This article may be in need of reorganization to comply with Wikipedia's layout guidelines. Please help by editing the article to make improvements to the overall structure. (November 2011)

Human embryonic stem cells in cell culture

Pluripotent: Embryonic stem cells are able to develop into any type of cell, excepting those of the placenta. Only embryonic stem cells of the morula are totipotent: able to develop into any type of cell, including those of the placenta.

Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo.^[1] Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the embryoblast or inner cell mass (ICM) results in destruction of the fertilized human embryo, which raises ethical issues. Those issues include whether or not a human life at the embryonic stage should be granted the moral status of a human being as it is with a child or an adult.^{[2] [3]}

Types[link]

Embryonic stem cells are distinguished by two distinctive properties:

• their pluripotency, and
• their ability to replicate indefinitely.^[4]

ES cells are pluripotent, that is, they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult body. Pluripotency distinguishes embryonic stem cells from adult stem cells found in adults; while embryonic stem cells can generate all cell types in the body, adult stem cells are multipotent and can produce only a limited number of cell types. Human ES cells measure approximately 14μm while mouse ES cells are closer to 8μm.^[5]

Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely. This allows embryonic stem cells to be employed as useful tools for both research and regenerative medicine, because they can produce limitless numbers of themselves for continued research or clinical use.

Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. Diseases that could potentially be treated by pluripotent stem cells include a number of blood and immune-system related genetic diseases, cancers, and disorders; juvenile diabetes; Parkinson's; blindness and spinal cord injuries. Besides the ethical concerns of stem cell therapy (see stem cell controversy), there is a technical problem of graft-versus-host disease associated with allogeneic stem cell transplantation. However, these problems associated with histocompatibility may be solved using autologous donor adult stem cells, therapeutic cloning, stem cell banks or more recently by reprogramming of somatic cells with defined factors (e.g. induced pluripotent stem cells). Other potential uses of embryonic stem cells include investigation of early human development, study of genetic disease and as in vitro systems for toxicology testing.

Research history and developments[link]

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.^[6] Although similarities in morphology and differentiating potential (pluripotency) led to the use of EC cells as the in vitro model for early mouse development,^[7] 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.

Martin Evans

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.^[8] 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”.^[9] 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 Wisconsin-Madison, first developed a technique to isolate and grow human embryonic stem cells in cell culture.^[10]

Techniques and Conditions for Embryonic Stem Cell Derivation and Culture[link]

Derivation of Human Embryonic Stem Cells:
In vitro fertilization generates multiple embryos. The surplus of embryos is not clinically used or is unsuitable for implantation into the patient, and therefore may be donated by the donor with consent. Human embryonic stem cells are derived from these donated embryos that would otherwise be discarded.^[11] The inner cell mass (cells of interest), from the blastocyst stage of the embryo, is separated from the trophectoderm, the cells that would differentiate into extra-embryonic tissue. Immunosurgery, the process in which antibodies are bound to the trophectoderm and removed by another solution, and mechanical dissection are performed to achieve separation. The resulting inner cell mass cells are plated onto cells that will supply support. The inner cell mass cells attach and expand further to form a human embryonic cell line, which are undifferentiated. These cells are fed daily and are enzymatically or mechanically separated every four to seven days. For differentiation to occur, the human embryonic stem cell line is removed from the supporting cells to form embryoid bodies, is co-cultured with a serum containing necessary signals, or is grafted in a three-dimensional scaffold to result.^[12]

Derivation of Embryonic Stem Cells from other animals:
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 mother’s ovaries and dosing her with progesterone, changing the hormone environment, which causes the embryos to remain free in the uterus. After 4–6 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, all of which indicating that the cells are pluripotent.^[8]

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.^[9]

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.^[13][14] 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.^[15] 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).^[16]

Contamination by reagents used in cell culture[link]

The online edition of Nature Medicine published a study on January 24, 2005, which stated that the human embryonic stem cells available for federally funded research are contaminated with non-human molecules from the culture medium used to grow the cells.^[17] It is a common technique to use mouse cells and other animal cells to maintain the pluripotency of actively dividing stem cells. The problem was discovered when non-human sialic acid in the growth media was found to compromise the potential uses of the embryonic stem cells in humans, according to scientists at the University of California, San Diego.^[18]

However, a study published in the online edition of Lancet Medical Journal on March 8, 2005 detailed information about a new stem cell line that was derived from human embryos under completely cell- and serum-free conditions. After more than 6 months of undifferentiated proliferation, these cells demonstrated the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem cell lines.^[19]

Potential Clinical Use[link]

Human embryonic stem cells have the ability to differentiate into various cell types, therefore useful for transplantation or tissue engineering(Langer, Robert).

Embryoid bodies 24 hours after formation.

Reducing donor-host rejection[link]

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[link]

• The major concern with the possible transplantation of ESC into patients as therapies is their ability to form tumors including teratoma.^[20] Safety issues prompted the FDA to place a hold on the first ESC clinical trial (see below), 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. 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 self-renewal and pluripotency,^[21] and potential strategies to improve safety by eliminating Myc expression are unlikely to preserve the cells' "stemness".

First clinical trial[link]

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.^[22] 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.^[23] 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.^[24] 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.^[25][26][27]

In October 2010 researchers enrolled and administered ESTs to the first patient at Shepherd Center in Atlanta.^[28] The makers of the stem cell therapy, Geron Corporation, estimate that it will take several months for the stem cells to replicate and for the GRNOPC1 therapy to be evaluated for success or failure. In November 2011 Geron announced it was dropping out of stem cell research for financial reasons, but would continue to monitor existing patients, and was attempting to find a partner that could continue their research.^[29]

Potential method for new cell line derivation[link]

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.^[30] 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.^[31]

Recently, it was shown that pluripotent stem cells highly similar to embryonic stem cells can be generated by the delivery of three genes (Oct4, Sox2, and Klf4) to differentiated cells.^[32] The delivery of these genes "reprograms" differentiated cells into pluripotent stem cells, allowing for the generation of pluripotent stem cells without the embryo. Because ethical concerns regarding embryonic stem cells typically are about their derivation from terminated embryos, it is believed that reprogramming to these "induced pluripotent stem cells" (iPS cells) may be less controversial. Both human and mouse cells can be reprogrammed by this methodology, generating both human pluripotent stem cells and mouse pluripotent stem cells without an embryo.^[33]

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.^[34]

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.^[35]

Use of human embryonic stem cells as models for human genetic disorders[link]

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.^[36]

See also[link]

• Embryonic Stem Cell Research Oversight (ESCRO) Committees
• Embryoid body
• Stem cell controversy
• Fetal tissue implant

References[link]

Langer, Robert. "Endothelial Cells Derived from Human Embryonic Stem Cells." Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences, 26 Mar. 2002. Web. 15 Apr. 2012. <http://www.pnas.org/content/99/7/4391.short>.

External links[link]

• Understanding Stem Cells: A View of the Science and Issues from the National Academies
• National Institutes of Health
• Fact sheet on embryonic stem cells
• Fact sheet on ethical issues in embryonic stem cell research
• The Center for Bioethics & Human Dignity
• Information & Alternatives to Embryonic Stem Cell Research
• A blog focusing specifically on ES cells and iPS cells including research, biotech, and patient-oriented issues

• Rappaport Institute of the Technion [1]
• insertech.simdif.com