Stem Cell Clinic

Embryonic stem cell – Wikipedia

Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo.[1][2] Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells. Isolating the embryoblast or inner cell mass (ICM) results in destruction of the blastocyst, which raises ethical issues, including whether or not embryos at the pre-implantation stage should be considered to have the same moral or legal status as more developed human beings.[3][4]

Human ES cells measure approximately 14 m while mouse ES cells are closer to 8 m.[5]

Embryonic stem cells, derived from the blastocyst stage early mammalian embryos, are distinguished by their ability to differentiate into any cell type and by their ability to propagate. Embryonic stem cell's properties include having a normal karyotype, maintaining high telomerase activity, and exhibiting remarkable long-term proliferative potential.[6]

Embryonic stem cells of the inner cell mass are pluripotent, that is, they are able to differentiate to generate primitive ectoderm, which ultimately differentiates during gastrulation 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. If the pluripotent differentiation potential of embryonic stem cells could be harnessed in vitro, it might be a means of deriving cell or tissue types virtually to order. This would provide a radical new treatment approach to a wide variety of conditions where age, disease, or trauma has led to tissue damage or dysfunction.

Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely in an undifferentiated state and have the capacity when provided with the appropriate signals to differentiate, presumably via the formation of precursor cells, to almost all mature cell phenotypes.[7] 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, embryonic stem 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 disease; 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). Embryonic stem cells provide hope that it will be possible to overcome the problems of donor tissue shortage and also, by making the cells immunocompatible with the recipient. 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.[6]

According to a 2002 article in PNAS, "Human embryonic stem cells have the potential to differentiate into various cell types, and, thus, may be useful as a source of cells for transplantation or tissue engineering."[8]

Current research focuses on differentiating ES into a variety of cell types for eventual use as cell replacement therapies (CRTs). Some of the cell types that have or are currently being developed include cardiomyocytes (CM), neurons, hepatocytes, bone marrow cells, islet cells and endothelial cells.[9] However, the derivation of such cell types from ESs is not without obstacles and hence current research is focused on overcoming these barriers. For example, studies are underway to differentiate ES in to tissue specific CMs and to eradicate their immature properties that distinguish them from adult CMs.[10]

Besides in the future becoming an important alternative to organ transplants, ES are also being used in field of toxicology and as cellular screens to uncover new chemical entities (NCEs) that can be developed as small molecule drugs. Studies have shown that cardiomyocytes derived from ES are validated in vitro models to test drug responses and predict toxicity profiles.[9] ES derived cardiomyocytes have been shown to respond to pharmacological stimuli and hence can be used to assess cardiotoxicity like Torsades de Pointes.[11]

ES-derived hepatocytes are also useful models that could be used in the preclinical stages of drug discovery. However, the development of hepatocytes from ES has proven to be challenging and this hinders the ability to test drug metabolism. Therefore, current research is focusing on establishing fully functional ES-derived hepatocytes with stable phase I and II enzyme activity.[12]

Researchers have also differentiated ES into dopamine-producing cells with the hope that these neurons could be used in the treatment of Parkinsons disease.[13][14] Recently, the development of ESC after Somatic Cell Nuclear Transfer (SCNT) of Olfactory ensheathing cells (OEC's) to a healthy Oocyte has been recommended for Neuro-degenerative diseases.[15] ESs have also been differentiated to natural killer (NK) cells and bone tissue.[16] Studies involving ES are also underway to provide an alternative treatment for diabetes. For example, DAmour et al. were able to differentiate ES into insulin producing cells[17] and researchers at Harvard University were able to produce large quantities of pancreatic beta cells from ES.[18]

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, 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.[19]

Scientists have discovered a new technique for deriving human embryonic stem cell (ESC). Normal ESC lines from different sources of embryonic material including morula and whole blastocysts have been established. These findings allows researchers to construct ESC lines from embryos that acquire different genetic abnormalities; therefore, allowing for recognition of mechanisms in the molecular level that are possibly blocked that could impede the disease progression. The ESC lines originating from embryos with genetic and chromosomal abnormalities provide the data necessary to understand the pathways of genetic defects.[20]

A donor patient acquires one defective gene copy and one normal, and only one of these two copies is used for reproduction. By selecting egg cell derived from embryonic stem cells that have two normal copies, researchers can find variety of treatments for various diseases. To test this theory Dr. McLaughlin and several of his colleagues looked at whether parthenogenetic embryonic stem cells can be used in a mouse model that has thalassemia intermedia. This disease is described as an inherited blood disorder in which there is a lack of hemoglobin leading to anemia. The mouse model used, had one defective gene copy. Embryonic stem cells from an unfertilized egg of the diseased mice were gathered and those stem cells that contained only healthy hemoglobin genes were identified. The healthy embryonic stem cell lines were then converted into cells transplanted into the carrier mice. After five weeks, the test results from the transplant illustrated that these carrier mice now had a normal blood cell count and hemoglobin levels.[21]

Differentiated somatic cells and ES cells use different strategies for dealing with DNA damage. For instance, human foreskin fibroblasts, one type of somatic cell, use non-homologous end joining (NHEJ), an error prone DNA repair process, as the primary pathway for repairing double-strand breaks (DSBs) during all cell cycle stages.[22] Because of its error-prone nature, NHEJ tends to produce mutations in a cells clonal descendants.

ES cells use a different strategy to deal with DSBs.[23] Because ES cells give rise to all of the cell types of an organism including the cells of the germ line, mutations arising in ES cells due to faulty DNA repair are a more serious problem than in differentiated somatic cells. Consequently, robust mechanisms are needed in ES cells to repair DNA damages accurately, and if repair fails, to remove those cells with un-repaired DNA damages. Thus, mouse ES cells predominantly use high fidelity homologous recombinational repair (HRR) to repair DSBs.[23] This type of repair depends on the interaction of the two sister chromosomes formed during S phase and present together during the G2 phase of the cell cycle. HRR can accurately repair DSBs in one sister chromosome by using intact information from the other sister chromosome. Cells in the G1 phase of the cell cycle (i.e. after metaphase/cell division but prior the next round of replication) have only one copy of each chromosome (i.e. sister chromosomes arent present). Mouse ES cells lack a G1 checkpoint and do not undergo cell cycle arrest upon acquiring DNA damage.[24] Rather they undergo programmed cell death (apoptosis) in response to DNA damage.[25] Apoptosis can be used as a fail-safe strategy to remove cells with un-repaired DNA damages in order to avoid mutation and progression to cancer.[26] Consistent with this strategy, mouse ES stem cells have a mutation frequency about 100-fold lower than that of isogenic mouse somatic cells.[27]

The major concern with the possible transplantation of ESC into patients as therapies is their ability to form tumors including teratoma.[28] 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,[29] and potential strategies to improve safety by eliminating Myc expression are unlikely to preserve the cells' "stemness".

In 1964, Lewis Kleinsmith and G. Barry Pierce Jr. isolated a single type of cell from a teratocarcinoma, a tumor now known to be derived from a germ cell.[30] These cells isolated from the teratocarcinoma replicated and grew in cell culture as a stem cell and are now known as embryonal carcinoma (EC) cells.[31] Although similarities in morphology and differentiating potential (pluripotency) led to the use of EC cells as the in vitro model for early mouse development,[32] 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.[33]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.[34] 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.[35]

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.[36] 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, founded by Michael D. West, PhD. 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.[37] The phase I clinical study was designed to enroll about eight to ten paraplegics who have had their injuries no longer than two weeks before the trial begins, since the cells must be injected before scar tissue is able to form. The researchers emphasized that the injections were not expected to fully cure the patients and restore all mobility. Based on the results of the rodent trials, researchers speculated that restoration of myelin sheathes and an increase in mobility might occur. This first trial was primarily designed to test the safety of these procedures and if everything went well, it was hoped that it would lead to future studies that involve people with more severe disabilities.[38] The trial was put on hold in August 2009 due to FDA concerns regarding a small number of microscopic cysts found in several treated rat models but the hold was lifted on July 30, 2010.[39]

In October 2010 researchers enrolled and administered ESTs to the first patient at Shepherd Center in Atlanta.[40] The makers of the stem cell therapy, Geron Corporation, estimated that it would 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 halting the trial and 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.[41] In 2013 BioTime (NYSEMKT:BTX), led by CEO Dr. Michael D. West, acquired all of Geron's stem cell assets, with the stated intention of restarting Geron's embryonic stem cell-based clinical trial for spinal cord injury research.[42]

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 can be derived from these donated embryos or additionally they can also be extracted from cloned embryos using a cell from a patient and a donated egg.[43] 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.[44]

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 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, all of which indicating that the cells are pluripotent.[33]

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 a medium 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 a medium containing serum and conditioned by ES 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.[34]

It is now known that the feeder cells provide leukemia inhibitory factor (LIF) and serum provides bone morphogenetic proteins (BMPs) that are necessary to prevent ES cells from differentiating.[45][46] 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.[47] 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).[48]

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.[49] 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.[50]

In 2007 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.[51] 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.[52]

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.[53][54]

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.[55]

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.[56] 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 medium was found to compromise the potential uses of the embryonic stem cells in humans, according to scientists at the University of California, San Diego.[57]

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.[58]

Visit link:
Embryonic stem cell - Wikipedia

Embryonic Stem Cells | stemcells.nih.gov

by Junying Yu* and James A. Thomson**

Human embryonic stem (ES) cells capture the imagination because they are immortal and have an almost unlimited developmental potential (Fig. 1.1: How hESCs are derived). After many months of growth in culture dishes, these remarkable cells maintain the ability to form cells ranging from muscle to nerve to bloodpotentially any cell type that makes up the body. The proliferative and developmental potential of human ES cells promises an essentially unlimited supply of specific cell types for basic research and for transplantation therapies for diseases ranging from heart disease to Parkinson's disease to leukemia. Here we discuss the origin and properties of human ES cells, their implications for basic research and human medicine, and recent research progress since August 2001, when President George W. Bush allowed federal funding of this research for the first time. A previous report discussed progress prior to June 17, 2001 (/info/scireport/.)

Figure 1.1. How Human Embryonic Stem Cells are Derived.

( 2006 Terese Winslow)

Embryonic stem cells are derived from embryos at a developmental stage before the time that implantation would normally occur in the uterus. Fertilization normally occurs in the oviduct, and during the next few days, a series of cleavage divisions occur as the embryo travels down the oviduct and into the uterus. Each of the cells (blastomeres) of these cleavage-stage embryos are undifferentiated, i.e. they do not look or act like the specialized cells of the adult, and the blastomeres are not yet committed to becoming any particular type of differentiated cell. Indeed, each of these blastomeres has the potential to give rise to any cell of the body. The first differentiation event in humans occurs at approximately five days of development, when an outer layer of cells committed to becoming part of the placenta (the trophectoderm) separates from the inner cell mass (ICM). The ICM cells have the potential to generate any cell type of the body, but after implantation, they are quickly depleted as they differentiate to other cell types with more limited developmental potential. However, if the ICM is removed from its normal embryonic environment and cultured under appropriate conditions, the ICM-derived cells can continue to proliferate and replicate themselves indefinitely and still maintain the developmental potential to form any cell type of the body (quot;pluripotencyquot;; see Fig. 1.2: Characteristics of ESCs). These pluripotent, ICM-derived cells are ES cells.

Figure 1.2.Characteristics of Embryonic Stem Cells.

( 2006 Terese Winslow)

The derivation of mouse ES cells was first reported in 1981,1,2 but it was not until 1998 that derivation of human ES cell lines was first reported.3 Why did it take such a long time to extend the mouse results to humans? Human ES cell lines are derived from embryos produced by in vitro fertilization (IVF), a process in which oocytes and sperm are placed together to allow fertilization to take place in a culture dish. Clinics use this method to treat certain types of infertility, and sometimes, during the course of these treatments, IVF embryos are produced that are no longer needed by the couples for producing children. Currently, there are nearly 400,000 IVF-produced embryos in frozen storage in the United States alone,4 most of which will be used to treat infertility, but some of which (~2.8%) are destined to be discarded. IVF-produced embryos that would otherwise have been discarded were the sources of the human ES cell lines derived prior to President Bush's policy decision of August 2001. These human ES cell lines are now currently eligible for federal funding. Although attempts to derive human ES cells were made as early as the 1980s, culture media for human embryos produced by IVF were suboptimal. Thus, it was difficult to culture single-cell fertilized embryos long enough to obtain healthy blastocysts for the derivation of ES cell lines. Also, species-specific differences between mice and humans meant that experience with mouse ES cells was not completely applicable to the derivation of human ES cells. In the 1990s, ES cell lines from two non-human primates, the rhesus monkey5 and the common marmoset,6 were derived, and these offered closer models for the derivation of human ES cells. Experience with non-human primate ES cell lines and improvements in culture medium for human IVF-produced embryos led rapidly to the derivation of human ES cell lines in 1998.3

Because ES cells can proliferate without limit and can contribute to any cell type, human ES cells offer an unprecedented access to tissues from the human body. They will support basic research on the differentiation and function of human tissues and provide material for testing that may improve the safety and efficacy of human drugs (Figure 1.3: Promise of SC Research).7,8 For example, new drugs are not generally tested on human heart cells because no human heart cell lines exist. Instead, researchers rely on animal models. Because of important species-specific differences between animal and human hearts, however, drugs that are toxic to the human heart have occasionally entered clinical trials, sometimes resulting in death. Human ES cell-derived heart cells may be extremely valuable in identifying such drugs before they are used in clinical trials, thereby accelerating the drug discovery process and leading to safer and more effective treatments.911 Such testing will not be limited to heart cells, but to any type of human cell that is difficult to obtain by other sources.

Figure 1.3: The Promise of Stem Cell Research.

( 2006 Terese Winslow)

Human ES cells also have the potential to provide an unlimited amount of tissue for transplantation therapies to treat a wide range of degenerative diseases. Some important human diseases are caused by the death or dysfunction of one or a few cell types, e.g., insulin-producing cells in diabetes or dopaminergic neurons in Parkinson's disease. The replacement of these cells could offer a lifelong treatment for these disorders. However, there are a number of challenges to develop human ES cell-based transplantation therapies, and many years of basic research will be required before such therapies can be used to treat patients. Indeed, basic research enabled by human ES cells is likely to impact human health in ways unrelated to transplantation medicine. This impact is likely to begin well before the widespread use of ES cells in transplantation and ultimately could have a more profound long-term effect on human medicine. Since August 2001, improvements in culture of human ES cells, coupled with recent insights into the nature of pluripotency, genetic manipulation of human ES cells, and differentiation, have expanded the possibilities for these unique cells.

Mouse ES cells and human ES cells were both originally derived and grown on a layer of mouse fibroblasts (called quot;feeder cellsquot;) in the presence of bovine serum. However, the factors that sustain the growth of these two cell types appear to be distinct. The addition of the cytokine, leukemia inhibitory factor (LIF), to serum-containing medium allows mouse ES cells to proliferate in the absence of feeder cells. LIF modulates mouse ES cells through the activation of STAT3 (signal transducers and activators of transcription) protein. In serum-free culture, however, LIF alone is insufficient to prevent mouse ES cells from differentiating into neural cells. Recently, Ying et al. reported that the combination of bone morphogenetic proteins (BMPs) and LIF is sufficient to support the self-renewal of mouse ES cells.12 The effects of BMPs on mouse ES cells involve induction of inhibitor of differentiation (Id) proteins, and inhibition of extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinases (MAPK).12,13 However, LIF in the presence of serum is not sufficient to promote the self-renewal of human ES cells,3 and the LIF/STAT3 pathway appears to be inactive in undifferentiated human ES cells.14,15 Also, the addition of BMPs to human ES cells in conditions that would otherwise support ES cells leads to the rapid differentiation of human ES cells.16,17

Several groups have attempted to define growth factors that sustain human ES cells and have attempted to identify culture conditions that reduce the exposure of human ES cells to non human animal products. One important growth factor, bFGF, allows the use of a serum replacement to sustain human ES cells in the presence of fibroblasts, and this medium allowed the clonal growth of human ES cells.18 A quot;feeder-freequot; human ES cell culture system has been developed, in which human ES cells are grown on a protein matrix (mouse Matrigel or Laminin) in a bFGF-containing medium that is previously quot;conditionedquot; by co-culture with fibroblasts.19 Although this culture system eliminates direct contact of human ES cells with the fibroblasts, it does not remove the potential for mouse pathogens being introduced into the culture via the fibroblasts. Several different sources of human feeder cells have been found to support the culture of human ES cells, thus removing the possibility of pathogen transfer from mice to humans.2023 However, the possibility of pathogen transfer from human to human in these culture systems still remains. More work is still needed to develop a culture system that eliminates the use of fibroblasts entirely, which would also decrease much of the variability associated with the current culture of human ES cells. Sato et al. reported that activation of the Wnt pathway by 6-bromoindirubin3'-oxime (BIO) promotes the self-renewal of ES cells in the presence of bFGF, Matrigel, and a proprietary serum replacement product.24 Amit et al. reported that bFGF, TGF, and LIF could support some human ES cell lines in the absence of feeders.25 Although there are some questions about how well these new culture conditions will work for different human ES cell lines, there is now reason to believe that defined culture conditions for human ES cells, which reduce the potential for contamination by pathogens, will soon be achieved*.

Once a set of defined culture conditions is established for the derivation and culture of human ES cells, challenges to improve the medium will still remain. For example, the cloning efficiency of human ES cellsthe ability of a single human ES cell to proliferate and become a colonyis very low (typically less than 1%) compared to that of mouse ES cells. Another difficulty is the potential for accumulation of genetic and epigenetic changes over prolonged periods of culture. For example, karyotypic changes have been observed in several human ES cell lines after prolonged culture, and the rate at which these changes dominate a culture may depend on the culture method.26,27 The status of imprinted (epigenetically modified) genes and the stability of imprinting in various culture conditions remain completely unstudied in human ES cells**. The status of imprinted genes can clearly change with culture conditions in other cell types.28,29 These changes present potential problems if human ES cells are to be used in cell replacement therapy, and optimizing medium to reduce the rate at which genetic and epigenetic changes accumulate in culture represents a long-term endeavor. The ideal human ES cell medium, then, (a) would be cost-effective and easy to use so that many more investigators can use human ES cells as a research tool; (b) would be composed entirely of defined components not of animal origin; (c) would allow cell growth at clonal densities; and (d) would minimize the rate at which genetic and epigenetic changes accumulate in culture. Such a medium will be a challenge to develop and will most likely be achieved through a series of incremental improvements over a period of years.

Among all the newly derived human ES cell lines, twelve lines have gained the most attention. In March 2004, a South Korean group reported the first derivation of a human ES cell line (SCNT-hES-1) using the technique of somatic cell nuclear transfer (SCNT). Human somatic nuclei were transferred into human oocytes (nuclear transfer), which previously had been stripped of their own genetic material, and the resultant nuclear transfer products were cultured in vitro to the blastocyst stage for ES cell derivation.30*** Because the ES cells derived through nuclear transfer contain the same genetic material as that of the nuclear donor, the intent of the procedure is that the differentiated derivatives would not be rejected by the donor's immune system if used in transplantation therapy. More recently, the same group reported the derivation of eleven more human SCNT-ES cell lines*** with markedly improved efficiency (16.8 oocytes/line vs. 242 oocytes/line in their previous report).31*** However, given the abnormalities frequently observed in cloned animals, and the costs involved, it is not clear how useful this procedure will be in clinical applications. Also, for some autoimmune diseases, such as type I diabetes, merely providing genetically-matched tissue will be insufficient to prevent immune rejection.

Additionally, new human ES cell lines were established from embryos with genetic disorders, which were detected during the practice of preimplantation genetic diagnosis (PGD). These new cell lines may provide an excellent in vitro model for studies on the effects that the genetic mutations have on cell proliferation and differentiation.32

* Editor's note: Papers published since this writing report defined culture conditions for human embryonic stem cells. See Ludwig et al., Nat. Biotech 24: 185187, 2006; and Lu et al., PNAS 103:56885693, 2006.08.14.

** Editor's note: Papers published since the time this chapter was written address this: see Maitra et al., Nature Genetics 37, 10991103, 2005; and Rugg-Gunn et al., Nature Genetics 37:585587, 2005.

*** Editor's note: Both papers referenced in 30 and 31 were later retracted: see Science 20 Jan 2006; Vol. 311. No. 5759, p. 335.

To date, more than 120 human ES cell lines have been established worldwide,33* 67 of which are included in the National Institutes of Health (NIH) Registry. As of this writing, 21 cell lines are currently available for distribution, all of which have been exposed to animal products during their derivation. Although it has been eight years since the initial derivation of human ES cells, it is an open question as to the extent that independent human ES cell lines differ from one another. At the very least, the limited number of cell lines cannot represent a reasonable sampling of the genetic diversity of different ethnic groups in the United States, and this has consequences for drug testing, as adverse reactions to drugs often reflect a complex genetic component. Once defined culture conditions are well established for human ES cells, there will be an even more compelling need to derive additional cell lines.

* Editor's note: One recent report now estimates 414 hESC lines, see Guhr et al., http://www.StemCells.com early online version for June 15, 2006: quot;Current State of Human Embryonic Stem Cell Research: An Overview of Cell Lines and their Usage in Experimental Work.quot;

The ability of ES cells to develop into all cell types of the body has fascinated scientists for years, yet remarkably little is known about factors that make one cell pluripotent and another more restricted in its developmental potential. The transcription factor Oct4 has been used as a key marker for ES cells and for the pluripotent cells of the intact embryo, and its expression must be maintained at a critical level for ES cells to remain undifferentiated.34 The Oct4 protein itself, however, is insufficient to maintain ES cells in the undifferentiated state. Recently, two groups identified another transcription factor, Nanog, that is essential for the maintenance of the undifferentiated state of mouse ES cells.35,36 The expression of Nanog decreased rapidly as mouse ES cells differentiated, and when its expression level was maintained by a constitutive promoter, mouse ES cells could remain undifferentiated and proliferate in the absence of either LIF or BMP in serum-free medium.12 Nanog is also expressed in human ES cells, though at a much lower level compared to that of Oct4, and its function in human ES cells has yet to be examined.

By comparing gene expression patterns between different ES cell lines and between ES cells and other cell types such as adult stem cells and differentiated cells, genes that are enriched in the ES cells have been identified. Using this approach, Esg-1, an uncharacterized ES cell-specific gene, was found to be exclusively associated with pluripotency in the mouse.37 Sperger et al. identified 895 genes that are expressed at significantly higher levels in human ES cells and embryonic carcinoma cell lines, the malignant counterparts to ES cells.38 Sato et al. identified a set of 918 genes enriched in undifferentiated human ES cells compared with their differentiated counterparts; many of these genes were shared by mouse ES cells.39 Another group, however, found 92 genes, including Oct4 and Nanog, enriched in six different human ES cell lines, which showed limited overlap with those in mouse ES cell lines.40 Care must be taken to interpret these data, and the considerable differences in the results may arise from the cell lines used in the experiments, methods to prepare and maintain the cells, and the specific methods used to profile gene expression.

Since establishing human ES cells in 1998, scientists have developed genetic manipulation techniques to determine the function of particular genes, to direct the differentiation of human ES cells towards specific cell types, or to tag an ES cell derivative with a certain marker gene. Several approaches have been developed to introduce genetic elements randomly into the human ES cell genome, including electroporation, transfection by lipid-based reagents, and lentiviral vectors.4144 However, homologous recombination, a method in which a specific gene inside the ES cells is modified with an artificially introduced DNA molecule, is an even more precise method of genetic engineering that can modify a gene in a defined way at a specific locus. While this technology is routinely used in mouse ES cells, it has recently been successfully developed in human ES cells (See chapter 4: Genetically Modified Stem Cells), thus opening new doors for using ES cells as vehicles for gene therapy and for creating in vitro models of human genetic disorders such as Lesch-Nyhan disease.45,46 Another method to test the function of a gene is to use RNA interference (RNAi) to decrease the expression of a gene of interest (see Figure 1.4: RNA interference). In RNAi, small pieces of double-stranded RNA (siRNA; small interfering RNA) are either chemically synthesized and introduced directly into cells, or expressed from DNA vectors. Once inside the cells, the siRNA can lead to the degradation of the messenger RNA (mRNA), which contains the exact sequence as that of the siRNA. mRNA is the product of DNA transcription and normally can be translated into proteins. RNAi can work efficiently in somatic cells, and there has been some progress in applying this technology to human ES cells.4749

Figure 1.4. How RNAi Can Be Used To Modify Stem Cells.

( 2006 Terese Winslow)

The pluripotency of ES cells suggests possible widespread uses for these cells and their derivatives. The ES cell-derived cells can potentially be used to replace or restore tissues that have been damaged by disease or injury, such as diabetes, heart attacks, Parkinson's disease or spinal cord injury. The recent developments in these particular areas are discussed in detail in other chapters, and Table 1 summarizes recent publications in the differentiation of specific cell lineages.

The differentiation of ES cells also provides model systems to study early events in human development. Because of possible harm to the resulting child, it is not ethically acceptable to experimentally manipulate the postimplantation human embryo. Therefore, most of what is known about the mechanisms of early human embryology and human development, especially in the early postimplantation period, is based on histological sections of a limited number of human embryos and on analogy to the experimental embryology of the mouse. However, human and mouse embryos differ significantly, particularly in the formation, structure, and function of the fetal membranes and placenta, and the formation of an embryonic disc instead of an egg cylinder.5052 For example, the mouse yolk sac is a well-vascularized, robust, extraembryonic organ throughout gestation that provides important nutrient exchange functions. In humans, the yolk sac also serves important early functions, including the initiation of hematopoiesis, but it becomes essentially a vestigial structure at later times or stages in gestation. Similarly, there are dramatic differences between mouse and human placentas, both in structure and function. Thus, mice can serve in a limited capacity as a model system for understanding the developmental events that support the initiation and maintenance of human pregnancy. Human ES cell lines thus provide an important new in vitro model that will improve our understanding of the differentiation of human tissues, and thus provide important insights into processes such as infertility, pregnancy loss, and birth defects.

Human ES cells are already contributing to the study of development. For example, it is now possible to direct human ES cells to differentiate efficiently to trophoblast, the outer layer of the placenta that mediates implantation and connects the conceptus to the uterus.17,53 Another use of human ES cells is for the study of germ cell development. Cells resembling both oocytes and sperm have been successfully derived from mouse ES cells in vitro.5456 Recently, human ES cells have also been observed to differentiate into cells expressing genes characteristic of germ cells.57 Thus it may also be possible to derive oocytes and sperm from human ES cells, allowing the detailed study of human gametogenesis for the first time. Moreover, human ES cell studies are not limited to early differentiation, but are increasingly being used to understand the differentiation and functions of many human tissues, including neural, cardiac, vascular, pancreatic, hepatic, and bone (see Table 1). Moreover, transplantation of ES-derived cells has offered promising results in animal models.5867

Although scientists have gained more insights into the biology of human ES cells since 2001, many key questions remain to be addressed before the full potential of these unique cells can be realized. It is surprising, for example, that mouse and human ES cells appear to be so different with respect to the molecules that mediate their self-renewal, and perhaps even in their developmental potentials. BMPs, for example, in combination with LIF, promote the self-renewal of mouse ES cells. But in conditions that would otherwise support undifferentiated proliferation, BMPs cause rapid differentiation of human ES cells. Also, human ES cells differentiate quite readily to trophoblast, whereas mouse ES cells do so poorly, if at all. One would expect that at some level, the basic molecular mechanisms that control pluripotency would be conserved, and indeed, human and mouse ES cells share the expression of many key genes. Yet we remain remarkably ignorant about the molecular mechanisms that control pluripotency, and the nature of this remarkable cellular state has become one of the central questions of developmental biology. Of course, the other great challenge will be to continue to unravel the factors that control the differentiation of human ES cells to specific lineages, so that ES cells can fulfill their tremendous promise in basic human biology, drug screening, and transplantation medicine.

We thank Lynn Schmidt, Barbara Lewis, Sangyoon Han and Deborah J. Faupel for proofreading this report.

Notes:

* Genetics and Biotechnology Building, Madison, WI 53706, Email: jyu@primate.wisc.edu.

** John D. MacArthur Professor, Department of Anatomy, University of WisconsinMadison Medical School, The Genome Center of Wisconsin, and The Wisconsin National Primate Research Center, Madison, WI 53715, Email: thomson@primate.wisc.edu.

Introduction|Table of Contents|Chapter 2

Originally posted here:
Embryonic Stem Cells | stemcells.nih.gov