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VIT University, Vellore

Animal physiology and Biotechnology Assignment On


Eembryonic Stem Cell Technology

(Winter Semester: 2011)

Submitted By: Swapnil Tiwari Reg.No. 09BBT174 B.Tech.(BioTech.)

Contents

1. Introduction

5. Use of human embryonic stem cells as model for human genetic disorders 6. Recent development and research 7. Conclusion 8. References

3. Techniques and conditions for embryonic stem cell derivation and culture 4. Potential clinical uses

2. Historical background and development

Embryonic Stem Cell Technology


1. Introduction:
Human Embryonic Stem Cells

Stem cells generally are self-renewing primitive cells that can develop into functional, differentiated cells. Human embryonic stem cells (hESCs), which are derived from very early stage embryos called blastocysts, are unique because:

they are pluripotent, which means they can develop into all cells and tissues in the body, and they self-renew indefinitely in the undifferentiated state because they express high levels of telomerase.

The ability of hESCs to divide indefinitely in the undifferentiated state without losing pluripotency is a unique characteristic that distinguishes them from all other stem cells discovered to date in humans. It has been demonstrated that hESCs express telomerase continuously, a characteristic of immortal cells. Other stem cells such as blood or gut stem cells express telomerase at very low levels or only periodically; they therefore age, limiting their use in research or therapeutic applications. hESCs can be expanded in culture indefinitely and hence can be banked for scaled product manufacture.
VistaGen, a biotechnology company based in South San Francisco, is one of the world's leading companies focused on using the power of stem cell technology to transform the ways drugs are discovered and tested. WARF is the private, non-profit patenting and licensing organization for the University of Wisconsin-Madison, one of the top-ranked public research universities in the U.S. The licensed patents result from the research of stem cell pioneer James Thompson of the University of Wisconsin Stem Cell and Regenerative Medicine Center and director of regenerative biology at the new Morgridge Institute for Research. The license will accelerate VistaGen's commercial programs focused on providing customized, next-generation, stem cell-based predictive toxicology and drug discovery screening assays to increase preclinical research and development (R&D) productivity for the pharmaceutical industry.

2. Historical background and development:


In 1964, researchers isolated a single type of cell from a teratocarcinoma, a tumor now known to be derived from a germ cell. These cells isolated from the teratocarcinoma replicated and grew in cell culture as a stem cell and are now known as embryonic carcinoma (EC) cells. Although similarities in morphology and differentiating potential (pluripotency) led to the use of EC cells as the in vitro

model for early mouse development, EC cells harbor genetic mutations and often abnormal karyotypes that accumulated during the development of the teratocarcinoma. These genetic aberrations further emphasized the need to be able to culture pluripotent cells directly from the inner cell mass. In 1981, embryonic stem cells (ES cells) were independently first derived from mouse embryos by two groups. Martin Evans and Matthew Kaufman from the Department of Genetics, University of Cambridge published first in July, revealing a new technique for culturing the mouse embryos in the uterus to allow for an increase in cell number, allowing for the derivation of ES cells from these embryos. Gail R. Martin, from the Department of Anatomy, University of California, San Francisco, published her paper in December and coined the term Embryonic Stem Cell. She showed that embryos could be cultured in vitro and that ES cells could be derived from these embryos. In 1998, a breakthrough occurred when researchers, led by James Thomson at the University of WisconsinMadison, first developed a technique to isolate and grow human embryonic stem cells in cell culture.

3. Techniques and conditions for embryonic stem cell derivation and culture
Embryonic stem cells are derived from the inner cell mass of the early embryo, which are harvested from the donor mother animal. Martin Evans and Matthew Kaufman reported a technique that delays embryo implantation, allowing the inner cell mass to increase. This process includes removing the donor mothers ovaries and dosing her with progesterone, changing the hormone environment, which causes the embryos to remain free in the uterus. After 46 days of this intrauterine culture, the embryos are harvested and grown in in vitro culture until the inner cell mass forms egg cylinder-like structures, which are dissociated into single cells, and plated on fibroblasts treated with mitomycin-c (to prevent fibroblast mitosis). Clonal cell lines are created by growing up a single cell. Evans and Kaufman showed that the cells grown out from these cultures could form teratomas and embryoid bodies, and differentiate in vitro, which all indicate the cells are pluripotent. Gail Martin derived and cultured her ES cells differently. She removed the embryos from the donor mother at approximately 76 hours after copulation and cultured them overnight in media containing serum. The following day, she removed the inner cell mass from the late blastocyst using microsurgery. The extracted inner cell mass was cultured on fibroblasts treated with mitomycin-c in media that containing serum and was conditioned by EC cells. After approximately one week, colonies of cells grew out. These cells grew in culture and demonstrated pluripotent characteristics, as demonstrated by the ability to form teratomas, differentiate in vitro, and form embryoid bodies. Martin referred to these cells as ES cells. It is now known that the feeder cells provide leukemic inhibitory factor (LIF) and serum provides bone morphogenetic proteins (BMPs) that are necessary to prevent ES cells from differentiating. These factors are extremely important for the efficiency of deriving ES cells.

Furthermore, it has been demonstrated that different mouse strains have different efficiencies for isolating ES cells. Current uses for mouse ES cells include the generation of transgenic mice, including knockout mice. For human treatment, there is a need for patient specific pluripotent cells. Generation of human ES cells is more difficult and faces ethical issues. So, in addition to human ES cell research, many groups are focused on the generation of induced pluripotent stem cells (iPS cells).

4. Potential clinical uses:


Reducing donor-host rejection There is also ongoing research to reduce the potential for rejection of the differentiated cells derived from ES cells once researchers are capable of creating an approved therapy from ES cell research. One of the possibilities to prevent rejection is by creating embryonic stem cells that are genetically identical to the patient via therapeutic cloning. An alternative solution for rejection by the patient to therapies derived from non-cloned ES cells is to derive many well-characterized ES cell lines from different genetic backgrounds and use the cell line that is most similar to the patient; treatment can then be tailored to the patient, minimizing the risk of rejection. Safety: reducing the risk of teratoma and other cancers as a side effect The major concern with the possible transplantation of ESC into patients as therapies is their ability to form tumors including teratoma. Safety issues prompted the FDA to place a hold on the first ESC clinical trial, however no tumors were observed. The main strategy to enhance the safety of ESC for potential clinical use is to differentiate the ESC into specific cell types (e.g. neurons, muscle, liver cells) that have reduced or eliminated ability to cause tumors. Following differentiation, the cells are subjected to sorting by flow cytometry for further purification. While ESC are predicted to be inherently safer than IPS cells because they are not genetically modified with genes such as c-Myc that are linked to cancer. Nonetheless ESC express very high levels of the iPS inducing genes and these genes including Myc are essential for ESC selfrenewal and pluripotency,[16] and potential strategies to improve safety by eliminating Myc expression are unlikely to preserve the cells' "stemness". First clinical trial On January 23, 2009, Phase I clinical trials for transplantation of oligodendrocytes (a cell type of the brain and spinal cord) derived from human ES cells into spinal cord-injured individuals received approval from the U.S. Food and Drug Administration (FDA), marking it the world's first human ES cell human trial. The study leading to this scientific

advancement was conducted by Hans Keirstead and colleagues at the University of California, Irvine and supported by Geron Corporation of Menlo Park, CA. A previous experiment had shown an improvement in locomotor recovery in spinal cord-injured rats after a 7-day delayed transplantation of human ES cells that had been pushed into an oligodendrocytic lineage. In the proposed phase I clinical study, about eight to ten paraplegics who have had their injuries no longer than two weeks before the trial begins, will be selected, since the cells must be injected before scar tissue is able to form. However, the researchers are emphasizing that the injections are not expected to fully cure the patients and restore all mobility. Based on the results of the rodent trials, researchers say restoration of myelin sheathes, and an increase in mobility is probable. This first trial is mainly testing the safety of these procedures and if everything goes well, it could lead to future studies that involve people with more severe disabilities. The trial had been put on hold in August 2009 due to concerns made by the FDA regarding a small number of microscopic cysts found in several treated rat models but the hold has been lifted as of July 30, 2010. Potential method for new cell line derivation On August 23, 2006, the online edition of Nature scientific journal published a letter by Dr. Robert Lanza (medical director of Advanced Cell Technology in Worcester, MA) stating that his team had found a way to extract embryonic stem cells without destroying the actual embryo. This technical achievement would potentially enable scientists to work with new lines of embryonic stem cells derived using public funding in the USA, where federal funding was at the time limited to research using embryonic stem cell lines derived prior to August 2001. In March, 2009, the limitation was lifted. This may enable the generation of patient specific ES cell lines that could potentially be used for cell replacement therapies. In addition, this will allow the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study those diseases. However, as a first indication that the induced pluripotent stem cell (iPS) cell technology can in rapid succession lead to new cures, it was used by a research team headed by Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, to cure mice of sickle cell anemia, as reported by Science journal's online edition on December 6, 2007. On January 16, 2008, a California based company, Stemagen, announced that they had created the first mature cloned human embryos from single skin cells taken from adults. These embryos can be harvested for patient matching embryonic stem cells. Use of human embryonic stem cells as models for human genetic disorders

In recent years there have been several reports regarding the potential use of human embryonic stem cells as models for human genetic diseases. This issue is especially important due to the species-specific nature of many genetic disorders. The relative inaccessibility of human primary tissue for research is another major hindrance. Several new studies have started to address this issue. This has been done either by genetically manipulating the cells, or more recently by deriving diseased cell lines identified by prenatal genetic diagnosis (PGD). This approach may very well prove invaluable at studying disorders such as Fragile-X syndrome, Cystic fibrosis, and other genetic maladies that have no reliable model system. Yury Verlinsky (Sept, 1, 1943 July 16, 2009), a Russian-American medical researcher who specialized in embryo and cellular genetics (genetic cytology), developed prenatal diagnosis testing methods to determine genetic and chromosomal disorders a month and a half earlier than standard amniocentesis. The techniques are now used by many pregnant women and prospective parents, especially those couples with a history of genetic abnormalities or where the woman is over the age of 35, when the risk of genetically-related disorders is higher. In addition, by allowing parents to select an embryo without genetic disorders, they have the potential of saving the lives of siblings that already had similar disorders and diseases using cells from the disease free offspring.

5. Use of human embryonic stem cells as model for human genetic disorders
In recent years there have been several reports regarding the potential use of human embryonic stem cells as models for human genetic diseases. This issue is especially important due to the species-specific nature of many genetic disorders. The relative inaccessibility of human primary tissue for research is another major hindrance. Several new studies have started to address this issue. This has been done either by genetically manipulating the cells, or more recently by deriving diseased cell lines identified by prenatal genetic diagnosis (PGD). This approach may very well prove invaluable at studying disorders such as Fragile-X syndrome, Cystic fibrosis, and other genetic maladies that have no reliable model system.Yury Verlinsky (Sept, 1, 1943 July 16, 2009), a Russian-American medical researcher who specialized in embryo and cellular genetics (genetic cytology), developed prenatal diagnosis testing methods to determine genetic and chromosomal disorders a month and a half earlier than standard amniocentesis. The techniques are now used by many pregnant women and prospective parents, especially those couples with a history of genetic abnormalities or where the woman is over the age of 35, when the risk of genetically-related disorders is higher. In addition, by allowing parents to select an embryo without genetic disorders, they have the potential of saving the lives of siblings that already had similar disorders and diseases using cells from the disease free offspring.

6. Recent development and research:


An MIT team has developed new technology that could jump-start scientists' ability to create specific cell types from human embryonic stem cells, a feat with implications for developing replacement organs and a variety of other tissue engineering applications.

The team developed robotic technology to deposit more than 1,700 spots of biomaterial (roughly 500 different materials in triplicate) on a glass slide measuring only 25 millimeters wide by 75 long. Twenty such slides, or microarrays, can be made in a single day. Exposure to ultraviolet light polymerizes the biomaterials, making each spot rigid and thus making the microarray ready for "seeding" with hES or other cells. (In the current work, the team seeded some arrays with hES and some with embryonic muscle cells.) Each seeded microarray can then be placed in a different solution, including such things as growth factors, to incubate. Another plus: the microarrays work with a minimal number of cells, growth factors and other media. Many of the media related to testing the cells, such as antibodies, are also expensive. In the current work, the scientists used an initial screening to find especially promising biomaterials for the differentiation of hES into epithelial cells. Additional experiments identified "a host of unexpected materials effects that offer new levels of control over hES cell behavior," the team writes, demonstrating the power of quick, easy screenings. This work was funded by the National Science Foundation through MIT's Biotechnology Process Engineering Center (http://web.mit.edu/bpec) and by the National Institutes of Health.

7. Conclusion:

Stem cell research is being pursued in the hope of achieving major medical breakthroughs. Recent scientific breakthroughs, celebrity patient advocates, and conflicting religious beliefs have come together to bring the state of stem cell research specifically embryonic stem cell research into the political crosshairs. Stem cells can develop into different cell types. They may offer a renewable source of replacement cells to treat diseases, conditions, and disabilities.

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References
1. Dudey R.C., A Textbook of Biotechnology, S.Chand & co.(2008) 2. Ranga M.M., Animal Biotechnology, Agarosbiose India 4. http://web.mit.edu/bpec 5. http://www.geron.com/stemcellprogram.aspx.htm 6. http://dels-old.nas.edu/bls/stemcells/

3. http://en.wikipedia.org/Embryonic_stem_cell.htm#cite_note-10

(adopted from: http://en.wikipedia.org/wiki/File:Stem_cells_diagram.png)

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