Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Human embryonic stem cells are currently produced from surplus embryos of in vitro fertilization procedures.
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 only produce a limited number of cell types.
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.
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, lead by James Thomson at the University of Wisconsin-Madison, first developed a technique to isolate and grow human embryonic stem cells in cell 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 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, 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).
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. 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.
However, a study published in the online edition of Lancet Medical Journal on March 8, 2005 detailed information about a new stem cell line which 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.
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.
On January 23, 2009, Phase I clinical trials for transplantation of a human-ES-derived cell population 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. The results of this experiment suggested an improvement in locomotor recovery in spinal cord-injured rats after a 7-day delayed transplantation of human ES cells that were pushed towards an oligodendrocytic lineage . In the proposed phase I clinical stidy, 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.. Unfortunately the trial is on hold since August 2009 due to concerns made by the FDA regarding a small number of microscopic cysts found in several treated rat models. If all goes well with Gerons follow-up experiments the clinical trial should resume by the end of 2010..
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.
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. 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
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 December 6.
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.
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.
Embryonic stem cells (ESCs), by definition, are primary cell cultures that have the ability to proliferate indefinitely with unlimited differentiation potential, both in vitro and vivo. Each ESC has the property of pluripotency, or rather the ability to differentiate into any cell type found in somatic tissues, including germ cells. All ESCs maintain the property of self-renewal, whereas each ESC can divide to form two clonal daughter cells, each with the exact same properties as the original ESC. An ESC may divide asymmetrically as well, meaning one clonal ESC is produced as well as another daughter cell destined for differentiation. With these combined properties, the current and potential applications of ESCs for use in biomedical and biotechnological applications is advancing rapidly.
Beginning in the mid-twentieth century, several investigators began working with an specific type of tumor. This tumor, termed a teratocarcinoma, contained within it many fully-developed tissues normally associated with embryonic and fetal development (including teeth, hair, gut-epithelial tissue, etc.). The disorganized tumor containing fully differentiated tissues suggested that certain primordial cell types were directing its aberrant growth. Eventually, it was discovered that these tumors were related to germ-cell tumors, specifically those arising from the testis. These germ-cell carcinomas led directly to modern embryonic stem cell basic research as a first-cause investigation into the origin of the programming that led to the tumors.
By the 1980's research into mouse embryogenesis was advanced enough to allow for the first isolation and characterization of mouse embryonic stem cells (mESCs) by Evans and Kaufman in the 1980's. These cells were isolation on a monolayer of mitotically-inactivated mouse primary fibroblast feeder cells, aka "feeders" or "MEFs". It was found that the addition of these support cells was necessary for the maintainence of the undifferentiated state of the mESCs in fetal bovine serum-containing media. The replicative and proliferative properties were noticed immediately by researchers, and so more thorough investigations into the mechanisms that allowed these cells to grow like cancer cell lines, but have the properties of primary cell cultures.
Shortly thereafter it was discovered by Dr. Austin Smith that the primary contribution of the feeders is Leukemia Inhibitory Factor (LIF), an IL-6 cytokine family member, that is secreted into the growth media and activates the LIF-JAK-STAT3 pathway to promote mESC self-renewal. These cells were further analyzed to express the canonical markers of pluripotency: Oct3/4, the master transcription factor responsible for the "stem-ness" of the cell; Sox-2, another transcription factor suggested to help re-model the epigenome; Nanog, like Oct3/4, a master pluripotency regulator, and Rex-1, a gene helps regulate the pluripotency network established by the previous three genes. LIF-supplemented culture medium has been sufficient thus far in maintaining most mouse ESC lines for lengthy passages (>P50). However, many "feeder-free" cell lines have adapted to the differing culture conditions by becoming karyotypically abnormal. This again, reflects the need to understand the fundamental self-renewal mechanism to better control these cells in vitro.
Recently, however it has been demonstrated that for feeder-dependent cells lines, LIF is not enough to maintain the pluripotent undifferentiated state once the feeders are removed from the culture system. This so-called mESC "crisis" was first described by Sir Martin Evans. Investigations have tried to identify another soluble factor that may promote self-renewal or actively block differentiation cues, however no known factor has been implicated as yet. Several studies have suggested that the cell-dependent contact is the missing factor, and so many studies have implicated the extra-cellular matrix as a likely candidate. So far, though, the elusive self-renewal factor in mESCs remains un-identified. For most basic research applications however, the current culture system, although hetereogenous, is satisfactory for conducting rigorous testing.
The first human embryonic stem cell (hESC) line was derived in 1998 by Dr. James Thomson at the University of Wisconsin. These cells, like their murine counterparts, require a feeder cell layer to remain undifferentiated. Unlike mouse cells however, is their insensitivity to LIF for self-renewal. hESCs require bFGF, and in some cases, Activin A. A consistent media formulation is still being being debated by different researchers, so there is considerable variation between laboratories in the manner in which the hESC lines are maintained. This debate has fueled the dialogue necessary to develop a standard media for both human and mouse ESCs. Scientists in the field are trying to rid the culture system of the feeders, in hopes of homogenizing culture conditions. But a limited understanding of which signal pathway(s) are involved in both mouse and human ESCs are hindering this movement. Another obvious problem is the lack of homology between human and mouse ESC lines. Other than a few pluripotency markers, such as Oct3/4, Sox-2, Nanog, and Rex-1, differentiation and self-renewal protocols used for each species is disparate at best. Yet, the potential applications for each are still better in comparison to the current tools available to researchers and clinicians now.