vitro. Polly and Molly represented the further step in which somatic cells were cultured in vitro, just as in the case with the previous sheep. However, in this case they were transfected with foreign DNA, and the transfected cells which stably integrated this new piece of genetic information were selected. The nuclei of these somatic cells was then transferred into an empty oocyte, as in the procedure of nuclear transfer, and this was used to produce several transgenic animals. A cell type PDFF was used. PDFF5 would produce male animals and were not transduced. Cell type PDFF2 produced female animals and were transduced. Of the gestations that occurred, three PDFF2 animals were born, two of which survived birth, 7LL8 and 7LL12. These animals were transfected but contained a marker gene  not  the cloned gene of interest. These were named "Holly" and "Olly". [ 4 ]  Two more subsets of female-producing PDFF2 cells, PDFF2-12 and PDFF2-13, also produced animals which had the cell of interest together with the marker. Of these lambs, 7LL12, 7LL15, and 7LL13 were born alive and healthy. Two of these were named Polly and Molly.


The transgene that was inserted in the donor somatic cells was designed to express the human clotting  factor IX  protein in the milk of sheep. This protein plays an essential role in blood coagulation, and deficiency leads to the disease  haemophilia B  of which treatment requires intravenous infusion of factor IX. The production of this protein in livestock milk, a process known as  pharming , would provide a source of this therapeutic protein that would reduce the cost and also would be free of potential infectious risk associated with the current source of this protein (human blood).


A  progenitor cell  is a  biological cell  that can  differentiate  into a specific cell type.  Stem cells  and progenitor cells have this ability in common. However, stem cells are less specified than progenitor cells. Progenitor cells can only differentiate into their "target" cell type. [ 1 ]  The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can divide only a limited number of times.  Controversy about the exact definition remains and the concept is still evolving. [ 2 ]


The terms "progenitor cell" and "stem cell" are sometimes equated. [ 3 ]


Most progenitors are identified as  oligopotent . In this point of view, they can compare to adult stem cells, but progenitors are said to be in a further stage of cell differentiation. They are "midway" between stem cells and fully differentiated cells. The kind of potency they have depends on the type of their "parent" stem cell and also on their niche. Some research found that progenitor cells were mobile and that these progenitor cells could move through the body and migrate towards the tissue where they are needed. [ 4 ]  Many properties are shared by adult stem cells and progenitor cells.


Progenitor cells have become a hub for research on a few different fronts. Current research on progenitor cells focuses on two different applications:  regenerative medicine  and cancer biology. Research on regenerative medicine has focused on progenitor cells, and stem cells, because their cellular  senescence  contributes largely to the process of aging. [ 5 ]  Research on cancer biology focuses on the impact of progenitor cells on cancer responses, and the way that these cells tie into the immune response. [ 6 ]


The natural aging of cells, called their cellular senescence, is one of the main contributors to aging on an organismal level. [ 7 ]  There are a few different ideas to the cause behind why aging happens on a cellular level.  Telomere  length has been shown to positively correlate to longevity. [ 8 ] [ 9 ]  Increased circulation of progenitor cells in the body has also positively correlated to increased longevity and regenerative processes. [ 10 ]   Endothelial progenitor cells  (EPCs) are one of the main focuses of this field. They are valuable cells because they directly precede endothelial cells, but have characteristics of stem cells. These cells can produce differentiated cells to replenish the supply lost in the natural process of aging, which makes them a target for aging therapy research. [ 11 ]  This field of regenerative medicine and aging research is still currently evolving.


Recent studies have shown that haematopoietic progenitor cells contribute to immune responses in the body. They have been shown to respond a range of  inflammatory cytokines . They also contribute to fighting infections by providing a renewal of the depleted resources caused by the stress of an infection on the immune system. Inflammatory cytokines and other factors released during infections will activate haematopoietic progenitor cells to differentiate to replenish the lost resources. [ 12 ]


The characterization or the defining principle of progenitor cells, in order to separate them from others, is  based on the different cell markers rather than their morphological appearance. [ 13 ]


Before embryonic day 40 (E40), progenitor cells generate other progenitor cells; after that period, progenitor cells produce only dissimilar mesenchymal stem cell daughters. The cells from a single progenitor cell form a proliferative unit that creates one cortical column; these columns contain a variety of neurons with different shapes. [ 20 ]


The  revival of the woolly mammoth  is a proposed  hypothetical  that frozen soft-tissue remains and  DNA  from  extinct   woolly mammoths  could be a means of regenerating the species. Several methods have been proposed to achieve this goal, including  cloning ,  artificial insemination , and  genome editing . Whether or not it is ethical to create a live mammoth is debated.


In 2003, the  Pyrenean ibex  was briefly revived, giving credence to the idea that the mammoth could be successfully revived.


In theory, preserved  genetic material  found in remains of  woolly mammoths  could be used to recreate living mammoths, due to advances in  molecular biology  techniques and the cloning of mammals, begun with  Dolly the Sheep  in 1996. [ 1 ] [ 2 ] [ 3 ]  Cloning of mammals has improved in the last two decades. To date, no viable mammoth tissue or its intact genome has been found to attempt cloning.


According to  Beth Shapiro , a scientist who has taken a central role in the sequencing of the mammoth genome, states in her 2015 book  How to Clone a Mammoth: The Science of De-Extinction , that a mammoth will never be cloned, at least not one that is pure mammoth. Nevertheless, the book concludes that we are likely, at some point, to see something that resembles a mammoth. [ 4 ]   Comparative genomics  shows that the mammoth genome matches 99% of the elephant genome, so researchers working in the field aim to engineer an elephant with mammoth genes, that code for the external appearance and traits of a mammoth. [ 5 ]  The outcome would be an elephant-mammoth hybrid with no more than 1% mammoth genes. [ 5 ]  Separate projects are working on gradually adding mammoth genes to elephant cells  in vitro . [ 1 ] [ 2 ] [ 6 ]


Colossal Biosciences , founded in 2021, is one  biotechnology  company that has publicly stated that its project is to genetically resurrect the  woolly mammoth , combining its genes with  Asian elephant   DNA . It has publicly stated that it intends to complete the project by 2027. [ 7 ] [ 8 ] [ 9 ] [ 10 ]


Cloning  involves removal of the DNA-containing  nucleus  of the  egg cell  of a female elephant, and replacement with a nucleus from woolly mammoth tissue, a process called  somatic cell nuclear transfer . For example, Akira Iritani, at the Kyoto University in Japan, reportedly planned to do this. [ 11 ]  The cell would then be stimulated into dividing, and implanted in a female elephant. The resulting calf would have the genes of the woolly mammoth. However, nobody as of date has found a viable mammoth cell to begin the cloning process, and most scientists doubt that any living cell could have survived freezing in the tundra of the  Arctic . [ 12 ] [ 13 ] [ 14 ] [ 15 ]  Because of their conditions of preservation, the DNA of frozen mammoths has deteriorated significantly over the millennia. [ 16 ] [ 17 ]


A second method involves  artificially inseminating  an elephant egg cell with  sperm  cells from a frozen woolly mammoth carcass. The resulting offspring would be an elephant–mammoth hybrid, and the process would have to be repeated, so more hybrids could be used in breeding. After several generations of cross-breeding these hybrids, an almost pure woolly mammoth would be produced. Whether the hybrid embryo would be carried through the two-year gestation is unknown; in one case, an Asian elephant and an African elephant produced a live calf named  Motty , but it died of defects at less than two weeks old. [ 18 ]  There is also another fact to consider, that sperm cells of modern mammals are viable for 15 years at most after deep-freezing. This makes this method unfeasible. [ 17 ]


In April 2015, Swedish scientists published the complete  genome  ( nuclear DNA  sequence) of the woolly mammoth. [ 1 ] [ 19 ]  Several projects are working on gradually replacing the genes in  elephant  cells with mammoth genes. [ 1 ] [ 2 ]  One such project is that of  Harvard University  geneticist  George M. Church , who is funded by the  Long Now Foundation , [ 1 ] [ 2 ]  is attempting to create a mammoth–elephant hybrid using DNA from frozen mammoth carcasses. According to the researchers, a mammoth cannot be recreated, but they will try to eventually grow a hybrid elephant with some woolly mammoth traits in an "artificial womb". [ 20 ] [ 21 ]
In 2017, George Church said "Actually it would be more like an elephant with a number of mammoth traits. We're not there yet, but it could happen in a couple of years." The creature, sometimes referred as a "mammophant", would be partly elephant, but with features such as small ears, subcutaneous fat, long shaggy hair and cold-adapted blood. [ 22 ]  The Harvard University team is attempting to study the animals' characteristics  in vitro  by replacing or  editing  some specific mammoth genes into Asian elephant skin cells called  fibroblasts  that have the potential to become  embryonic stem cells . [ 23 ]  By March 2015 and using the new  CRISPR  DNA editing technique, Church's team had some woolly mammoth genes edited into the genome of an Asian elephant; focusing on cold-resistance initially, [ 12 ]  the target genes are for the external ear size, subcutaneous fat,  hemoglobin , and hair attributes. [ 23 ] [ 24 ]  By February 2017, Church's team had made 45 substitutions to the elephant genome. [ 20 ]  So far his work focuses solely on single cells. In 2021, Church received $15 million in funding and spun off a new company called Colossal. [ 25 ]


The Mammoth Genome Project at  Pennsylvania State University  is also researching the modification of African elephant DNA to create a mammoth–elephant hybrid. [ 26 ]  If a viable hybrid embryo is obtained by  gene editing  procedures, implanting it into a female Asian elephant housed in a zoo may be possible, [ 1 ]  but with the current knowledge and technology, whether the hybrid embryo would be carried through the two-year gestation is unknown. [ 27 ] [ 28 ]


If any method is ever successful, a suggestion has been made to introduce the hybrids to a wildlife reserve in  Siberia  called the  Pleistocene Park , [ 29 ]  but some biologists question the ethics of such recreation attempts. [ 30 ]  In addition to the technical problems, not much habitat is left that would be suitable for mammoth–elephant hybrids. Because both species are [were] social and  gregarious , creating a few specimens would not be ideal. The time and resources required would be enormous, and the scientific benefits would be unclear, suggesting these resources should instead be used to preserve extant elephant species which are endangered. [ 17 ] [ 31 ]  The ethics of using elephants as surrogate mothers in hybridisation attempts has also been questioned, as most embryos would not survive, and knowing the exact needs of a hybrid mammoth–elephant calf would be impossible. [ 14 ]


Researchers from the company Colossal confirmed that their primary goal when trying to revive the woolly mammoth is to better the environment and  climate change  itself. [ 32 ]


The  Roslin Institute  is an  animal sciences  research institute at Easter Bush,  Midlothian , Scotland, part of the  University of Edinburgh , and is funded by the  Biotechnology and Biological Sciences Research Council .


It is best known for creating  Dolly the sheep  in 1996, the first  mammal  to be successfully  cloned  from an adult cell.


The Roslin Institute has its roots in the University of Edinburgh's Institute of Animal Genetics (IAG), which was founded in 1917 under the direction of  Francis Albert Eley Crew . [ 1 ]


The Poultry Research Centre (PRC) was founded in 1947 by the  Agricultural Research Council (ARC) . [ 2 ]  The new institute used expertise and material from the IAG, and its laboratories were located adjacent to the IAG's building on the university's  King's Buildings  campus. A second site housing larger experiments was located on the Bush Estate, south of Edinburgh. [ 3 ]


In 1971, the institute's experimental facility moved from the Bush Estate to a larger site near the village of  Roslin , and the main laboratories moved to the same site in 1980. [ 3 ]


The Animal Breeding Research Organisation (ABRO) was founded at the same time as the PRC in 1947, again using the IAG's expertise. Its research focused mainly on genetic improvement of cattle, pigs and sheep. [ 4 ]


In the 1980s, under the direction of John King and  Roger Land , ABRO's research began a shift towards  molecular biology , which was key in laying the groundwork for the institute's work on cloning in the 1990s. [ 5 ]


In 1986, the Poultry Research Centre and the Animal Breeding Research Organisation merged with the Institute of Animal Physiology, based in  Babraham , Cambridgeshire, to form the Institute of Animal Physiology and Genetics Research (IAPGR). [ 4 ]  The PRC's buildings in Roslin became the IAPGR's Edinburgh Research Station, with the former ABRO facilities progressively relocating there between 1986 and 1989. [ 6 ]


The IAPGR's sites at Babraham and Roslin became two independent institutes owned by the  Biotechnology and Biological Sciences Research Council  in 1993 – the  Babraham Institute  and the Roslin Institute. Animal genetics research had been gradually consolidating on the Roslin site since 1986, [ 6 ]  and all agricultural research at Babraham had ceased by 1998.


The institute became a company limited by guarantee and a charity registered in Scotland, with the BBSRC as its sponsor, in 1995. [ 7 ]


In 2006, the BBSRC announced that the institute would move to a new site on the University of Edinburgh's Easter Bush campus, under the direction of David Hume. [ 8 ]  As part of the plans, the Roslin Institute merged with the Neuropathogenesis Unit of the  Institute for Animal Health , well known for its role in deciphering the biology of  transmissible spongiform encephalopathies  and this was headed by  Jean Manson .


In April 2008, the combined institute became part of the University of Edinburgh's  Royal (Dick) School of Veterinary Studies , and the institute's 197 staff members became University of Edinburgh employees on 1 May. [ 7 ] [ 9 ]  The move to Easter Bush was completed in March 2011. [ 10 ]  Under the original plans, the new institute was to be known as EBRC, [ 11 ]  but the institute ultimately retained the Roslin name.


In February 2020, Bruce Whitelaw became interim director of the institute, replacing  Eleanor Riley , who had been director since 2017. [ 12 ]


In 1996, the institute won international fame when  Ian Wilmut ,  Keith Campbell , and their colleagues created  Dolly the sheep , the first  mammal  to be successfully  cloned  from an adult cell, at the institute. [ 13 ] [ 14 ] [ 15 ]  A year later, two other sheep named  Polly and Molly  were cloned, each of which contained a human gene.


Roslin has made many other contributions to animal science and biotechnology research, especially in the area of livestock improvement and welfare through the application of  quantitative genetics . In 2007, a Roslin team developed  genetically modified  chickens capable of laying eggs containing proteins needed to make cancer-fighting drugs.


The Roslin Institute aims to enhance the lives of animals and humans through world-class research in animal biology. The principal objectives are to:


Research at the Roslin Institute is categorised into four scientific divisions: [ 16 ]


Three Institute Strategic Programmes, which are funded by the  Biotechnology and Biological Sciences Research Council , span the Divisions of the institute.


S1909/A2840  is a bill that was passed by the  New Jersey Legislature  in December 2003, and signed into law by Governor  James McGreevey  on January 4, 2004, that permits human  cloning  for the purpose of developing and harvesting human  stem cells .  Specifically, it legalizes the process of cloning a human embryo, and implanting the clone into a womb, provided that the clone is then  aborted  and used for medical research. The legislation was sponsored by Senators  Richard Codey  (D-Essex) and  Barbara Buono , and Assembly members  Neil M. Cohen  (D-Union),  John F. McKeon ,  Mims Hackett  (D-Essex), and  Joan M. Quigley  (D-Hudson). The enactment of this law will enable researchers to find cures for debilitating and deadly diseases.


Supporters of the legislation hailed it as promoting medical progress through science, giving hope for the development of treatments for debilitating diseases such as Parkinson's disease,  Alzheimer's disease ,  cancer , and  diabetes . Assemblyman Neil Cohen lauded it as "not the most significant law we'll write this session—but this century."  Paralyzed actor  Christopher Reeve , who believed that such legislation may hasten the development of methods to reverse paralysis, testified in support of the bill.


However, Congressmen  Chris Smith ,  Mike Ferguson , and  Scott Garrett  assailed it, saying, "This legislation will launch New Jersey blindly into the vanguard of terrible human-rights violations and grisly human experimentation."  They also claim that, in practice, once a clone is developing in a womb, there is nothing that will prevent it from leading to "the world's first human clone being born and starting a horrible new era of human history."  New Jersey's  Catholic  bishops condemned the newly legalized process as violating "a central tenet of all civilized codes on human experimentation beginning with the  Nuremberg Code ...[It approves] doing deadly harm to a member of the human species solely for the sake of potential benefit to others."


SCNT is currently legal for research purposes in the United Kingdom, having been incorporated into the 1990 Human Fertilisation and Embryology Act in 2001. Permission must be obtained from the Human Fertilisation 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.


In 2005, the United Nations adopted a proposal submitted by Costa Rica, calling on member states to "prohibit all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life." This phrase 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 Dignity of the Human Being with regard to the Application of Biology and Medicine, on the Prohibition of Cloning Human Being appear to ban SCNT. Of the council's 45 member states, the convention has been signed by 31 and ratified by 18. The Additional Protocol has been signed by 29 member nations and ratified by 14.


Richard Griffith Seed  (May 22, 1928 – November 17, 2013) was an American physicist and businessman best known for forcing a national debate on  human cloning  in the late 1990s. [ 1 ]


Seed was born in  Chicago, Illinois  on May 22, 1928. He graduated from  Oak Park and River Forest High School  in Illinois before attending  Harvard University , earning his undergraduate degree there in 1949. [ 2 ]  He later received a master's degree, as well as a Ph.D. in physics from Harvard in 1953. His interests soon shifted to the new frontier of  biomedicine . In the 1970s Seed co-founded a company that commercialized a technique for transferring embryos in cattle. Later, he and his brother, Chicago surgeon Randolph Seed, started another company, Fertility & Genetics Research Inc., to help infertile women conceive children using the same technique. [ 3 ]  These efforts to transplant a  human embryo  from one woman to an infertile  surrogate mother  were published in 1984. The cumbersome procedure involved flushing embryos out of the  uterus  of the egg donor—and was soon eclipsed by  in-vitro fertilization . Ultimately the venture failed.


On December 5, 1997, Seed announced that he planned to clone a human being before any federal laws could be enacted to ban the process. [ 4 ]  Seed's announcement added fuel to the raging ethical debate on human cloning that had been sparked by  Ian Wilmut 's creation of  Dolly the sheep , the first clone obtained from adult cells. [ 5 ]  Seed's plans were to use the same technique used by the Scottish team. [ 6 ]  Seed's announcement went against President Clinton's 1997 proposal for a voluntary private moratorium against human cloning. In the media frenzy that followed, the story of a 69-year-old eccentric, and maverick scientist emerged, but Seed possessed impressive credentials and was not dismissed immediately. [ 7 ]  While virtually no mainstream scientist believed Seed would succeed, there began a subtle shift in attitudes after Seed made his announcement. [ 8 ]


Retired at the time of his announcement to clone the first human, Seed was reported to have dabbled in ill-fated ventures in the past. He claimed at one time to have commitments for $800,000 toward a goal of $2.5 million needed to clone the first human before 2000. Seed first said that he was going to make little baby clones for infertile couples. Later, "to defuse criticism that I'm taking advantage of desperate women," he announced that he would first clone himself. Still later he announced that he would re-create his wife Gloria. "God made man in his own image," he told National Public Radio correspondent  Joe Palca  in December 1997. "God intended for man to become one with God. Cloning, is the first serious step in becoming one with God." In a later interview on CNN, Seed elaborated: "Man," he said, "will develop the technology and the science and the capability to have an indefinite life span."


Seed was awarded the 1998  Ig Nobel Prize  in economics, [ 9 ]  and a performance titled  The Seedy Opera  debuted at the event. [ 10 ]


Richard Seed died in  Hobart, Indiana  on November 17, 2013, at the age of 85. [ 11 ]


The  Shanghai Stem Cell Institute  is an institute in  Shanghai ,  People's Republic of China  dedicated to  stem cell research .


The institute, located within the  Shanghai Jiao Tong University  under the  School of Medicine  faculty, is entirely funded by the government of the People's Republic of China. [ 1 ]


In 2007, the first Shanghai International Symposium on Stem Cell Research took place at Shanghai Jiatong University. [ citation needed ]


On July 24, 2009, the first publication of a successful breakthrough in  Stem cell research  was released, [ 2 ] [ 3 ]  where Chinese researchers from the Shanghai Stem Cell Institute, led by Professor Fanyi Zeng, successfully reprogrammed  adult stem cells  to be able to differentiate into any body cell, as in the case with standard  embryonic stem cells , the cells in question known as " induced pluripotent stem cells " (IPS cells). The IPS cells were obtained by genetically reprogramming the skin cells of mice to acts like  embryonic stem cells , which then were able to differentiate into all forms of body tissue. [ 4 ]  The researchers have managed to use the IPS cells to create every type of cell in a mouse, creating entire mouse pups using the technique. This is the first time the technique has been used to make an entire mouse.


This breakthrough, published in the journals  Nature  and  Cell Stem Cell  and developed independently by two teams in China, may possibly depreciate the usage of stem cells obtained from human embryos. [ 5 ]  The oldest living mice created by the technique are nine months old and are reproducing, albeit showing signs of abnormalities. "This gives us hope for future therapeutic intervention using patients' own re-programmed cells in our far future," according to Professor  Zeng Fanyi . [ 5 ]  A total of 27 mice were successfully born from the first generation of mice created from the IPS cells which were able to reproduce without any issues. [ 4 ]


Sinogene Biotechnology  is a Chinese biotechnology company, focusing on  animal cloning  technology for consumers. [ 1 ] [ 2 ]  Their pet cloning services include: dog, [ 3 ] [ 4 ]  cat, [ 5 ] [ 6 ]  cow, and horse cloning. [ 7 ]


In 2022, Sinogene made history by being the first to clone a wild Arctic wolf. [ 8 ]


Sinogene Biotechnology began offering dog cloning services in 2017 [ 3 ] [ 4 ] [ 9 ]  and later introduced cat cloning in 2019. [ 5 ] [ 6 ] [ 10 ]


In 2022, Sinogene became the first company to successfully clone an  Arctic wolf , [ 11 ]  and started horse cloning in 2023. [ 12 ] [ 7 ]  The donor cell came from a wild female Arctic wolf, the  oocyte  was from a female dog, and the surrogate was a  beagle . [ 8 ]  The company transferred 85  embryos  into seven beagles and one Arctic wolf was born. [ 13 ]  In June of the same year, Sinogene cloned a male horse using skin cells from a horse born in 1995. [ 14 ]


Sinogene partnered with Beijing Wildlife Park in 2022 to work together on improving breeding for endangered animals as well as improving ways to protect endangered animals. [ 13 ] [ 15 ]


Sinogene clients can harvest cells from their living pets, to one day use in the cloning process after their pet dies. [ 7 ]  Customers receive their cloned animals three months after they are born. [ 7 ]


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In  cellular biology , a  somatic cell  (from  Ancient Greek     σῶμα  (sôma)  'body'), or  vegetal cell , is any  biological cell  forming the body of a  multicellular organism  other than a  gamete ,  germ cell ,  gametocyte  or undifferentiated  stem cell . [ 1 ]  Somatic cells compose the body of an organism and divide through  mitosis .


In contrast,  gametes  derive from  meiosis  within the  germ cells  of the  germline  and they fuse during  sexual reproduction .  Stem cells  also can divide through  mitosis , but are different from somatic in that they  differentiate  into diverse specialized cell types.


In  mammals , somatic cells make up all the internal organs, skin, bones, blood and  connective tissue , while mammalian germ cells give rise to  spermatozoa  and  ova  which fuse during  fertilization  to produce a cell called a  zygote , which divides and differentiates into the cells of an  embryo . There are approximately 220 types of somatic cell in the human body. [ 1 ]


Theoretically, these cells are not germ cells (the source of gametes); they transmit their  mutations , to their cellular descendants (if they have any), but not to the organism's descendants. However, in  sponges , non-differentiated somatic cells form the germ line and, in  Cnidaria , differentiated somatic cells are the source of the germline. Mitotic cell division is only seen in  diploid  somatic cells. Only some cells like germ cells take part in reproduction. [ 2 ]


As  multicellularity  was theorized to be evolved many times, [ 3 ]  so did sterile somatic cells. [ citation needed ]  The evolution of an immortal  germline  producing specialized somatic cells involved the emergence of  mortality , and can be viewed in its simplest version in  volvocine  algae. [ 4 ]  Those species with a separation between sterile somatic cells and a germline are called  Weismannists . Weismannist development is relatively rare (e.g.,  vertebrates ,  arthropods ,  Volvox ), as many species have the capacity for  somatic embryogenesis  (e.g.,  land plants , most  algae , and numerous  invertebrates ). [ 5 ] [ 6 ]


Like all cells, somatic cells contain  DNA  arranged in  chromosomes . If a somatic cell contains chromosomes arranged in pairs, it is called  diploid  and the organism is called a diploid organism. The gametes of diploid organisms contain only single unpaired chromosomes and are called  haploid . Each pair of chromosomes comprises one chromosome inherited from the father and one inherited from the mother. In humans, somatic cells contain 46  chromosomes  organized into 23 pairs. By contrast, gametes of diploid organisms contain only half as many chromosomes. In humans, this is 23 unpaired chromosomes. When two gametes (i.e. a spermatozoon and an ovum) meet during conception, they fuse together, creating a  zygote . Due to the fusion of the two gametes, a human zygote contains 46 chromosomes (i.e. 23 pairs). [ citation needed ]


A large number of  species  have the chromosomes in their somatic cells arranged in fours (" tetraploid ") or even sixes (" hexaploid "). Thus, they can have diploid or even triploid germline cells. An example of this is the modern cultivated species of  wheat ,  Triticum aestivum L. , a hexaploid species whose somatic cells contain six copies of every  chromatid . [ citation needed ]


The frequency of spontaneous  mutations  is significantly lower in advanced male  germ cells  than in somatic cell types from the same individual. [ 7 ]  Female germ cells also show a mutation frequency that is lower than that in corresponding somatic cells and similar to that in male germ cells. [ 8 ]  These findings appear to reflect employment of more effective mechanisms to limit the initial occurrence of spontaneous mutations in germ cells than in somatic cells. Such mechanisms likely include elevated levels of  DNA repair  enzymes that ameliorate most potentially mutagenic  DNA damages . [ 8 ]


In recent years, the technique of  cloning  whole organisms has been developed in mammals, allowing almost identical genetic clones of an animal to be produced. One method of doing this is called " somatic cell nuclear transfer " and involves removing the  nucleus  from a somatic cell, usually a skin cell. This nucleus contains all of the genetic information needed to produce the organism it was removed from. This nucleus is then injected into an  ovum  of the same species which has had its own genetic material removed. [ 9 ]  The ovum now no longer needs to be fertilized, because it contains the correct amount of genetic material (a  diploid  number of  chromosomes ). In theory, the ovum can be implanted into the  uterus  of a same-species animal and allowed to develop. The resulting animal will be a nearly genetically identical clone to the animal from which the nucleus was taken. The only difference is caused by any  mitochondrial  DNA that is retained in the ovum, which is different from the cell that donated the nucleus. In practice, this technique has so far been problematic, although there have been a few high-profile successes, such as  Dolly the Sheep  (July 5, 1996 - February 14, 2003) [ 10 ]  and, more recently,  Snuppy  (April 24, 2005 - May 2015), the first cloned  dog . [ 11 ]


Somatic cells have also been collected in the practice of biobanking. The  cryoconservation of animal genetic resources  is a means of conserving animal genetic material in response to decreasing ecological biodiversity. [ 12 ]  As populations of living organisms fall so does their genetic diversity. This places species long-term survivability at risk. Biobanking aims to preserve biologically viable cells through long-term storage for later use. Somatic cells have been stored with the hopes that they can be reprogrammed into induced pluripotent stem cells (iPSCs), which can then differentiate into viable reproductive cells. [ 13 ]


Development of  biotechnology  has allowed for the genetic manipulation of somatic cells, whether for the modelling of chronic disease or for the prevention of malaise conditions. [ 14 ] [ 15 ]  Two current means of gene editing are the use of  transcription activator-like effector nucleases  (TALENs) or  clustered regularly interspaced short palindromic repeats  (CRISPR). [ citation needed ]


Genetic engineering of somatic cells has resulted in some  controversies , [ 16 ]  although the International Summit on Human Gene Editing has released a statement in support of genetic modification of somatic cells, as the modifications thereof are not passed on to offspring. [ 17 ]


In mammals a high level of repair and maintenance of cellular DNA appears to be beneficial early in life.  However, some types of cell, such as those of the brain and muscle, undergo a transition from mitotic cell division to a post-mitotic (non-dividing) condition during early development, and this transition is accompanied by a reduction in  DNA repair  capability. [ 18 ] [ 19 ] [ 20 ]  This reduction may be an evolutionary adaptation permitting the diversion of cellular resources that were earlier used for DNA repair, as well as for  DNA replication  and  cell division , to higher priority neuronal and muscular functions.  An effect of these reductions is to allow increased accumulation of  DNA damage  likely contributing to cellular aging.


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 a  denucleated   oocyte  (egg cell) and implanting a donor  nucleus  from a somatic (body) cell. It is used in both therapeutic and reproductive  cloning . In 1996,  Dolly the sheep  became famous for being the first successful case of the reproductive cloning of a mammal. [ 1 ]  In January 2018, a team of scientists in  Shanghai  announced the successful cloning of two female  crab-eating macaques  (named  Zhong Zhong and Hua Hua ) from foetal nuclei. [ 2 ]


" 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  (ESCs) and the destruction of viable embryos for medical use, though questions remain on how  homologous  the two cell types truly are.


Somatic cell nuclear transfer is a technique for cloning in which the nucleus of a somatic cell is transferred to the cytoplasm of an enucleated egg. After the somatic cell transfers, the cytoplasmic factors affect the nucleus to become a zygote. The blastocyst stage is developed by the egg to help create embryonic stem cells from the inner cell mass of the blastocyst. [ 3 ]  The first mammal to be developed by this technique was Dolly the sheep, in 1996. [ 4 ]


Although Dolly is generally recognized as the first animal to be cloned using this technique, earlier instances of SCNT exist as early as the 1950s. In particular, the research of  Sir John Gurdon  in 1958 entailed the cloning of  Xenopus laevis  utilizing the principles of SCNT. [ 5 ]  In short, the experiment consisted of inducing a female specimen to ovulate, at which point her eggs were harvested. From here, the egg was enucleated using ultra-violet irradiation to disable the egg's pronucleus. At this point, the prepared egg cell and nucleus from the donor cell were combined, and then incubation and eventual development into a tadpole proceeded. [ 5 ]  Gurdon's application of SCNT differs from more modern applications and even applications used on other model systems of the time (i.e.,  Rana pipiens ) due to his usage of UV irradiation to enucleate the egg instead of using a pipette to remove the nucleus from the egg. [ 6 ]


The process of somatic cell nuclear transfer involves two different cells. The first being a female gamete, known as the ovum (egg/oocyte). In human SCNT experiments, 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 genetic material of the donor egg cell is removed and discarded, leaving it 'deprogrammed.' What is left is a somatic cell and an enucleated egg cell. These are then fused by inserting the somatic cell into the 'empty' ovum. [ 7 ]  After being inserted into the egg, the somatic cell nucleus is  reprogrammed  by its host egg cell. The ovum, now containing the somatic cell's 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 necessary genetic information from just one parent. Development will ensue normally and after many mitotic divisions, the single cell forms a  blastocyst  (an early stage  embryo  with about 100 cells) with an identical genome to the original organism (i.e. a clone). [ 8 ]  Stem cells can then be obtained by the destruction of this clone embryo for use in therapeutic cloning or in the case of reproductive cloning the clone embryo is implanted into a host mother for further development and brought to term.


Conventional SCNT requires the use of  micromanipulators , which are expensive machines used to accurately manipulate cells. [ 9 ]  Using the micromanipulator, a scientist makes an opening in the  zona pellucida  and sucks out the egg cell's original nucleus using a pipette. They then make another opening to a different pipette to inject the donor nucleus. [ 10 ]  Alternatively, electric energy can be applied to fuse the empty egg cell with a donor cell containing a nucleus. [ 9 ]


An alternative technique called "handmade cloning" was described by Indian scientists in 2001. This technique requires no use of a micromanipulator and has been used for the cloning of several livestock species. [ 11 ]  Removal of the nucleus can be done chemically, by centrifuge, or with the use of a blade. The empty egg is glued to the donor cell with  phytohaemagglutinin , then fused using electricity. (If a blade is used, two fusion steps would be required: the first fusion is between the donor and an empty half-egg, the second between the half-size "demi-embryo" and another empty half-egg.) [ 9 ]


Somatic cell nuclear transplantation has become a focus of study in  stem cell research . The aim of carrying out 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 patient specific pluripotent cells, which could then be used in therapies or disease research. [ 12 ]


Embryonic stem cells  are undifferentiated cells of an embryo. These cells are deemed to have a pluripotent potential because they have the ability to give rise to all of the tissues found in an adult organism. This ability allows stem cells to create any cell type, which could then be transplanted to replace damaged or destroyed cells.  Controversy  surrounds human ESC work due to the destruction of viable human embryos, leading scientists to seek alternative methods of obtaining pluripotent stem cells, SCNT is one such method.


A potential use of stem cells genetically matched to a patient would be to create cell lines that have genes linked to a patient's particular disease. By doing so, an  in vitro  model could be created, would be useful for studying that particular disease, potentially discovering its pathophysiology, and discovering therapies. [ 13 ]  For example, if a person with  Parkinson's disease  donated their somatic cells, the stem cells resulting from SCNT would have genes that contribute to Parkinson's disease. The disease specific stem cell lines could then be studied in order to better understand the condition. [ 14 ]


Another application of SCNT stem cell research is using the patient specific stem cell lines to generate tissues or even organs for transplant into the specific patient. [ 15 ]  The resulting cells would be genetically identical to the somatic cell donor, thus avoiding any complications from  immune system rejection . [ 14 ] [ 16 ]


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 , [ 17 ]   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 . [ 18 ]  In the  United Kingdom , the  Human Fertilisation and Embryology Authority  has granted permission to research groups at the  Roslin Institute  and the  Newcastle Centre for Life . [ 19 ]  SCNT may also be occurring in China. [ 20 ]


Though there has been numerous successes with cloning animals, questions remain concerning the mechanisms of reprogramming in the ovum. Despite many attempts, success in creating human nuclear transfer embryonic stem cells has been limited. There lies a problem in the human cell's ability to form a blastocyst; the cells fail to progress past the eight cell stage of development. This is thought to be a result from the somatic cell nucleus being unable to turn on embryonic genes crucial for proper development. These earlier experiments used procedures developed in non-primate animals with little success.


A research group from the  Oregon Health & Science University  demonstrated SCNT procedures developed for primates successfully using skin 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 enucleated eggs are hampered in their reprogramming ability. It is theorized the critical embryonic genes are physically 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.


Taking this into account the research group applied their new technique in an attempt to produce human 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 improved SCNT procedure, human clone embryos were successfully produced. These embryos were of poor quality, lacking a substantial inner cell mass and poorly constructed  trophectoderm . The imperfect embryos prevented the acquisition of human ESC. The addition of caffeine during the removal of the ovum's nucleus and fusion of the somatic cell and the egg improved blastocyst formation and ESC isolation. The ESC obtain were found to be capable of producing teratomas, expressed pluripotent transcription factors, and expressed a normal 46XX karyotype, indicating these SCNT were in fact ESC-like. [ 17 ]  This was the first instance of successfully using SCNT to reprogram human somatic cells. This study used fetal and infantile somatic cells to produce their ESC.


In April 2014, an international research team expanded on this break through. There remained the question of whether the same success could be accomplished using adult somatic cells. Epigenetic and age related changes were thought to possibly hinder an adult somatic cells ability to be reprogrammed. Implementing the procedure pioneered by the Oregon research group they indeed were able to grow stem cells generated by SCNT using adult cells from two donors aged 35 and 75, indicating that age does not impede a cell's ability to be reprogrammed. [ 21 ] [ 22 ]


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 type 1 diabetic. 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 successfully formed. The most significant of these cells, were those who expressed insulin and were capable of secreting the hormone. [ 23 ]  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 decreased by the development and improvement of alternative methods of generating stem cells. Methods to reprogram normal body cells into  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 cell lines that have all genes linked to various diseases. [ 24 ]  Some scientists working on SCNT-based stem cell research have recently moved to the new methods of induced pluripotent stem cells. Though recent studies have put in question how similar iPS cells are to 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 using only the yamanaka factors will be more efficient at differentiating into blood cells, while it will be less efficient at creating a neuron. [ 25 ]  Recent studies indicate however that changes to the epigenetic memory of iPSCs using small molecules can reset them to an almost naive state of pluripotency. [ 26 ] [ 27 ]  Studies have even shown  that via tetraploid complementation, an entire viable organism can be created solely from iPSCs. [ 28 ]  SCNT stem cells have been found to have similar challenges. The cause for low yields in bovine SCNT cloning has, in recent years, been attributed to the previously hidden epigenetic memory of the somatic cells that were being introduced into the oocyte. [ 29 ]


This technique is currently the basis for  cloning  animals (such as the famous  Dolly the sheep ), [ 30 ]  and has been 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, reproductive cloning in humans is proscribed in more than 30 countries. [ 31 ]  Most researchers believe that in the foreseeable future it will not be possible to use the current cloning technique to produce a human clone that will develop to term. It remains a possibility, though critical adjustments will be required to overcome current limitations during early embryonic development in human SCNT. [ 32 ] [ 33 ]


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 into 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 father providing a sperm cell, one mother providing the egg nucleus, and another mother providing the enucleated egg cell. [ 15 ]


In 2018, the first successful  cloning  of  primates  using somatic cell nuclear transfer, the same method as  Dolly the sheep , with the birth of two live female clones ( crab-eating macaques  named  Zhong Zhong  and  Hua Hua ) was reported. [ 2 ] [ 34 ] [ 35 ] [ 36 ] [ 37 ]


Interspecies nuclear transfer (iSCNT) is a means of somatic cell nuclear transfer being used to facilitate the rescue of endangered species, or even to restore species after their 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 and a donor 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 successfully with a domestic cow,  Bos taurus . [ 38 ]


In 2017, the first cloned  Bactrian camel  was born through iSCNT, using oocytes of  dromedary camel  and skin fibroblast cells of an adult Bactrian camel as donor nuclei. [ 39 ]


Somatic cell nuclear transfer (SCNT) can be inefficient due to stresses placed on both the egg cell and the introduced nucleus. This can result in a low percentage of successfully reprogrammed cells. For example, in 1996 Dolly the sheep was born after 277 eggs were used for SCNT, which created 29 viable embryos, giving it a measly 0.3% efficiency. [ 40 ]  Only three of these embryos survived until birth, and only one survived to adulthood. [ 30 ]  Millie, the offspring that survived, took 95 attempts to produce. [ 40 ]  Because the procedure was not automated and had to be performed manually under a  microscope , SCNT was very resource intensive. Another reason why there is such high mortality rate with the cloned offspring is due to the fetus being larger than even other large offspring, resulting in death soon after birth. [ 40 ]  The  biochemistry  involved in reprogramming the  differentiated  somatic cell nucleus and activating the recipient egg was also far from understood. Another limitation is trying to use one-cell embryos during the SCNT. When using just one-cell cloned embryos, the experiment has a 65% chance to fail in the process of making morula or blastocyst. The biochemistry also has to be extremely precise, as most late term cloned fetus deaths are the result of inadequate placentation. [ 40 ]  However, by 2014, researchers were reporting success rates of 70-80% with cloning pigs [ 41 ]  and in 2016 a Korean company, Sooam Biotech, was reported to be producing 500 cloned embryos a day. [ 42 ]


In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's  mitochondria  that contain their own  mitochondrial DNA  are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that 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 and organs for therapy, as there may be an immuno-response to the non-self mtDNA after transplant. Additionally, the genes found in the mitochondria’s genome need to communicate with the cell’s genome and a failure of somatic cell nuclear reprogramming can lead to non communication to the cell’s genome causing SCNT to fail. [ 43 ]


Epigenetic factors play an important role in the success or failure of SCNT attempts. The varying gene expression of a previously activated cell and its mRNAs may lead to overexpression, underexpression, or in some cases non functional genes which will affect the developing fetus. [ 44 ]  One such example of epigenetic limitations to SCNT is regulating histone methylation. Differing regulation of these histone methylation genes can directly affect the transcription of the developing genome, causing failure of the SCNT. [ 45 ]  Another contributing factor to failure of SCNT includes the X chromosome inactivation in early development of the embryo. A non coding gene called XIST is responsible for inactivating one X chromosome during development, however in SCNT this gene can have abnormal regulation causing mortality to the developing fetus. [ 45 ]


Nuclear transfer techniques present a different set of ethical considerations than those associated with the use of other stem cells like embryonic stem cells which are  controversial  for their requirement to destroy an embryo. These different considerations have led to some individuals and organizations who are  not  opposed to human embryonic stem cell research to be concerned about, or opposed to, SCNT research. [ 46 ] [ 47 ] [ 48 ]


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 transferred embryo, most likely via SCNT. Those who hold this concern often advocate for strong regulation of SCNT to preclude implantation of any derived products for the intention of human reproduction, [ 49 ]  or its prohibition. [ 46 ]


A second important concern is the appropriate source of the eggs that are needed. SCNT requires human  egg cells , which can only be obtained from women. The most common source of these eggs today are eggs that are produced and in excess of the clinical need during 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 custom stem cell line would consist of a collection of identical stem cells each carrying the patient's own DNA, thus reducing or eliminating any problems with rejection when the stem cells were transplanted for treatment. 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 donated eggs in order to successfully create a single custom therapeutic stem cell line. Such large numbers of donated eggs would exceed the number of eggs currently left over and available from couples trying to have children through  assisted reproductive technology . Therefore, healthy young women would need to be induced to sell eggs to be used in the creation of custom stem cell lines that could then 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 egg donations would occur in a developed country because of the unknown long-term public health effects of treating large numbers of healthy young women with heavy doses of hormones in order to induce hyper-ovulation (ovulating several eggs at once). Although such treatments have been performed for several decades now, the long-term effects have not been studied or declared safe to use on a large scale on otherwise healthy women. Longer-term treatments with much lower doses of hormones are known to increase the rate of cancer decades later. Whether hormone treatments to induce hyper-ovulation could have similar effects is unknown. There are also ethical questions surrounding paying for eggs. In general, marketing body parts is considered unethical and is banned in most countries. [ why? ]  Human eggs have been a notable exception to this rule for some time.


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.


SCNT involving human cells is currently legal for research purposes in the  United Kingdom , having been incorporated into the  Human Fertilisation and Embryology Act 1990 . [ 50 ] [ 7 ]  Permission must be obtained from the  Human Fertilisation 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. [ 51 ]  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 cannot be federally funded. [ 52 ]  American scholars have recently argued that because the product of SCNT is a clone embryo, rather than a human embryo, these policies are morally wrong and should be revised. [ 53 ]


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." [ 54 ]  This phrase 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 Dignity of the Human Being with regard to the Application of Biology and Medicine, on the Prohibition of Cloning Human Being  appear to ban SCNT of human beings. Of the Council's 45 member states, the  Convention  has been signed by 31 and ratified by 18. The  Additional Protocol  has been signed by 29 member nations and ratified by 14. [ 55 ]


In  multicellular organisms ,  stem cells  are  undifferentiated  or partially differentiated  cells  that can change into various  types of cells  and  proliferate  indefinitely to produce more of the same stem cell. They are the earliest type of cell in a  cell lineage . [ 1 ]  They are found in both  embryonic  and adult organisms, but they have slightly different properties in each. They are usually distinguished from  progenitor cells , which cannot divide indefinitely, and  precursor  or blast cells, which are usually committed to differentiating into one cell type.


In  mammals , roughly 50 to 150 cells make up the  inner cell mass  during the  blastocyst  stage of  embryonic development , around days 5–14. These have stem-cell capability.  In vivo , they eventually differentiate into all of the body's cell types (making them  pluripotent ). This process starts with the differentiation into the three  germ layers  – the  ectoderm ,  mesoderm  and  endoderm  – at the  gastrulation  stage. However, when they are isolated and  cultured   in vitro , they can be kept in the stem-cell stage and are known as  embryonic stem cells  (ESCs).


Adult stem cells  are found in a few select locations in the body, known as  niches , such as those in the  bone marrow  or  gonads . They exist to replenish rapidly lost cell types and are  multipotent  or unipotent, meaning they only differentiate into a few cell types or one type of cell. In mammals, they include, among others,  hematopoietic stem cells , which replenish blood and immune cells,  basal cells , which maintain the skin  epithelium , and  mesenchymal stem cells , which maintain bone,  cartilage , muscle and fat cells. Adult stem cells are a small minority of cells; they are vastly outnumbered by the progenitor cells and terminally differentiated cells that they differentiate into. [ 1 ]


Research into stem cells grew out of findings by Canadian biologists  Ernest McCulloch ,  James Till  and Andrew J. Becker at the  University of Toronto  and the Ontario Cancer Institute in the 1960s. [ 2 ] [ 3 ]  As of 2016 [update] , the only established  medical therapy using stem cells  is  hematopoietic stem cell transplantation , [ 4 ]  first performed in 1958 by French oncologist  Georges Mathé . Since 1998 however, it has been possible to culture and differentiate human embryonic stem cells (in  stem-cell lines ). The process of isolating these cells has been  controversial , because it typically results in the destruction of the embryo. Sources for isolating ESCs have been  restricted  in some European countries and Canada, but others such as the UK and China have promoted the research. [ 5 ]   Somatic cell nuclear transfer  is a  cloning  method that can be used to create a cloned embryo for the use of its embryonic stem cells in stem cell therapy. [ 6 ]  In 2006, a Japanese team led by  Shinya Yamanaka  discovered a method to convert mature body cells back into stem cells. These were termed  induced pluripotent stem cells  (iPSCs). [ 7 ]


The term  stem cell  was coined by  Theodor Boveri  and  Valentin Haecker  in late 19th century. [ 8 ]  Pioneering works in theory of blood stem cell were conducted in the beginning of 20th century by  Artur Pappenheim ,  Alexander A. Maximow ,  Franz Ernst Christian Neumann . [ 8 ]


The key properties of a stem cell were first defined by  Ernest McCulloch  and  James Till  at the University of Toronto and the Ontario Cancer Institute in the early 1960s. They discovered the blood-forming stem cell, the  hematopoietic stem cell  (HSC), through their pioneering work in mice. McCulloch and Till began a series of experiments in which bone marrow cells were injected into irradiated mice. They observed lumps in the spleens of the mice that were linearly proportional to the number of bone marrow cells injected. They hypothesized that each lump (colony) was a clone arising from a single marrow cell (stem cell). In subsequent work, McCulloch and Till, joined by graduate student  Andrew John Becker  and senior scientist  Louis Siminovitch , confirmed that each lump did in fact arise from a single cell. Their results were published in  Nature  in 1963. In that same year, Siminovitch was a lead investigator for studies that found colony-forming cells were capable of self-renewal, which is a key defining property of stem cells that Till and McCulloch had theorized. [ 9 ]


The first therapy using stem cells was a  bone marrow transplant  performed by French oncologist  Georges Mathé  in 1956 on five workers at the  Vinča Nuclear Institute  in  Yugoslavia  who had been affected by a  criticality accident . The workers all survived. [ 10 ]


In 1981, embryonic stem (ES) cells were first isolated and successfully cultured using mouse blastocysts by British biologists  Martin Evans  and  Matthew Kaufman . This allowed the formation of murine genetic models, a system in which the genes of mice are deleted or altered in order to study their function in pathology. In 1991, a process that allowed the human stem cell to be isolated was patented by Ann Tsukamoto. By 1998, human embryonic stem cells were first isolated by American biologist  James Thomson , which made it possible to have new transplantation methods or various cell types for testing new treatments. In 2006,  Shinya Yamanaka 's team in Kyoto, Japan converted fibroblasts into pluripotent stem cells by modifying the expression of only four genes. The feat represents the origin of induced pluripotent stem cells, known as iPS cells. [ 7 ]


In 2011, a female  maned wolf , run over by a truck, underwent stem cell treatment at the  Zoo Brasília  [ pt ] , this being the first recorded case of the use of stem cells to heal injuries in a wild animal. [ 11 ] [ 12 ]


The classical definition of a stem cell requires that it possesses two properties:


Two mechanisms ensure that a stem cell population is maintained (does not shrink in size):


1.  Asymmetric cell division : a stem cell divides into one mother cell, which is identical to the original stem cell, and another daughter cell, which is differentiated.


When a stem cell self-renews, it divides and disrupts the undifferentiated state. This self-renewal demands control of cell cycle as well as upkeep of multipotency or pluripotency, which all depends on the stem cell. [ 13 ]


H.


Stem cells use  telomerase , a protein that restores  telomeres , to protect their DNA and extend their cell division limit (the  Hayflick limit ). [ 14 ]


Potency  specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell. [ 15 ]


In practice, stem cells are identified by whether they can regenerate tissue. For example, the defining test for bone marrow or  hematopoietic stem cells  (HSCs) is the ability to transplant the cells and save an individual without HSCs. This demonstrates that the cells can produce new blood cells over a long term. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.


Properties of stem cells can be illustrated  in vitro , using methods such as  clonogenic assays , in which single cells are assessed for their ability to differentiate and self-renew. [ 18 ] [ 19 ]  Stem cells can also be isolated by their possession of a distinctive set of cell surface markers. However,  in vitro  culture conditions can alter the behavior of cells, making it unclear whether the cells shall behave in a similar manner  in vivo . There is considerable debate as to whether some proposed adult cell populations are truly stem cells. [ 20 ]


Embryonic stem cells  (ESCs) are the cells of the  inner cell mass  of a  blastocyst , formed prior to  implantation  in the uterus. [ 21 ]  In  human embryonic development  the  blastocyst  stage is reached 4–5 days after  fertilization , at which time it consists of 50–150 cells. ESCs are  pluripotent  and give rise during development to all derivatives of the three  germ layers :  ectoderm ,  endoderm  and  mesoderm . In other words, they can develop into each of the more than 200 cell types of the adult  body  when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the  extraembryonic membranes  or to the  placenta .


During embryonic development the cells of the inner cell mass continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as ' neurectoderm ', which will become the future  central nervous system  (CNS). [ 22 ]  Later in development,  neurulation  causes the neurectoderm to form the  neural tube .  At the neural tube stage, the anterior portion undergoes  encephalization  to generate or 'pattern' the basic form of the brain.  At this stage of development, the principal cell type of the CNS is considered a  neural stem cell .


The neural stem cells self-renew and at some point transition into  radial glial progenitor cells  (RGPs). Early-formed RGPs self-renew by symmetrical division to form a reservoir group of  progenitor cells . These cells transition to a  neurogenic  state and start to divide  asymmetrically  to produce a large diversity of many different neuron types, each with unique gene expression, morphological, and functional characteristics. The process of generating neurons from radial glial cells is called  neurogenesis . The radial glial cell, has a distinctive bipolar morphology with highly elongated processes spanning the thickness of the neural tube wall. It shares some  glial  characteristics, most notably the expression of  glial fibrillary acidic protein  (GFAP). [ 23 ] [ 24 ]   The radial glial cell is the primary neural stem cell of the developing  vertebrate  CNS, and its cell body resides in the  ventricular zone , adjacent to the developing  ventricular system .  Neural stem cells are committed to the neuronal lineages ( neurons ,  astrocytes , and  oligodendrocytes ), and thus their potency is restricted. [ 22 ]


Nearly all research to date has made use of mouse embryonic stem cells (mES) or  human embryonic stem cells    (hES)  derived from the early inner cell mass.  Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of  gelatin  as an  extracellular matrix  (for support) and require the presence of  leukemia inhibitory factor  (LIF) in serum media. A drug cocktail containing inhibitors to  GSK3B  and the  MAPK/ERK pathway , called 2i, has also been shown to maintain pluripotency in stem cell culture. [ 25 ]  Human ESCs are grown on a feeder layer of mouse embryonic  fibroblasts  and require the presence of basic fibroblast growth factor (bFGF or FGF-2). [ 26 ]  Without optimal culture conditions or genetic manipulation, [ 27 ]  embryonic stem cells will rapidly differentiate.


A human embryonic stem cell is also defined by the expression of several transcription factors and cell surface proteins. The transcription factors  Oct-4 ,  Nanog , and  Sox2  form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. [ 28 ]   The cell surface antigens most commonly used to identify hES cells are the glycolipids  stage specific embryonic antigen 3  and 4, and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research. [ 29 ]


By using human embryonic stem cells to produce specialized cells like nerve cells or heart cells in the lab, scientists can gain access to adult human cells without taking tissue from patients. They can then study these specialized adult cells in detail to try to discern complications of diseases, or to study cell reactions to proposed new drugs.


Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for  regenerative medicine  and tissue replacement after injury or disease., [ 30 ]  however, there are currently no approved treatments using ES cells. The first human trial was approved by the US Food and Drug Administration in January 2009. [ 31 ]  However, the human trial was not initiated until October 13, 2010 in Atlanta for  spinal cord injury research . On November 14, 2011 the company conducting the trial ( Geron Corporation ) announced that it will discontinue further development of its stem cell programs. [ 32 ]  Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. [ 33 ]  Embryonic stem cells, being pluripotent, require specific signals for correct differentiation – if injected directly into another body, ES cells will differentiate into many different types of cells, causing a  teratoma . Ethical considerations regarding the use of unborn human tissue are another reason for the lack of approved treatments using embryonic stem cells. Many nations currently have  moratoria  or limitations on either human ES cell research or the production of new human ES cell lines.


Mesenchymal stem cells (MSC) or mesenchymal stromal cells, also known as medicinal signaling cells are known to be multipotent, which can be found in adult tissues, for example, in the muscle, liver, bone marrow and adipose tissue. Mesenchymal stem cells usually function as structural support in various organs as mentioned above, and control the movement of substances. MSC can differentiate into numerous cell categories as an illustration of adipocytes, osteocytes, and chondrocytes, derived by the mesodermal layer. [ 34 ]  Where the mesoderm layer provides an increase to the body's skeletal elements, such as relating to the cartilage or bone. The term "meso" means middle, infusion originated from the Greek, signifying that mesenchymal cells are able to range and travel in early embryonic growth among the ectodermal and endodermal layers. This mechanism helps with space-filling thus, key for repairing wounds in adult organisms that have to do with mesenchymal cells in the dermis (skin), bone, or muscle. [ 35 ]


Mesenchymal stem cells are known to be essential for regenerative medicine. They are broadly studied in  clinical trials . Since they are easily isolated and obtain high yield, high plasticity, which makes able to facilitate inflammation and encourage cell growth, cell differentiation, and restoring tissue derived from immunomodulation and immunosuppression. MSC comes from the bone marrow, which requires an aggressive procedure when it comes to isolating the quantity and quality of the isolated cell, and it varies by how old the donor. When comparing the rates of MSC in the bone marrow aspirates and bone marrow stroma, the aspirates tend to have lower rates of MSC than the stroma. MSC are known to be heterogeneous, and they express a high level of pluripotent markers when compared to other types of stem cells, such as embryonic stem cells. [ 34 ]  MSCs injection leads to wound healing primarily through stimulation of angiogenesis. [ 36 ]


Embryonic stem cells (ESCs) have the ability to divide indefinitely while keeping their  pluripotency , which is made possible through specialized mechanisms of  cell cycle  control. [ 37 ]  Compared to proliferating  somatic cells , ESCs have unique cell cycle characteristics—such as rapid cell division caused by shortened  G1 phase , absent  G0 phase , and modifications in  cell cycle checkpoints —which leaves the cells mostly in  S phase  at any given time. [ 37 ] [ 38 ]  ESCs' rapid division is demonstrated by their short doubling time, which ranges from 8 to 10 hours, whereas somatic cells have doubling time of approximately 20 hours or longer. [ 39 ]  As cells differentiate, these properties change: G1 and G2 phases lengthen, leading to longer cell division cycles. This suggests that a specific cell cycle structure may contribute to the establishment of pluripotency. [ 37 ]


Particularly because G1 phase is the phase in which cells have increased sensitivity to differentiation, shortened G1 is one of the key characteristics of ESCs and plays an important role in maintaining undifferentiated  phenotype . Although the exact molecular mechanism remains only partially understood, several studies have shown insight on how ESCs progress through G1—and  potentially other phases—so rapidly. [ 38 ]


The cell cycle is regulated by complex network of  cyclins ,  cyclin-dependent kinases  (Cdk),  cyclin-dependent kinase inhibitors  (Cdkn), pocket proteins of the retinoblastoma (Rb) family, and other accessory factors. [ 39 ]  Foundational insight into the distinctive regulation of ESC cell cycle was gained by studies on mouse ESCs (mESCs). [ 38 ]  mESCs showed a cell cycle with highly abbreviated G1 phase, which enabled cells to rapidly alternate between M phase and S phase. In a somatic cell cycle, oscillatory activity of Cyclin-Cdk complexes is observed in sequential action, which controls crucial regulators of the cell cycle to induce unidirectional transitions between phases:  Cyclin D  and Cdk4/6 are active in the G1 phase, while  Cyclin E  and  Cdk2  are active during the late G1 phase and S phase; and  Cyclin A  and Cdk2 are active in the S phase and G2, while  Cyclin B  and  Cdk1  are active in G2 and M phase. [ 39 ]  However, in mESCs, this typically ordered and oscillatory activity of Cyclin-Cdk complexes is absent. Rather, the Cyclin E/Cdk2 complex is constitutively active throughout the cycle, keeping  retinoblastoma protein  (pRb)  hyperphosphorylated  and thus inactive. This allows for direct transition from M phase to the late G1 phase, leading to absence of D-type cyclins and therefore a shortened G1 phase. [ 38 ]  Cdk2 activity is crucial for both cell cycle regulation and cell-fate decisions in mESCs; downregulation of Cdk2 activity prolongs G1 phase progression, establishes a somatic cell-like cell cycle, and induces expression of differentiation markers. [ 40 ]


In human ESCs (hESCs), the duration of G1 is dramatically shortened. This has been attributed to high mRNA levels of G1-related Cyclin D2 and Cdk4 genes and low levels of cell cycle regulatory proteins that inhibit cell cycle progression at G1, such as  p21 CipP1 , p27 Kip1 , and p57 Kip2 . [ 37 ] [ 41 ]  Furthermore, regulators of Cdk4 and Cdk6 activity, such as members of the Ink family of inhibitors (p15, p16, p18, and p19), are expressed at low levels or not at all. Thus, similar to mESCs, hESCs show high Cdk activity, with Cdk2 exhibiting the highest kinase activity. Also similar to mESCs, hESCs demonstrate the importance of Cdk2 in G1 phase regulation by showing that G1 to S transition is delayed when Cdk2 activity is inhibited and G1 is arrest when Cdk2 is knocked down. [ 37 ]  However unlike mESCs, hESCs have a functional G1 phase. hESCs show that the activities of Cyclin E/Cdk2 and Cyclin A/Cdk2 complexes are cell cycle-dependent and the Rb checkpoint in G1 is functional. [ 39 ]


ESCs are also characterized by G1 checkpoint non-functionality, even though the G1 checkpoint is crucial for maintaining genomic stability. In response to  DNA damage , ESCs do not stop in G1 to repair DNA damages but instead, depend on S and G2/M checkpoints or undergo apoptosis. The absence of G1 checkpoint in ESCs allows for the removal of cells with damaged DNA, hence avoiding potential mutations from inaccurate DNA repair. [ 37 ]  Consistent with this idea, ESCs are hypersensitive to DNA damage to minimize mutations passed onto the next generation. [ 39 ]


The primitive stem cells located in the organs of  fetuses  are referred to as fetal stem cells. [ 42 ]


There are two types of fetal stem cells:


Adult stem cells, also called  somatic  (from Greek σωματικóς, "of the body") stem cells, are stem cells which maintain and repair the tissue in which they are found. [ 44 ]


There are three known accessible sources of  autologous  adult stem cells in humans:


Stem cells can also be taken from  umbilical cord blood  just after birth. Of all stem cell types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one may bank their own blood for elective surgical procedures. [ citation needed ]


Pluripotent adult stem cells are rare and generally small in number, but they can be found in umbilical cord blood and other tissues. [ 48 ]   Bone marrow  is a rich source of adult stem cells, [ 49 ]  which have been used in treating several conditions including liver cirrhosis, [ 50 ]  chronic limb ischemia [ 51 ]  and endstage heart failure. [ 52 ]  The quantity of bone marrow stem cells declines with age and is greater in males than females during reproductive years. [ 53 ]  Much adult stem cell research to date has aimed to characterize their potency and self-renewal capabilities. [ 54 ]    DNA damage accumulates with age in both stem cells and the cells that comprise the stem cell environment.  This accumulation is considered to be responsible, at least in part, for increasing stem cell dysfunction with aging (see  DNA damage theory of aging ). [ 55 ]


Most adult stem cells are lineage-restricted ( multipotent ) and are generally referred to by their tissue origin ( mesenchymal stem cell , adipose-derived stem cell,  endothelial stem cell ,  dental pulp stem cell , etc.). [ 56 ] [ 57 ]   Muse cells  (multi-lineage differentiating stress enduring cells) are a recently discovered pluripotent stem cell type found in multiple adult tissues, including adipose, dermal fibroblasts, and bone marrow. While rare, muse cells are identifiable by their expression of  SSEA-3 , a marker for undifferentiated stem cells, and general mesenchymal stem cells markers such as CD90,  CD105 . When subjected to single cell suspension culture, the cells will generate clusters that are similar to embryoid bodies in morphology as well as gene expression, including canonical pluripotency markers  Oct4 ,  Sox2 , and  Nanog . [ 58 ]


Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants. [ 59 ]  Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses. [ 60 ]