Group 10 Project - Somatic Cell Nuclear Transfer

From CellBiology

Introduction

Figure 1: Schematic of the SCNT Technique

Somatic Cell Nuclear Transfer (SCNT) is a technique, which allows the harvesting of embryonic stem cells (ES cells). ES cells are pluripotent, because they can undergo continual self-renewal without becoming differentiated like other body (somatic) cells.[1] These can then be used as treatment in human and veterinary medicine such as patient-tailored tissue grafts, as well as allowing the possibility of cloning entire organisms such as Dolly the Sheep. SCNT avoids or at least, minimises the potential ethical issues that other embryo sources, such as surplus in-vitro fertilisation embryos, have encountered in many countries including Australia (see Stem Cell Controversy).

SCNT involves taking any differentiated somatic cell's nucleus, for example, skin and muscle cells, and placing that nucleus into an unfertilised egg cell. The egg's own nucleus has already been removed (see Figure 1). This initiates a process called nuclear reprogramming, which causes the donor nucleus to become pluripotent again by certain chemicals in the egg's cytoplasm.[2] The combined hybrid egg can develop into an embryo without normal fertilisation, and the EScells can be extracted.

Stem Cells in General

Figure 2: Student-drawn diagram of the human blastocyst (Carnegie Stage 3)

As noted before, ES cells are pluripotent, which means they are the first cells to form in embryoes. Subsequently, they will undergo continual division to become all the cell types in our bodies. Their pluripotent property is similar to cancer cells, however the stem cell process is tightly controlled, unlike the random growth of tumours[3].

Stem cells can be found in two places in a human:

(1) In a developing embryo called a blastocyst (see Figure 2) - only the inner cell mass (ICM) contains stem cells. The surrounding shell of trophoblast cells do not - they give rise to the placenta, instead.

(2) Adult and child tissues such as bone marrow and skin contain stem cells, which produce new cells to regularly replace old/dead cells. However they are rarer and difficult to identify amongst the crowded somatic cells, and do not survive long enough outside the human body for potential therapeutic use.


Timeline for Stem Cell & SCNT Research

1981 Martin Evans at the University of Cambridge[4] and Gail Martin at the University of California - San Francisco[5] successfully cultured pluripotent mouse ES cells from ICMs of late blastocysts derived from an established teratocarcinoma stem cell line.


1995 James Thomson and his colleagues at the University of Wisconsin[6] isolate the first embryonic stem cells from rhesus macaque monkeys. The research shows it is possible to derive embryonic stem cells from primates, including humans.


1997 Ian Wilmut and colleagues announced the creation of the first cloned mammal using SCNT - a baby lamb that was subsequently named Dolly.[7] Dolly the Sheep was groundbreaking and highly controversial, raising debates amongst laymen, theologians and government officials on the seemingly inevitable cloning of human beings.


1998 Two research groups independently announce that they have derived stem cell lines from embryonic tissue, however their research is controversial due to the sources of the used embryos:

  • James Thomson's group at the University of Wisconsin[8] used ICMs from in-vitro fertilization clinic surpluses.
  • Michael Shamblott's group at the Johns Hopkins University School of Medicine[9] used primordial germ cells from aborted fetuses.
  • Jose Cibelli and Michael West publicize their experiments. [10] The experiment was conducted at the University of Massachusetts and allowed the university to receive a patent in 1999 for the technology.


2001 US President George W. Bush authorises selected ES cell lines to be used for human research, however other cell lines are not allowed. Most of these cell lines are subsequently found to be non-viable and of no use.[11]


2004 South Korean researchers, Hwang Woo-Suk and Moon Shin-Yong, publish that they are the first to clone a human embryo using SCNT from a female cumulus cell and have it reach the 100-150 cell stage.[12] Viable clones were only obtained when the donor and recipient was the same person. Cells propagated for one year and could develop into cartilage, bone, and muscle when implanted in a severely immunodeficient (SCID) mouse.


2005 Two research teams publish reports online that attempt to address the loss of life issue associated with the derivation of ESCs:

  • Robert Lanza’s group established ES cell lines using only one cell from an eight-cell mouse blastomere, which maintained the embryo’s viability in the womb.[13]
  • Alexander Meissner and Rudolf Jaenisch used altered nuclear transfer in mice to create mutant embryos incapable of uterine implantation.[14]

2006

US President George W. Bush vetoed the Stem Cell Research Enhancement Act (see H.R. 810) and approved the Fetus Farming Prohibition Act (see Public Law 109-242). This effectively banned any human stem cell research in the public sector, leaving the majority of embryonic stem cell research up to private funds.

Two research papers[15][16] by Hwang Woo-Suk (see Figure 4) and his colleagues, submitted to the journal Science in 2005, were editorially retracted for consisting largely of fabricated data.[17]


2009 The US Food and Drug Administration (FDA) approves the first human clinical trials for complete spinal injury patients to determine the safety of injecting ES cells into such patients.[18] These ES cells can be used, because they had previously been authorised in 2001


2010 The US FDA approves human clinical trials, conducted by the Medical College of Georgia, for children with cerebral palsy. These trials do not use SCNT, but rely on ES cells present in umbilical cord blood.[19]


Methodology

The actual techniques in SCNT are common between different researchers, however they differ by the materials and equipment used, for example, specific growth chemicals and electric fusion voltages. Apart from this, they all are common centrally - they involve the nucleus of a somatic cell (eg. a normal body cell such as a blood cell, heart cell/cardiocyte, skin cell/fibroblast; the sperm and egg are germ cells not somatic cells) being physically transferred into an unfertilised egg cell that has had its own nucleus removed (referred to as 'enucleation').

Stated below is an outline of the steps you would take in order to perform SCNT for humans.


Step 1: Preparation of the somatic cell

The somatic cell, as stated, can be any type of normal cell in the body apart from the sperm or egg. Most researchers appear to favour skin fibroblasts, because the skin is easy to access, non-invasive and fairly painless. However, cells from the breast/mammary gland[20] and cumulus cells[21] have also been used.

A tiny amount of skin is cut and placed in a trypsin enzyme-buffer solution that frees the target fibroblasts from the extracellular matrix. The mixture is placed on a serum medium and incubated for three weeks, in order to obtain a single layer of fibroblasts without any other cell types.


Figure 5: Step 2 - Generating cytoplasts by oocyte enucleation.


Step 2: Preparation of the egg/oocyte

Typically, researchers will select the target egg that is in the antral stage and exhibits the 1st polar body. When the researchers can see follicles at least 18mm wide, human chorionic gonadotropin (hCG) is injected into the female donor.[22] hCG is used since it is a strong inducer of ovulation, and allows a more 'comfortable' way of obtaining the egg without any invasive and direct surgical procedure to the ovaries and the donors themselves. Consequently, the ovulated egg is collected by ultrasound-guided transvaginal needle aspiration in a procedure similar to in-vitro fertilisation.

Once the egg is extracted, it is placed in liquid human serum.[23]

Fluorescent tags are bound to the oocyte's DNA, allowing the researchers to check all the oocyte's DNA/nucleus has been removed when exposing the egg to UV light.[24]

The egg's nucleus is removed using an inverted microscope, UV light and a glass needle. This setup minimises damage to the delicate egg as it can cut through the thick zona pellucida shell, and is fairly easy to manipulate. At this point with its nucleus removed, the oocyte is called a cytoplast (see Figure 5).


Figure 6: Step 3 - An adult cell nucleus is injected into a enucleated egg.

Step 3: Nuclear Transfer

Both fibroblast and egg are placed in a thin human serum solution[25] with cytochalasin B.

Once the donor fibroblast's nucleus is extracted from the fibroblast with a pipette, it is called a karyoplast. Subsequently, this karyoplast is injected into the egg/cytoplast past the zona pellucida (see Figure 6).

Link: Video of Nuclear Transfer provided by Howard Hughes Medical Institute

At this point, the karyoplast and cytoplast are still functionally separate (see Figure 7 at 0 minutes), therefore a few electric pulses are given to the entire solution causing fusion between the two entities (see Figure 7 at 10 minutes).


Figure 7: Step 3/4 - Timelapse images of fusion between fibroblast (arrowed) and cytoplast/egg

Step 4: Post Nuclear Transfer Procedures

The complete process of nuclear transfer is completed approximately 35-45 hours after the original hCG was administered to the female donor. However, it takes an additional three hours before cleavage can be seen if the transfer and activation has been successful.

Finally, the egg is incubated in a culture medium at 37°C in highly humidified conditions. This could be both an artificial attempt and natural requirement that replicates the uterine conditions, which are conducive to embryonic development. After this activation, in approximately four days for human donors, cleavage of the egg can be clearly seen.

It has been noted by Dominko and colleagues[26] that the oocyte can be from any mammal if using a mammalian nucleus. It does not necessarily mean a human fibroblast must require a human egg for use. This is, because the initial development of all mammal eggs undergo a similar process, and it is only later in the morphogenesis of the embryo that the nucleus's DNA actually starts taking control of the process. The consequence of using a cow egg with a human fibroblast or other species' fibroblast is the first 2 cleavages correlated to bovine development time, while after these two divisions, the growth rate and timing of the embryo matched the donor species.[27] Therefore, if human nucleus and oocyte were used, the entire process would mirror the normal human rate.


Figure 8: Step 5 - Timelapse images of embryo development of blastocyst (arrowed) after activation


Step 5: Embryogenesis

Once the hybrid egg has developed into a blastocyst, what happens to it from this point depends on its application:

  • For reproductive cloning (creating an entire organism): the blastocyst is implanted into a surrogate mother who carries the developing embryo like a normal pregnancy.
Figure 9: A human ES cell colony (large centre) on layer of mice feeder cells (background)
  • For applications in regenerative medicine (obtaining specific cell/tissue types that can be surgically grafted for a patient): the ICM is harvested from the blastocyst onto mice-derived feeder cells for nutrients (see Figure 9) and differentiated into the required tissue/cell types, using certain growth and differentiation factors over two days.[28]

The actual differentiation factors required for specific somatic cells has been determined over the years by many different researchers, for example, stem cells exposed to dimethyl sulfoxide would diffentiate into different proportions of muscle cells, while stem cells exposed to retinoic acid would become neurons[29]

Apart from the differentiation factors, the removal of the feeder cells or the cells' chemical messengers (cytokines) would also be required to signal the embryonic stem cells to differentiate.[30][31]


Current Situation

Unfortunately, current research has not been successful in producing human embryoes that develop the blastocoele which is essential for later gastrulation and morphogenesis. It appears the cells remain alive and viable as the cell numbers keep increasing, but they fail to organise themselves into any identifiable embryonic structures. In other primates such as monkeys the production of embryonic stem cells has also been unsuccessful - apparently as a result of large amounts of genetic instability.[32]

There is a high rate of blastocysts that are transplanted into surrogate mothers that result in none reaching full term. Instead, the fallopian tubes, where the embryo was transplanted, develop large sacs of fluid surrounding the dead blastocyst.

Due to ethical issues, the transplantation of human blastocysts in reproductive cloning has not occurred to date. However, blastocyst transplants of other animals such as cows have been successful in producing undamaged and living offspring.[33]


Applications of SCNT

Veterinary, Animal Science

  • Mass production of animals: As farm animals are being used for human use, SCNT can be used to produce high quality farm animals in infinite number. Cloning technology can be applied, without compromising animal welfare, if integrated in breeding programs and these transgenic clones will be delivering the expected products.[34]. Researches show that, somatic cell cloned cattle reportedly were physiologically, immunologically, and behaviorally normal and this makes use of SCNT useful for mass production.[9]
  • Conserving wild animals for next generations: Another area where SCNT can be useful is conservation. This use can be effective to preserve and propagate endangered species that are being produced poorly in the zoos.[35] With effective reproduction, these species can be reintroduced to the wild again, allowing maintenance of genetic diversity of species by introducing new genes. The use of SCNT can also be helpful to even create the extinct species, if any tissues or cells are available.[36] The idea of producing mammoth is being considered as an intact animal was discovered frozen in the tundra. The close relative of the mammoth, the elephant, could be used both as a surrogate mother and an oocyte donor.

As also mentioned by Holt et al, [10], Reproductive cloning, by nuclear transfer, is often regarded as having potential for conserving endangered species. Cloning non-mammalian vertebrates can be more practical than using conventional reproductive methods. As cloning technology has made a good progress in amphibians, it may be possible to breed threatened amphibians and even reproduce extinct amphibian species.

  • Disease resistant animal production: By using SCNT, genes causing diseases can be manipulated in order to have healthier farm animals that live a lot longer.[37]

Human Medicine

  • Human therapeutic proteins: Human proteins are needed and in demand for the treatment of diseases. Purifying proteins from blood is an expensive procedure and also carries the risk of contamination by Hepatitis C or HIV. Proteins can be produced in human cell culture but the output is small and it is also an expensive procedure. But also, Ng et al. [38]mentions that human proteins can be produced in the milk of transgenic sheep, goats and cattle. The output can be as high as 40 g per litre of milk and the cost of the procedure is not as high. By using nuclear transfer, it is possible to insert human genes at specific points in the genome, improving the reliability of their expression; deleting, substituting and adding of genes that are missing in the patient to lead them have better lives, possible as well.
  • Xenotransplantation: Shortage of organs is a big problem considering the amount of patients needing them. Transplant organs can be a solution for this. Genetically modified animals such as pigs are being developed as a solution but so far the modifications are limited to adding genes. By using SCNT, deleting genes that are responsible for rejection from pigs is possible and this way it is aimed to avoid rejection of an organ transplanted from a normal pig to a human patient.[39]
  • Animal model for diseases: experimental animals with altered disease-causing genes can be tailor generated using SCNT, allowing better understanding of the complex pathogenesis of the diseases, eg. good SCNT-mouse models would allow cystic fibrosis between the lung and intestine to be understood better[40][41]
  • Cell Therapy using dedifferentiated stem cells: This use is being developed for a range of diseases including heart attack, stroke and diabetes etc. Patient’s own cell can be used as transplanted cells are likely to be rejected. Cloning of adult animals shows that egg and the embryo have the capability of reprogramming. This use may make it possible to reprogramming patient’s own cells without creating and destroying embryos. The differentiating stem cells can then be grown into several hundred or thousand cells and surgically transported into the patient where they will produce the required tissue.[42]. To give an example, a child's problem of severe immnunodefficiency due to chemotheraphy and whole body radiation because of having Hodgkin's Lymphoma, can be corrected as child's skin fibroblast can be obtained and embryonic stem cells obtained as per the procedures mentioned above. Transcription factors would differentiate the stem cells into bone marrow cells, which can be transplanted into the marrow cavities of the child, and they would gradually rebuild the child's haemopoietic system and also, their immune system.

SCNT can also be used in treatment for Cardiovascular and Nervous System Disorders. A study by Eschenhagen et al,[43], shows that tissue engineering for cardiac development and cardiac repair by SCNT is possible, that is creating contracting heart mucles from patients own stem cells and use them to decrease the intensity of the disease.

Developmental Biology

  • Events during fertilization and pre-implantation embryos: knowledge of the complex mechanisms and the various controlling factors during embryonic development will be understood better with SCNT.[44]


Advantages and Disadvantages of SCNT

Advantages of SCNT Disadvantages of SCNT
Retains genetic code of the donor nucleus: Resultant tissue that is transplanted into the donor patient will not be exposed to potentially fatal immune rejection.[45] Adverse effects of somatic nucleus choice: Somatic nucleus adversely determines cloned offspring's post-birth growth, eg. cumulus cell lead to morbidly obese adults, and Sertoli cells lead to premature deaths of offspring.[46]
Common Procedures and Methods: Relatively similar and uniform between different researchers - the main difference appears to be the choice of culture mediums which do not seem to affect the actual outcome.[47] Inefficient steps: ie. maximum 4% non-human embryoes become live offspring, while others display fatal abnormalities resulting in spontaneous loss with current techniques and technology.[48]
Easy Access: Easily obtainable equipment and growth substrates via purchases from biotechnology and biological supply companies. Difficulty in inducing the re-expression of differentiated genes: This is especially the case where the donor nuclei have been obtained from adults as opposed to fetal or newborn stage.[49][50] This phenomenon appears to be in common with in vitro fertilisation in human and non-human species, where higher rates of spontaneous pregnancy terminations occur with adult-age nuclei, as opposed to higher term births with fetal/embryonic nuclei.[51]
Electric Fusion damage: Eiges and colleagues[52] have suggested electroporation, which has been demonstrated to be fatal to stem cell survival in other non-SCNT techniques, may disrupt the delicate interaction between the fusing oocyte and somatic nucleus, thereby preventing them from communicating properly. Other methods for cytoplast-karyoplast fusion in SCNT have not been assessed yet.
Cross-species incompatibilities: Gurdon et al.[53] suggests irreconcilable genetic differences between egg and nucleus species leads to low success rate; that is, mice eggs should not be mixed with human nuclei. Though amphibian and mammal sources were used by Gurdon and colleagues, there has been no other evidence to suggest why this incompatibility could not affect human-only procedures as well.


Future Directions

Fulka and colleagues[54] has suggested issues that need to be solved for SCNT in order for progress to be made such as:

  • Cytoplast age
  • Cytoplast cell-cycle stage
  • Activation procedure: methods of activating the karyoplast-cytoplast so that the embryo is not harmed (as noted in the Disadvantages) need to be found.
  • Source of karyoplasts and their degree of differentiation
  • Karyoplast cell cycle stage: in the case of regenerative medicine, the donor nucleus age would be very important since many patients who could potentially benefit from this therapy would be quite elderly. However, the low success of obtaining viable ICM have not allowed researchers to determine the effects of age on the donor nucleus, if any.[55]
  • Karyoplast-cytoplast (nuclear-cytoplasmic) interactions
  • Species-specific differences
  • Technical aspects: for example, the currently used feeder fibroblast cells are derived from mice, which introduces the possibility and already reported occurrences of cross-contamination of human embryonic cells by mouse disease-causing pathogens.[56] This contamination issue can be addressed by using screened human fibroblast feeder cells or eliminate feeder cells,[57] and use serum-free medium only, especially if the cultured cells are for therapeutic use.[58]

According to Trounson[59], the questions of whether the cells undergo differentiation or transdifferentiation during offspring development and also how these changes are controlled are sources of ongoing debates. Regardless of the outcome of this debate, using SCNT has a place in future in research. In the future the uses of SCNT could be:

  • production of transgenic mice
  • rapid production of genetically modified herds or elite livestock individuals with desirable traits in agriculture
  • production of patient-specific embryonic stem cells in human medicine

In recognition of the stalled progress in SCNT, Maherali et al.[60]suggested the use of four transcription factors (c-Myc, Sox2, Oct4 and Klf4) can cause somatic cells to revert into embryonic-like stem cells, referred to as induced pluripotent stem cells (iPS cells), without needing the entire SCNT procedure. However, Okita and colleagues[61] expressed concern such cells display an increased likelihood to develop into cancerous cells as a result of both using c-Myc factor and reactivating the c-Myc transgene. As a result, Yu et al.[62] have indicated the use of a different set of transcription factors (Oct4, Sox2, Nanog and Lin28) are both sufficient and without the side-effects of c-Myc to obtain the iPS cells. In 2007, Byrne et al.[63] successfully obtained sustainable iPS cell lines from the rhesus macaque monkeys. This has so far not been accomplished by SCNT in human or other primates.

It is plainly evident that researchers require more progress into the field of SCNT for humans. There is still much to be learnt and understood about the actual mechanisms that occur during the fusion of the oocyte and somatic cell.[64] Hall and Stojkovic [65] theorise, though the future will bring more improvements and uses for SCNT, human SCNT may remain clouded in ethical, moral, and religious controversies.


Ethics

stduent drawn diagram of Ethics & SCNT

SCNT is considered a new technology. Due to being new, all uses of this technology have not been explored yet and it is not certain yet which uses of this technology can be acceptable or not. Safety and also inefficiency of the procedure is another concern as relatively few births have resulted from many attempts in cloning technology and this has brought some ethical and moral concerns. Morally, seeing scientists in the role of ‘God’ have been a question in minds as this idea, cloning a human, created a big debate amongst lawmakers, religious leaders, academicians and professional societies. The idea of SCNT, has violated moral values and traditions and this raised question marks which contributed to the passage of restrictive laws in several nations and to proposals for restrictive legislation in USA.


A positive idea defending the use of reproductive SCNT is that the cloning technology can give a positive progress in medicine and other fields to improve quality and conditions of life as SCNT can be used for therapeutic purposes and produce embryonic stem cells for people who need organ and tissue transplants and also in infertile couples provided that the safety of procedure can be guaranteed.

The Religous and Legal Issues:

World religions have different approaches to stem cell and hES research. Here are perspectives of major religions according to Lori P.Knowles, [11] :

Greek Orthodox and Roman Catholic Churches: Authorities of Greek Orthodox and Roman Catholic have come in favor of stem cell research using adult stem cells but they have opposed hES research as illegal and immoral as they see it as human person begins at conception and the human embryo has the same moral status as human persons. So, the reserach on human embryos, including hES is seen as willful destruction and is homicide.

Judaism: Unlike Greek Orthodox and Roman Catholic Churches, The rules of the Jewish culture do not see use of hES as immoral as they say the fetus does not become a person until the head emerges from the womb.

Islam: Islamic belief is against human suffering and illness, means that the use of surplus IVF embryos for stem cell research is relatively uncontroversial. But creating embryos for the purposes of research is still argued.

Legal Issues

With Prohibi-tion of Human Reproductive Cloning and the Regulation of Human Research Amendment Bill 2006 through parliament, Australia has joined the nations that fund and regulate Somatic Cell Nuclear Transfer research, [12]

SCNT Research in Australia

Funding was granted by NSW and Victorian Governments to two research centers in 2008, to encourage research in development of use of SCNT. One of them is research center based at Fertility East in Sydney collaborating with the Monash Institute of Medical Research and the other one is Sydney IVF collaborating with the Australian Stem Cell Center. Outcomes of the research studies are expected soon.

Despite the ambiguity of SCNT research for human use, SCNT research in other animals has been thriving in Australia - for example, Lee and his colleagues at the University of New South Wales were able to demonstrate that enriched stem cells placed in chemotherapy-treated muscle tissue were able to regenerate more optimally than without the enriched stem cells.[66] Though the experiments were performed in mice and also have not been licensed for any human clinical trials, the study allows us to understand possible mechanisms and properties that will be encountered in regenerative medicine - a large applicative field of SCNT.

Government's funding will be supporting Victorian stem cell reserachers collaborating with Californian scientists. The funding is focusing on research into Stem Cell Transplantation Immunology which is aimed at ensuring human immune tolerance of stem cell derived cell and tissues, as skin, bone and organ tissues. [13]

Links to Research Laboratories and Researchers

International Society for Stem Cell Research (ISSCR)[14]

The International Society for Stem Cell Research (ISSCR) is an independent, nonprofit organization formed in 2002 to foster the exchange of information on stem cell research. Learn more about this exciting new organization.

Fertility East [15] is a new independent Fertility and IVF unit started in June 2007 collaborationg with Monash Institute of Medical Research [16]and both granted by the government in 2008 in reasearch of SCNT.

Sydney IVF [17] and Australian Stem Cell Center [18] are collaborating together in development of use of SCNT and granted by the government in 2008.


Further Reading

  • Mollard R, Nuclear Transfer--Stem Cells or Somatic Cell Nuclear Transfer (SCNT) [19]
  • Wilmut I, Beaujean N, de Sousa PA, et al., Somatic cell nuclear transfer,Nature, vol.419(6907):583–6,October 2002, pmid=12374931 [20]
  • Kikyo N, Wolffe AP, Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons, J. Cell. Science, vol.113(1):11–20, January 2000, pmid=10591621 [[21]]


Glossary

Antral Stage: a very mature oocyte-containing follice in the female ovary; usually ready to undergo ovulation.

Blastomere: is a type of cell produced by division of the egg after fertilization.

Blastocyst: A thin-walled hollow structure in early embryo development that contains a group of cells called the inner cell mass from which the embryo arises.

Cleavage: The act or state of splitting or dividing of a cell, particularly during the telophase of (animal) cell division.

Cumulus cell: cumulus oophorus granulosa cells, cumulus oophorus granulosa cells, At one part of the mature follicle, the cells of the membrana granulosa are collected into a mass which projects into the cavity of the follicle.

Cytochalasin B:fungal metabolites that have the ability to bind to actin filaments and block polymerization and the elongation of actin; permeate cell membranes, prevent cellular translocation and cause cells to enucleate.cytochalasin A and cytochalasin B can also inhibit the transport of monosaccharides across the cell membrane

Cytoplast: the inner part of the cell without cell wall and plasma membrane. It includes cytroskeleton, organelles and cytosol.

Cytokines: are any of a number of small proteins that are secreted by specific cells of the immune system and that carry signals locally between cells, and thus have an effect on other.

Dolly: the first mammal cloned from differentiated cells

Fetal cell serum: Similar to fetal bovine serum[22] since it is essentially blood with all cells/platelets have been removed, however can be obtained by coagulating donated human blood. Both human and bovine serum can be used in SCNT with no side effects.

Human chorionic gonadotropin: produced by the female to sustain the early stages of pregnancy; to stimulate ovulation.

Karyoplast:A cell nucleus surrounded by a narrow band of cytoplasm and a plasma membrane. (Retrieved from [23])

Luteinising Hormone: A hormone produced by the anterior lobe of the pituitary gland that stimulates ovulation and the development of the corpus luteum in the female and the production of testosterone by the interstitial cells of the testis in the male. (Retrieved from [24])

Plasma estradiol concentrations: represents the major estrogen in humans; Estradiol has not only a critical impact on reproductive and sexual functioning, but also affects other organs including the bones.

Somatic cell:any cells forming the body of an organism, as opposed to germline cells.

Transvaginal needle aspiration: or oocyte retrieval (OCR) is a technique used in in vitro fertilization in order to remove oocytes from the ovary of the female, enabling fertilization outside the body. It is commonly known as "egg collection."

Trophoblast: The outermost cell layer of the blastocyst that attaches the fertilized ovum to the uterine wall and conducts nutrients from mother to developing child. (Retrieved from [25])

Trypsin: a natural enzyme that can be found in the human gut; breaks down proteins/peptides; used experimentally to inactivate any protein/enzymes/bacteria in the medium.

Xenotransplantation : The surgical transfer of cells, tissues, or especially whole organs from one species to another, such as from pigs to humans. (Retrieved from [26])

Zona pellucida:a glycoprotein membrane surrounding the plasma membrane of an oocyte.

References

  1. Semb H. Human embryonic stem cells: origin, properties and applications. APMIS. 2005 Nov-Dec; 113(11-12):743-50. PMID 16480446
  2. Gurdon JB, Byrne JA, Simonsson S. Nuclear reprogramming and stem cell creation. Proc Natl Acad Sci U S A. 2003 Sep 30;100 Suppl 1:11819-22. Epub 2003 Aug 14. Review. PMID 12920185
  3. Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev. 2005 Apr;85(2):635-78. Review. PMID 15788707
  4. Evans MJ & Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos Nature. 1981 Jul 9;292(5819):154-6. [1]
  5. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8. PMID 6950406
  6. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7844-8. PMID 7544005
  7. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997 Feb 27;385(6619):810-3. PMID 9039911
  8. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7. [2]
  9. Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13726-31. PMID 9811868
  10. Cibelli JB, Yoon SY etl. Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development.J Clin Invest. 2008 Nov;118(11):3671-81. Epub 2008 Oct 16.[3]
  11. Marlene Cimons, Bush policy: Attention turns to existing human ES cells.Nature Medicine 7, 981 - 982 (2001)[4]
  12. Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, Lee BC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. PMID 14963337
  13. Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature. 2006 Jan 12;439(7073):216-9. Epub 2005 Oct 16. PMID 16227970
  14. Meissner A, Jaenisch R. Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature. 2006 Jan 12;439(7073):212-5. Epub 2005 Oct 16. PMID 16227971
  15. Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, Lee BC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science. 2004 Mar 12;303(5664):1669-74. Epub 2004 Feb 12. PMID 14963337
  16. Hwang WS, Roh SI, Lee BC, Kang SK, Kwon DK, Kim S, Kim SJ, Park SW, Kwon HS, Lee CK, Lee JB, Kim JM, Ahn C, Paek SH, Chang SS, Koo JJ, Yoon HS, Hwang JH, Hwang YY, Park YS, Oh SK, Kim HS, Park JH, Moon SY, Schatten G. Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science. 2005 Jun 17;308(5729):1777-83. Epub 2005 May 19. PMID 15905366
  17. Kennedy D. Editorial retraction. Science. 2006 Jan 20;311(5759):335. Epub 2006 Jan 12. PMID 16410485
  18. Falco M. FDA approves human embryonic stem cell study. CNN (January 23, 2009) Retrieved May 19, 2010 from [5]
  19. Medical College of Georgia (2010, February 11). First FDA-approved stem cell trial in pediatric cerebral palsy. ScienceDaily. Retrieved May 19, 2010, from http://www.sciencedaily.com/releases/2010/02/100211121812.htm
  20. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997 Feb 27;385(6619):810-3. PMID 9039911
  21. Collas P, Barnes FL. Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei. Mol Reprod Dev. 1994 Jul;38(3):264-7. PMID 7917277
  22. French AJ, Adams CA, Anderson LS, Kitchen JR, Hughes MR, Wood SH. Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts. Stem Cells. 2008 Feb;26(2):485-93. Epub 2008 Jan 17. PMID 18202077
  23. Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E, McKusick B, First NL. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod. 1999 Jun;60(6):1496-502. PMID 10330111
  24. Heindryckx B, De Sutter P, Gerris J, Dhont M, Van der Elst J. Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes. Hum Reprod. 2007 Jul;22(7):1982-90. Epub 2007 May 18. PMID 17513316
  25. Cibelli JB, Kiessling AA, Cunniff K, Richards C, Lanza RP, West MD (2001), 'Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development', e-biomed: The Journal of Regenerative Medicine. Vol. 2, No. 5, pp. 25-31 [6] PMID not available.
  26. Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E, McKusick B, First NL. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod. 1999 Jun;60(6):1496-502. PMID 10330111
  27. Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E, McKusick B, First NL. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod. 1999 Jun;60(6):1496-502. PMID 10330111
  28. Chen Y, He ZX, Liu A, Wang K, Mao WW, Chu JX, Lu Y, Fang ZF, Shi YT, Yang QZ, Chen da Y, Wang MK, Li JS, Huang SL, Kong XY, Shi YZ, Wang ZQ, Xia JH, Long ZG, Xue ZG, Ding WX, Sheng HZ. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 2003 Aug;13(4):251-63. PMID 12974615
  29. Dinsmore J, Ratliff J, Deacon T, Pakzaban P, Jacoby D, Galpern W, Isacson O. Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant. 1996 Mar-Apr;5(2):131-43. PMID 8689027
  30. Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, Wobus AM. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res. 2002 Aug 9;91(3):189-201. PMID 12169644
  31. Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji Guanju, Fleischmann B, Katus HA, Hescheler J, Franz WM. Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol. 1997 Jun;29(6):1525-39. PMID 9220339
  32. Simerly C, Dominko T, Navara C, Payne C, Capuano S, Gosman G, Chong KY, Takahashi D, Chace C, Compton D, Hewitson L, Schatten G. Molecular correlates of primate nuclear transfer failures. Science. 2003 Apr 11;300(5617):297. PMID 12690191
  33. Collas P, Barnes FL. Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei. Mol Reprod Dev. 1994 Jul;38(3):264-7. PMID 7917277
  34. Brophy B, Smolenski G, Wheeler T, Wells D, L'Huillier P, Laible G. Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein. Nat Biotechnol. 2003 Feb;21(2):157-62. Epub 2003 Jan 27. PMID 12548290
  35. Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD, Damiani P. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning. 2000;2(2):79-90. PMID 16218862
  36. Kato H, Anzai M, Mitani T, Morita M, Nishiyama Y, Nakao A, Kondo K, Lazarev PA, Ohtani T, Shibata Y, Iritani A. Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(7):240-7. PMID 19644224
  37. Cyranoski D. Koreans rustle up madness-resistant cows. Nature. 2003 Dec 18;426(6968):743. PMID 14685182
  38. Ng et al. Somatic Cell Nuclear Transfer (Cloning) – Science. 2001[7]
  39. Torben Greve. Xenotransplantation: Biotechnological Aspects and Current Attitudes. Acta Veterinaria Scandinavica 2004, 45(Suppl 1):S13-S17 PMID 1534714
  40. Griesenbach U, Alton EW. Cystic fibrosis: ferreting with fibroblasts for cystic fibrosis. Gene Ther. 2009 Jan;16(1):1-2. Epub 2008 Oct 2. PMID 18830274
  41. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503-12. PMID 2822260
  42. Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med. 2003 Jul 17;349(3):275-86. PMID 12867612
  43. http://circres.ahajournals.org/cgi/reprint/97/12/1220
  44. Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev. 2005 Apr;85(2):635-78. PMID 15788707
  45. Colman A, Kind A. Therapeutic cloning: concepts and practicalities. Trends Biotechnol. 2000 May;18(5):192-6. PMID 10758513
  46. Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN, Young LE. Somatic cell nuclear transfer. Nature. 2002 Oct 10;419(6907):583-6. PMID 12374931
  47. Colman A, Kind A. Therapeutic cloning: concepts and practicalities. Trends Biotechnol. 2000 May;18(5):192-6. PMID 10758513
  48. Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN, Young LE. Somatic cell nuclear transfer. Nature. 2002 Oct 10;419(6907):583-6. PMID 12374931
  49. Kasinathan P, Knott JG, Wang Z, Jerry DJ, Robl JM. Production of calves from G1 fibroblasts. Nat Biotechnol. 2001 Dec;19(12):1176-8. PMID 11731789
  50. Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN, Young LE. Somatic cell nuclear transfer. Nature. 2002 Oct 10;419(6907):583-6. PMID 12374931
  51. Heyman Y, Chavatte-Palmer P, LeBourhis D, Camous S, Vignon X, Renard JP. Frequency and occurrence of late-gestation losses from cattle cloned embryos. Biol Reprod. 2002 Jan;66(1):6-13. PMID 11751257
  52. Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol. 2001 Apr 3;11(7):514-8. PMID 11413002
  53. Gurdon JB, Byrne JA, Simonsson S. Nuclear reprogramming and stem cell creation. Proc Natl Acad Sci U S A. 2003 Sep 30;100 Suppl 1:11819-22. Epub 2003 Aug 14. PMID 12920185
  54. Fulka J Jr, First NL, Loi P, Moor RM. Cloning by somatic cell nuclear transfer. Bioessays. 1998 Oct;20(10):847-51. PMID 9819572
  55. Chen Y, He ZX, Liu A, Wang K, Mao WW, Chu JX, Lu Y, Fang ZF, Shi YT, Yang QZ, Chen da Y, Wang MK, Li JS, Huang SL, Kong XY, Shi YZ, Wang ZQ, Xia JH, Long ZG, Xue ZG, Ding WX, Sheng HZ. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 2003 Aug;13(4):251-63. PMID 12974615
  56. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol. 2002 Sep;20(9):933-6. Epub 2002 Aug 5. PMID 12161760
  57. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J. Human feeder layers for human embryonic stem cells. Biol Reprod. 2003 Jun;68(6):2150-6. Epub 2003 Jan 22. PMID 12606388
  58. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod. 2004 Mar;70(3):837-45. Epub 2003 Nov 19. PMID 14627547
  59. Cyranoski D. Koreans rustle up madness-resistant cows. Nature. 2003 Dec 18;426(6968):743. PMID 14685182
  60. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution Cell Stem Cell. 2007 Jun 7;1(1):55-70. PMID 18371336
  61. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007 Jul 19;448(7151):313-7. Epub 2007 Jun 6. PMID 17554338
  62. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec 21;318(5858):1917-20. Epub 2007 Nov 20. PMID 18029452
  63. Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S, Wolf DP, Mitalipov SM. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature. 2007 Nov 22;450(7169):497-502. Epub 2007 Nov 14. PMID 18004281
  64. Rathjen PD, Lake J, Whyatt LM, Bettess MD, Rathjen J. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. Reprod Fertil Dev. 1998;10(1):31-47. PMID 9727591
  65. Hall VJ, Stojkovic M. The Status of Human Nuclear Transfer. Stem Cells Review. 2006;2(4):301-8[8]
  66. Lee AS, Kahatapitiya P, Kramer B, Joya JE, Hook J, Liu R, Schevzov G, Alexander IE, McCowage G, Montarras D, Gunning PW, Hardeman EC. Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle. Stem Cells. 2009 May;27(5):1098-108. PMID 19415780


2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer