Stem cell – Wikipedia

Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cellsectoderm, endoderm and mesoderm (see induced pluripotent stem cells)but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.

There are three known accessible sources of autologous adult stem cells in humans:

Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one may bank his or her own blood for elective surgical procedures.

Adult stem cells are frequently used in various medical therapies (e.g., bone marrow transplantation). Stem cells can now be artificially grown and transformed (differentiated) into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic stem cells generated through somatic cell nuclear transfer or dedifferentiation have also been proposed as promising candidates for future therapies.[1] Research into stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.[2][3]

The classical definition of a stem cell requires that it possess two properties:

Two mechanisms exist to ensure that a stem cell population is maintained:

Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.[4]

In practice, stem cells are identified by whether they can regenerate tissue. For example, the defining test for bone marrow or hematopoietic stem cells (HSCs) is the ability to transplant the cells and save an individual without HSCs. This demonstrates that the cells can produce new blood cells over a long term. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.

Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, in which single cells are assessed for their ability to differentiate and self-renew.[7][8] Stem cells can also be isolated by their possession of a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells shall behave in a similar manner in vivo. There is considerable debate as to whether some proposed adult cell populations are truly stem cells.[citation needed]

Embryonic stem (ES) cells are the cells of the inner cell mass of a blastocyst, an early-stage embryo.[9] Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

During embryonic development these inner cell mass cells continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as 'neurectoderm', which will become the future central nervous system.[10] Later in development, neurulation causes the neurectoderm to form the neural tube. At the neural tube stage, the anterior portion undergoes encephalization to generate or 'pattern' the basic form of the brain. At this stage of development, the principal cell type of the CNS is considered a neural stem cell. These neural stem cells are pluripotent, as they can generate a large diversity of many different neuron types, each with unique gene expression, morphological, and functional characteristics. The process of generating neurons from stem cells is called neurogenesis. One prominent example of a neural stem cell is the radial glial cell, so named because it has a distinctive bipolar morphology with highly elongated processes spanning the thickness of the neural tube wall, and because historically it shared some glial characteristics, most notably the expression of glial fibrillary acidic protein (GFAP).[11][12] The radial glial cell is the primary neural stem cell of the developing vertebrate CNS, and its cell body resides in the ventricular zone, adjacent to the developing ventricular system. Neural stem cells are committed to the neuronal lineages (neurons, astrocytes, and oligodendrocytes), and thus their potency is restricted.[10]

Nearly all research to date has made use of mouse embryonic stem cells (mES) or human embryonic stem cells (hES) derived from the early inner cell mass. Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin as an extracellular matrix (for support) and require the presence of leukemia inhibitory factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2).[13] Without optimal culture conditions or genetic manipulation,[14] embryonic stem cells will rapidly differentiate.

A human embryonic stem cell is also defined by the expression of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency.[15] The cell surface antigens most commonly used to identify hES cells are the glycolipids stage specific embryonic antigen 3 and 4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. By using human embryonic stem cells to produce specialized cells like nerve cells or heart cells in the lab, scientists can gain access to adult human cells without taking tissue from patients. They can then study these specialized adult cells in detail to try and catch complications of diseases, or to study cells reactions to potentially new drugs. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.[16]

There are currently no approved treatments using embryonic stem cells. The first human trial was approved by the US Food and Drug Administration in January 2009.[17] However, the human trial was not initiated until October 13, 2010 in Atlanta for spinal cord injury research. On November 14, 2011 the company conducting the trial (Geron Corporation) announced that it will discontinue further development of its stem cell programs.[18] ES cells, being pluripotent cells, require specific signals for correct differentiationif injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face.[19] Due to ethical considerations, many nations currently have moratoria or limitations on either human ES cell research or the production of new human ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.

Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer

The primitive stem cells located in the organs of fetuses are referred to as fetal stem cells.[20] There are two types of fetal stem cells:

Adult stem cells, also called somatic (from Greek , "of the body") stem cells, are stem cells which maintain and repair the tissue in which they are found.[22] They can be found in children, as well as adults.[23]

Pluripotent adult stem cells are rare and generally small in number, but they can be found in umbilical cord blood and other tissues.[24] Bone marrow is a rich source of adult stem cells,[25] which have been used in treating several conditions including liver cirrhosis,[26] chronic limb ischemia [27] and endstage heart failure.[28] The quantity of bone marrow stem cells declines with age and is greater in males than females during reproductive years.[29] Much adult stem cell research to date has aimed to characterize their potency and self-renewal capabilities.[30] DNA damage accumulates with age in both stem cells and the cells that comprise the stem cell environment. This accumulation is considered to be responsible, at least in part, for increasing stem cell dysfunction with aging (see DNA damage theory of aging).[31]

Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, dental pulp stem cell, etc.).[32][33]

Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants.[34] Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.[35]

The use of adult stem cells in research and therapy is not as controversial as the use of embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. Additionally, in instances where adult stem cells are obtained from the intended recipient (an autograft), the risk of rejection is essentially non-existent. Consequently, more US government funding is being provided for adult stem cell research.[36]

Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic, endothelial, hepatic and also neuronal lines.[37] Amniotic stem cells are a topic of active research.

Use of stem cells from amniotic fluid overcomes the ethical objections to using human embryos as a source of cells. Roman Catholic teaching forbids the use of embryonic stem cells in experimentation; accordingly, the Vatican newspaper "Osservatore Romano" called amniotic stem cells "the future of medicine".[38]

It is possible to collect amniotic stem cells for donors or for autologuous use: the first US amniotic stem cells bank [39][40] was opened in 2009 in Medford, MA, by Biocell Center Corporation[41][42][43] and collaborates with various hospitals and universities all over the world.[44]

These are not adult stem cells, but rather adult cells (e.g. epithelial cells) reprogrammed to give rise to pluripotent capabilities. Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue.[45][46][47]Shinya Yamanaka and his colleagues at Kyoto University used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4[45] in their experiments on human facial skin cells. Junying Yu, James Thomson, and their colleagues at the University of WisconsinMadison used a different set of factors, Oct4, Sox2, Nanog and Lin28,[45] and carried out their experiments using cells from human foreskin.

As a result of the success of these experiments, Ian Wilmut, who helped create the first cloned animal Dolly the Sheep, has announced that he will abandon somatic cell nuclear transfer as an avenue of research.[48]

Frozen blood samples can be used as a source of induced pluripotent stem cells, opening a new avenue for obtaining the valued cells.[49]

To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation diagram). Symmetric division gives rise to two identical daughter cells both endowed with stem cell properties. Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.[50]

An alternative theory is that stem cells remain undifferentiated due to environmental cues in their particular niche. Stem cells differentiate when they leave that niche or no longer receive those signals. Studies in Drosophila germarium have identified the signals decapentaplegic and adherens junctions that prevent germarium stem cells from differentiating.[51][52]

Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is a form of stem cell therapy that has been used for many years without controversy. No stem cell therapies other than bone marrow transplant are widely used.[53][54]

Stem cell treatments may require immunosuppression because of a requirement for radiation before the transplant to remove the person's previous cells, or because the patient's immune system may target the stem cells. One approach to avoid the second possibility is to use stem cells from the same patient who is being treated.

Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type. It is also difficult to obtain the exact cell type needed, because not all cells in a population differentiate uniformly. Undifferentiated cells can create tissues other than desired types.[55]

Some stem cells form tumors after transplantation;[56] pluripotency is linked to tumor formation especially in embryonic stem cells, fetal proper stem cells, induced pluripotent stem cells. Fetal proper stem cells form tumors despite multipotency.[citation needed]

Some of the fundamental patents covering human embryonic stem cells are owned by the Wisconsin Alumni Research Foundation (WARF) they are patents 5,843,780, 6,200,806, and 7,029,913 invented by James A. Thomson. WARF does not enforce these patents against academic scientists, but does enforce them against companies.[57]

In 2006, a request for the US Patent and Trademark Office (USPTO) to re-examine the three patents was filed by the Public Patent Foundation on behalf of its client, the non-profit patent-watchdog group Consumer Watchdog (formerly the Foundation for Taxpayer and Consumer Rights).[57] In the re-examination process, which involves several rounds of discussion between the USTPO and the parties, the USPTO initially agreed with Consumer Watchdog and rejected all the claims in all three patents,[58] however in response, WARF amended the claims of all three patents to make them more narrow, and in 2008 the USPTO found the amended claims in all three patents to be patentable. The decision on one of the patents (7,029,913) was appealable, while the decisions on the other two were not.[59][60] Consumer Watchdog appealed the granting of the '913 patent to the USTPO's Board of Patent Appeals and Interferences (BPAI) which granted the appeal, and in 2010 the BPAI decided that the amended claims of the '913 patent were not patentable.[61] However, WARF was able to re-open prosecution of the case and did so, amending the claims of the '913 patent again to make them more narrow, and in January 2013 the amended claims were allowed.[62]

In July 2013, Consumer Watchdog announced that it would appeal the decision to allow the claims of the '913 patent to the US Court of Appeals for the Federal Circuit (CAFC), the federal appeals court that hears patent cases.[63] At a hearing in December 2013, the CAFC raised the question of whether Consumer Watchdog had legal standing to appeal; the case could not proceed until that issue was resolved.[64]

Diseases and conditions where stem cell treatment is being investigated include:

Research is underway to develop various sources for stem cells, and to apply stem cell treatments for neurodegenerative diseases and conditions, diabetes, heart disease, and other conditions.[80]

In more recent years, with the ability of scientists to isolate and culture embryonic stem cells, and with scientists' growing ability to create stem cells using somatic cell nuclear transfer and techniques to create induced pluripotent stem cells, controversy has crept in, both related to abortion politics and to human cloning.

Hepatotoxicity and drug-induced liver injury account for a substantial number of failures of new drugs in development and market withdrawal, highlighting the need for screening assays such as stem cell-derived hepatocyte-like cells, that are capable of detecting toxicity early in the drug development process.[81]

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Stem cell - Wikipedia

Stem-cell therapy – Wikipedia

This article is about the medical therapy. For the cell type, see Stem cell.

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition.

Bone marrow transplant is the most widely used stem-cell therapy, but some therapies derived from umbilical cord blood are also in use. Research is underway to develop various sources for stem cells, and to apply stem-cell treatments for neurodegenerative diseases and conditions such as diabetes, heart disease, and other conditions.

Stem-cell therapy has become controversial following developments such as the ability of scientists to isolate and culture embryonic stem cells, to create stem cells using somatic cell nuclear transfer and their use of techniques to create induced pluripotent stem cells. This controversy is often related to abortion politics and to human cloning. Additionally, efforts to market treatments based on transplant of stored umbilical cord blood have been controversial.

For over 30 years, bone marrow has been used to treat cancer patients with conditions such as leukaemia and lymphoma; this is the only form of stem-cell therapy that is widely practiced.[1][2][3] During chemotherapy, most growing cells are killed by the cytotoxic agents. These agents, however, cannot discriminate between the leukaemia or neoplastic cells, and the hematopoietic stem cells within the bone marrow. It is this side effect of conventional chemotherapy strategies that the stem-cell transplant attempts to reverse; a donor's healthy bone marrow reintroduces functional stem cells to replace the cells lost in the host's body during treatment. The transplanted cells also generate an immune response that helps to kill off the cancer cells; this process can go too far, however, leading to graft vs host disease, the most serious side effect of this treatment.[4]

Another stem-cell therapy called Prochymal, was conditionally approved in Canada in 2012 for the management of acute graft-vs-host disease in children who are unresponsive to steroids.[5] It is an allogenic stem therapy based on mesenchymal stem cells (MSCs) derived from the bone marrow of adult donors. MSCs are purified from the marrow, cultured and packaged, with up to 10,000 doses derived from a single donor. The doses are stored frozen until needed.[6]

The FDA has approved five hematopoietic stem-cell products derived from umbilical cord blood, for the treatment of blood and immunological diseases.[7]

In 2014, the European Medicines Agency recommended approval of Holoclar, a treatment involving stem cells, for use in the European Union. Holoclar is used for people with severe limbal stem cell deficiency due to burns in the eye.[8]

In March 2016 GlaxoSmithKline's Strimvelis (GSK2696273) therapy for the treatment ADA-SCID was recommended for EU approval.[9]

Stem cells are being studied for a number of reasons. The molecules and exosomes released from stem cells are also being studied in an effort to make medications.[10]

Research has been conducted on the effects of stem cells on animal models of brain degeneration, such as in Parkinson's, Amyotrophic lateral sclerosis, and Alzheimer's disease.[11][12][13] There have been preliminary studies related to multiple sclerosis.[14][15]

Healthy adult brains contain neural stem cells which divide to maintain general stem-cell numbers, or become progenitor cells. In healthy adult laboratory animals, progenitor cells migrate within the brain and function primarily to maintain neuron populations for olfaction (the sense of smell). Pharmacological activation of endogenous neural stem cells has been reported to induce neuroprotection and behavioral recovery in adult rat models of neurological disorder.[16][17][18]

Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons and oligodendrocytes within the brain. A small clinical trial was underway in Scotland in 2013, in which stem cells were injected into the brains of stroke patients.[19]

Clinical and animal studies have been conducted into the use of stem cells in cases of spinal cord injury.[20][21][22]

The pioneering work[23] by Bodo-Eckehard Strauer has now been discredited by the identification of hundreds of factual contradictions.[24] Among several clinical trials that have reported that adult stem-cell therapy is safe and effective, powerful effects have been reported from only a few laboratories, but this has covered old[25] and recent[26] infarcts as well as heart failure not arising from myocardial infarction.[27] While initial animal studies demonstrated remarkable therapeutic effects,[28][29] later clinical trials achieved only modest, though statistically significant, improvements.[30][31] Possible reasons for this discrepancy are patient age,[32] timing of treatment[33] and the recent occurrence of a myocardial infarction.[34] It appears that these obstacles may be overcome by additional treatments which increase the effectiveness of the treatment[35] or by optimizing the methodology although these too can be controversial. Current studies vary greatly in cell-procuring techniques, cell types, cell-administration timing and procedures, and studied parameters, making it very difficult to make comparisons. Comparative studies are therefore currently needed.

Stem-cell therapy for treatment of myocardial infarction usually makes use of autologous bone-marrow stem cells (a specific type or all), however other types of adult stem cells may be used, such as adipose-derived stem cells.[36] Adult stem cell therapy for treating heart disease was commercially available in at least five continents as of 2007.[citation needed]

Possible mechanisms of recovery include:[11]

It may be possible to have adult bone-marrow cells differentiate into heart muscle cells.[11]

The first successful integration of human embryonic stem cell derived cardiomyocytes in guinea pigs (mouse hearts beat too fast) was reported in August 2012. The contraction strength was measured four weeks after the guinea pigs underwent simulated heart attacks and cell treatment. The cells contracted synchronously with the existing cells, but it is unknown if the positive results were produced mainly from paracrine as opposed to direct electromechanical effects from the human cells. Future work will focus on how to get the cells to engraft more strongly around the scar tissue. Whether treatments from embryonic or adult bone marrow stem cells will prove more effective remains to be seen.[37]

In 2013 the pioneering reports of powerful beneficial effects of autologous bone marrow stem cells on ventricular function were found to contain "hundreds" of discrepancies.[38] Critics report that of 48 reports there seemed to be just five underlying trials, and that in many cases whether they were randomized or merely observational accepter-versus-rejecter, was contradictory between reports of the same trial. One pair of reports of identical baseline characteristics and final results, was presented in two publications as, respectively, a 578 patient randomized trial and as a 391 patient observational study. Other reports required (impossible) negative standard deviations in subsets of patients, or contained fractional patients, negative NYHA classes. Overall there were many more patients published as having receiving stem cells in trials, than the number of stem cells processed in the hospital's laboratory during that time. A university investigation, closed in 2012 without reporting, was reopened in July 2013.[39]

One of the most promising benefits of stem cell therapy is the potential for cardiac tissue regeneration to reverse the tissue loss underlying the development of heart failure after cardiac injury.[40]

Initially, the observed improvements were attributed to a transdifferentiation of BM-MSCs into cardiomyocyte-like cells.[28] Given the apparent inadequacy of unmodified stem cells for heart tissue regeneration, a more promising modern technique involves treating these cells to create cardiac progenitor cells before implantation to the injured area.[41]

The specificity of the human immune-cell repertoire is what allows the human body to defend itself from rapidly adapting antigens. However, the immune system is vulnerable to degradation upon the pathogenesis of disease, and because of the critical role that it plays in overall defense, its degradation is often fatal to the organism as a whole. Diseases of hematopoietic cells are diagnosed and classified via a subspecialty of pathology known as hematopathology. The specificity of the immune cells is what allows recognition of foreign antigens, causing further challenges in the treatment of immune disease. Identical matches between donor and recipient must be made for successful transplantation treatments, but matches are uncommon, even between first-degree relatives. Research using both hematopoietic adult stem cells and embryonic stem cells has provided insight into the possible mechanisms and methods of treatment for many of these ailments.[citation needed]

Fully mature human red blood cells may be generated ex vivo by hematopoietic stem cells (HSCs), which are precursors of red blood cells. In this process, HSCs are grown together with stromal cells, creating an environment that mimics the conditions of bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stem cells to complete terminal differentiation into red blood cells.[42] Further research into this technique should have potential benefits to gene therapy, blood transfusion, and topical medicine.

In 2004, scientists at King's College London discovered a way to cultivate a complete tooth in mice[43] and were able to grow bioengineered teeth stand-alone in the laboratory. Researchers are confident that the tooth regeneration technology can be used to grow live teeth in human patients.

In theory, stem cells taken from the patient could be coaxed in the lab turning into a tooth bud which, when implanted in the gums, will give rise to a new tooth, and would be expected to be grown in a time over three weeks.[44] It will fuse with the jawbone and release chemicals that encourage nerves and blood vessels to connect with it. The process is similar to what happens when humans grow their original adult teeth. Many challenges remain, however, before stem cells could be a choice for the replacement of missing teeth in the future.[45][46]

Research is ongoing in different fields, alligators which are polyphyodonts grow up to 50 times a successional tooth (a small replacement tooth) under each mature functional tooth for replacement once a year.[47]

Heller has reported success in re-growing cochlea hair cells with the use of embryonic stem cells.[48]

Since 2003, researchers have successfully transplanted corneal stem cells into damaged eyes to restore vision. "Sheets of retinal cells used by the team are harvested from aborted fetuses, which some people find objectionable." When these sheets are transplanted over the damaged cornea, the stem cells stimulate renewed repair, eventually restore vision.[49] The latest such development was in June 2005, when researchers at the Queen Victoria Hospital of Sussex, England were able to restore the sight of forty patients using the same technique. The group, led by Sheraz Daya, was able to successfully use adult stem cells obtained from the patient, a relative, or even a cadaver. Further rounds of trials are ongoing.[50]

In April 2005, doctors in the UK transplanted corneal stem cells from an organ donor to the cornea of Deborah Catlyn, a woman who was blinded in one eye when acid was thrown in her eye at a nightclub. The cornea, which is the transparent window of the eye, is a particularly suitable site for transplants. In fact, the first successful human transplant was a cornea transplant. The absence of blood vessels within the cornea makes this area a relatively easy target for transplantation. The majority of corneal transplants carried out today are due to a degenerative disease called keratoconus.

The University Hospital of New Jersey reports that the success rate for growth of new cells from transplanted stem cells varies from 25 percent to 70 percent.[51]

In 2014, researchers demonstrated that stem cells collected as biopsies from donor human corneas can prevent scar formation without provoking a rejection response in mice with corneal damage.[52]

In January 2012, The Lancet published a paper by Steven Schwartz, at UCLA's Jules Stein Eye Institute, reporting two women who had gone legally blind from macular degeneration had dramatic improvements in their vision after retinal injections of human embryonic stem cells.[53]

In June 2015, the Stem Cell Ophthalmology Treatment Study (SCOTS), the largest adult stem cell study in ophthalmology ( http://www.clinicaltrials.gov NCT # 01920867) published initial results on a patient with optic nerve disease who improved from 20/2000 to 20/40 following treatment with bone marrow derived stem cells.[54]

Diabetes patients lose the function of insulin-producing beta cells within the pancreas.[55] In recent experiments, scientists have been able to coax embryonic stem cell to turn into beta cells in the lab. In theory if the beta cell is transplanted successfully, they will be able to replace malfunctioning ones in a diabetic patient.[56]

Human embryonic stem cells may be grown in cell culture and stimulated to form insulin-producing cells that can be transplanted into the patient.

However, clinical success is highly dependent on the development of the following procedures:[11]

Clinical case reports in the treatment orthopaedic conditions have been reported. To date, the focus in the literature for musculoskeletal care appears to be on mesenchymal stem cells. Centeno et al. have published MRI evidence of increased cartilage and meniscus volume in individual human subjects.[57][58] The results of trials that include a large number of subjects, are yet to be published. However, a published safety study conducted in a group of 227 patients over a 3-4-year period shows adequate safety and minimal complications associated with mesenchymal cell transplantation.[59]

Wakitani has also published a small case series of nine defects in five knees involving surgical transplantation of mesenchymal stem cells with coverage of the treated chondral defects.[60]

Stem cells can also be used to stimulate the growth of human tissues. In an adult, wounded tissue is most often replaced by scar tissue, which is characterized in the skin by disorganized collagen structure, loss of hair follicles and irregular vascular structure. In the case of wounded fetal tissue, however, wounded tissue is replaced with normal tissue through the activity of stem cells.[61] A possible method for tissue regeneration in adults is to place adult stem cell "seeds" inside a tissue bed "soil" in a wound bed and allow the stem cells to stimulate differentiation in the tissue bed cells. This method elicits a regenerative response more similar to fetal wound-healing than adult scar tissue formation.[61] Researchers are still investigating different aspects of the "soil" tissue that are conducive to regeneration.[61]

Culture of human embryonic stem cells in mitotically inactivated porcine ovarian fibroblasts (POF) causes differentiation into germ cells (precursor cells of oocytes and spermatozoa), as evidenced by gene expression analysis.[62]

Human embryonic stem cells have been stimulated to form Spermatozoon-like cells, yet still slightly damaged or malformed.[63] It could potentially treat azoospermia.

In 2012, oogonial stem cells were isolated from adult mouse and human ovaries and demonstrated to be capable of forming mature oocytes.[64] These cells have the potential to treat infertility.

Destruction of the immune system by the HIV is driven by the loss of CD4+ T cells in the peripheral blood and lymphoid tissues. Viral entry into CD4+ cells is mediated by the interaction with a cellular chemokine receptor, the most common of which are CCR5 and CXCR4. Because subsequent viral replication requires cellular gene expression processes, activated CD4+ cells are the primary targets of productive HIV infection.[65] Recently scientists have been investigating an alternative approach to treating HIV-1/AIDS, based on the creation of a disease-resistant immune system through transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and progenitor cells (GM-HSPC).[66]

On 23 January 2009, the US Food and Drug Administration gave clearance to Geron Corporation for the initiation of the first clinical trial of an embryonic stem-cell-based therapy on humans. The trial aimed evaluate the drug GRNOPC1, embryonic stem cell-derived oligodendrocyte progenitor cells, on patients with acute spinal cord injury. The trial was discontinued in November 2011 so that the company could focus on therapies in the "current environment of capital scarcity and uncertain economic conditions".[67] In 2013 biotechnology and regenerative medicine company BioTime (NYSEMKT:BTX) acquired Geron's stem cell assets in a stock transaction, with the aim of restarting the clinical trial.[68]

Scientists have reported that MSCs when transfused immediately within few hours post thawing may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth(fresh), so cryopreserved MSCs should be brought back into log phase of cell growth in invitro culture before these are administered for clinical trials or experimental therapies, re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various clinical trials on MSCs have failed which used cryopreserved product immediately post thaw as compared to those clinical trials which used fresh MSCs.[69]

Research currently conducted on horses, dogs, and cats can benefit the development of stem cell treatments in veterinary medicine and can target a wide range of injuries and diseases such as myocardial infarction, stroke, tendon and ligament damage, osteoarthritis, osteochondrosis and muscular dystrophy both in large animals, as well as humans.[70][71][72][73] While investigation of cell-based therapeutics generally reflects human medical needs, the high degree of frequency and severity of certain injuries in racehorses has put veterinary medicine at the forefront of this novel regenerative approach.[74] Companion animals can serve as clinically relevant models that closely mimic human disease.[75][76]

There is widespread controversy over the use of human embryonic stem cells. This controversy primarily targets the techniques used to derive new embryonic stem cell lines, which often requires the destruction of the blastocyst. Opposition to the use of human embryonic stem cells in research is often based on philosophical, moral, or religious objections.[110] There is other stem cell research that does not involve the destruction of a human embryo, and such research involves adult stem cells, amniotic stem cells, and induced pluripotent stem cells.

Stem-cell research and treatment was practiced in the People's Republic of China. The Ministry of Health of the People's Republic of China has permitted the use of stem-cell therapy for conditions beyond those approved of in Western countries. The Western World has scrutinized China for its failed attempts to meet international documentation standards of these trials and procedures.[111]

State-funded companies based in the Shenzhen Hi-Tech Industrial Zone treat the symptoms of numerous disorders with adult stem-cell therapy. Development companies are currently focused on the treatment of neurodegenerative and cardiovascular disorders. The most radical successes of Chinese adult stem cell therapy have been in treating the brain. These therapies administer stem cells directly to the brain of patients with cerebral palsy, Alzheimer's, and brain injuries.[citation needed]

Since 2008 many universities, centers and doctors tried a diversity of methods; in Lebanon proliferation for stem cell therapy, in-vivo and in-vitro techniques were used, Thus this country is considered the launching place of the Regentime[112] procedure. http://www.researchgate.net/publication/281712114_Treatment_of_Long_Standing_Multiple_Sclerosis_with_Regentime_Stem_Cell_Technique The regenerative medicine also took place in Jordan and Egypt.[citation needed]

Stem-cell treatment is currently being practiced at a clinical level in Mexico. An International Health Department Permit (COFEPRIS) is required. Authorized centers are found in Tijuana, Guadalajara and Cancun. Currently undergoing the approval process is Los Cabos. This permit allows the use of stem cell.[citation needed]

In 2005, South Korean scientists claimed to have generated stem cells that were tailored to match the recipient. Each of the 11 new stem cell lines was developed using somatic cell nuclear transfer (SCNT) technology. The resultant cells were thought to match the genetic material of the recipient, thus suggesting minimal to no cell rejection.[113]

As of 2013, Thailand still considers Hematopoietic stem cell transplants as experimental. Kampon Sriwatanakul began with a clinical trial in October 2013 with 20 patients. 10 are going to receive stem-cell therapy for Type-2 diabetes and the other 10 will receive stem-cell therapy for emphysema. Chotinantakul's research is on Hematopoietic cells and their role for the hematopoietic system function in homeostasis and immune response.[114]

Today, Ukraine is permitted to perform clinical trials of stem-cell treatments (Order of the MH of Ukraine 630 "About carrying out clinical trials of stem cells", 2008) for the treatment of these pathologies: pancreatic necrosis, cirrhosis, hepatitis, burn disease, diabetes, multiple sclerosis, critical lower limb ischemia. The first medical institution granted the right to conduct clinical trials became the "Institute of Cell Therapy"(Kiev).

Other countries where doctors did stem cells research, trials, manipulation, storage, therapy: Brazil, Cyprus, Germany, Italy, Israel, Japan, Pakistan, Philippines, Russia, Switzerland, Turkey, United Kingdom, India, and many others.

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Stem-cell therapy - Wikipedia

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

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Embryonic Stem Cells | stemcells.nih.gov

Embryonic Stem Cells | Stem Cells Freak

As their name suggests, embryonic stem cells (ESCs) are stem cells that are derived from embryos. If we wanted to be more scientific, we would say that ESCs are pluripotent stem cells derived from a blastocyst, an embryo in a very early stage (4-5 days of age).A blastocyst is consisted of 50-150 cells. ESCs measure approximately 14m in diameter.

The use of human embryonic stem cells is highly controversial, as their extraction requires the destruction of a human embryo, raising a great number of ethical issues. The main one is whether a blastocyst can be considered a living person or not. Check our article, Stem Cell Controversy for more info on this topic

Embryonic Stem cell properties There are two important attributes that distinguish stem cells from any other typical cell:

Embryonic stem cells are pluripotent, having the capacity to differentiate and develop into almost all kinds of cells belonging to thethree primary germ layers:

As for self-renewal, ES cells have the capacity to replicate indefinitely. In other words they have the ability, under the proper conditions, to produce infinite numbers of daughter cells just from one or a few father cells.

Human Embryonic Stem Cell Extraction And Culture First the inner cell mass (ICM) of the blastocyst is separated from the trophectoderm. Then the cells of the ICM are placed on aplastic laboratory culture dish that contains a nutrient broth called the "culture medium". Typically the inner surface of the dish is coated with what is called a "feeder layer", consisting of reprogrammed embryonic mouse skin cells that don't divide. These mouse cells lay in the bottom of the dish and act as a support for the hESCs. The feeder layer not only provides support, but it also releases all the needed nutrients for thehESCs to grow and replicate. Recently, scientists have devised new ways for culturing hESCs without the need of a mouse feeder cell, a really important advance as there is always the danger of viruses being transmitted from the mouse cells to the human embryonic stem cells.

It should be noted that the process described above isn't always successful, and many times the cells fail to replicate and/or survive. If on the other hand, the hESCs do manage to survive and multiply enough so that the dish is "full", they have to be removed and plated into several dishes. This replating and subculturing process can be done again and again for many months. This way we can get millions and millions of hESCs from the handful ones we had at the beginning.

At any stage of the process, a batch of hESCs can be frozen for future use or to be sent somewhere else for further culturing and experimentation.

How are human embryonic stem cells induced to differentiate ? There are various options for researchers to choose from, if they decide to differentiate the cultured cells.

The easiest one, is to simply allow the cells to replicate until the disc is "full". Once the disc is full, they start to clump together forming embryoid bodies(rounded collections of cells ). These embryoid bodies contain all kinds of cells including muscle, nerve, blood and heart cells. As said before, although this is easiest method to induce differentiation, it is the most inefficient and unpredictable as well.

In order to induce differentiation to a specific type of cell, researchers have to change the environment of the dish by employingone of the ways below:

Human Embryonic Stem Cells, potential uses Many researchers believe that studying hESCs is crucial for fully understanding the complex events happening during the fetal development. This knowledge would also include all the complex mechanisms that trigger undifferentiated stem cells to develop into tissues and organs. A deeper understanding of all these mechanisms would in return give scientists a deeper understanding of what sometimes goes wrong and as a result tumours,birth defects and other genetic conditions occur, thus helping them to come up with effective treatments.

Several new studies also address the fact that human embryonicstem cells can be used as models for human genetic disorders that currently have no reliable model system. Two examples are the Fragile-X syndromeandCystic fibrosis.

As of now, there has been only one human clinical trial ,involving embryonic stem cells, with the officialapproval of the U.S. Food and Drug Administration (FDA).The trial started on January 23, 2009, and involved the transplantation ofoligodendrocytes (a cell type of the brain and spinal cord) derived from human embryonic stem cells. During phase I of the trial, 8 to 10paraplegics with fresh spinal cord injuries (two weeks or less) were supposed to participate.

In August 2009,the trial wasput on hold, due to concerns made by the FDA, regarding a small number of microscopic cysts found in several treated rat models. InJuly 30, 2010 the hold was lifted and researchers enrolled the first patient and administered him with the stem cell therapy.

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Embryonic Stem Cells | Stem Cells Freak

Embryonic stem cell research – alsa.org

Overview

Stem cells have the ability to divide for indefinite periods in culture and give rise to multiple specialized cell types. They can develop into blood, neurons, bone, muscle, skin and other cell types. They have emerged as a major tool for research into the causes of ALS, and in the search of new treatments.

Types of Stem Cells:

The field of stem cell research is progressing rapidly, and The ALS Association is spearheading work on several critical fronts. The research portfolio supports innovative projects using IPSCs for drug development and disease modeling. The Association is supporting an IPSC core at Cedars-Sinai Medical Center providing access to lines for researchers globally. Several of the big data initiatives are collecting skin cells or blood for IPSC generation, such as Genomic Translation for ALS Clinical Care (GTAC), Project MinE, NeuroLINCS and Answer ALS. The ALS Association also sponsors pre-clinical studies and pilot clinical trials using stem cell transplant approaches to develop the necessary tools for stem cell transplant studies and to improve methods for safety and efficiency. We also support studies that involve isolating IPSCs to develop biomarkers for clinical trials through ALS ACT. In addition, the retigabine clinical trial that we sponsor uses iPSCs derived from participants in parallel with clinical data to help test whether the drug has the desired effect.

Stem cells are being used in many laboratories today for research into the causes of and treatments for ALS. Most commonly, researchers use iPSCs to make a unique source of motor neurons from individual ALS patients to try to understand why and how motor neurons die in ALS. Two types of motor neurons are affected in ALS are upper coriticospinal motor neurons, that when damaged, cause muscle spasticity (uncontrolled movement), and lower motor neurons, that when damaged, cause muscle weakness. Both types can be made from iPSCs to cover the range of pathology and symptoms found in ALS. Astrocytes, a type of support cell, called glia, of the central nervous system (CNS), are also being generated from iPSCs. It is well established that glia play a role in disease process and contribute to motor neuron death.

Motor neurons created from iPSCs have many uses. The availability of large numbers of identical neurons, made possible by iPSCs, has dramatically expanded the ability to search for new treatments. For example, they can also be used to screen for drugs that can alter the disease process. Motor neurons derived from iPSCs can be genetically modified to produce colored fluorescent markers that allow clear visualization under a microscope. The health of individual motor neurons can be tracked over time to understand if a test compound has a positive or negative effect.

Because iPSCs can be made from skin samples or blood of any person, researchers have begun to make cell lines derived from dozens of individuals with ALS. One advantage of iPSCs are that they capture a persons exact genetic material and provide an unlimited supply of cells that can be studied in a dish, which is like persons own avatar. Comparing the motor neurons derived from these cells lines allows them to ask what is common, and what is unique, about each case of ALS, leading to further understanding of the disease process. They are also used to correlate patients clinical parameters, such as site of onset and severity with any changes in the same patients motor neurons.

Stem cells may also have a role to play in treating the disease. The most likely application may be to use stem cells or cells derived from them to deliver growth factors or protective molecules to motor neurons in the spinal cord. Clinical trials of such stem cell transplants are in the early stages, but appear to be safe. In addition, transplantation of healthy astrocytes have the potential to be beneficial in supporting motor neurons in the brain and spinal cord.

While the idea of replacing dying motor neurons with new ones derived from stem cells is appealing, using stem cells as a delivery tool to provide trophic factors to motor neurons is a more realistic and feasible approach. The significant challenge to replacing dying motor neurons is making the appropriate connections between muscles and surrounding neurons.

Isolation of IPSCs from people with ALS in clinical trials is extremely valuable for the identification of unique signatures in the presence or absence of a specific treatment approach and as a read out to test whether a drug or test compound has an impact on the health of motor neurons and/or astrocytes. A positive result gives researchers confidence to move forward to more advanced clinical trials. For example, The ALS Association is currently funding a clinical trial to test the effects of retigabine on motor neurons, which use the enrolled patients individual iPSCs lines derived from collected skin samples and testing whether there is a change in the excitability of motor neurons in people with ALS. (see above).

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Embryonic stem cell research - alsa.org

Pros and Cons of Stem Cell Research – thebalance.com

Debates over the ethics of embryonic stem cell research continue to divide scientists, politicians and religious groups. However, promising developments in other areas of stem cell research might lead to solutions that bypass these ethical issues. These new developments could help win stem cell research more support from those against embryonic stem cell research, since they don't require the destruction of blastocysts.

Latest Developments

The most recent research has shown that there are many options available other than working with embryonic stem cells. Stem cells can be obtained from cord blood or derived by manipulating differentiated cells (i.e. skin cells) to revert them to a pluripotent state. These are alternatives that may help broaden the acceptance of stem cell research.

Background

In November 1998 the first published research paper reported that stem cells could be taken from human embryos. Subsequent research led to the ability to maintain undifferentiated stem cell lines (pluripotent cells) and techniques for differentiating them into cells specific to various tissues and organs.

The debates over the ethics of stem cell research began almost immediately in 1999, despite reports that stem cells cannot grow into complete organisms.

In 2000 2001, governments worldwide were beginning to draft proposals and guidelines in an effort to control stem cell research and the handling of embryonic tissues, and reach universal policies to prevent brain-drains (emigration of top scientists) between countries.

The CIHR (Canadian Institute of Health Sciences) drafted a list of recommendations for stem cell research in 2001. The Clinton administration drafted guidelines for stem cell research in 2000, but Clinton left office prior to them being released. The Bush government has had to deal with the issue throughout his administration.

Australia, Germany, UK and other countries have also formulated policies.

Pros

The excitement about stem cell research is primarily due to the medical benefits in areas of regenerative medicine and therapeutic cloning. Stem cells provide huge potential for finding treatments and cures to a vast array of diseases including different cancers, diabetes, spinal cord injuries, Alzheimers, MS, Huntingtons, Parkinsons and more.

There is endless potential for scientists to learn about human growth and cell development from studying stem cells.

Use of adult-derived stem cells, from blood, cord blood, skin and other tissues, known as IPSCs, has been demonstrated to be effective for treating different diseases in animal models. Umbilical-cord-derived stem cells (obtained from the cord blood) have also been isolated and utilized for various experimental treatments. Another option is use of uniparental stem cells. Although these cells lines have some disadvantages or shortcomings compared to embryonic cell lines (they are shorter-lived), there is vast potential if enough money is invested in researching them further, and they are not technically considered individual living beings by pro-life advocates.

Cons

Use of embryonic stem cells for reasearch involves the destruction of blastocysts formed from laboratory-fertilized human eggs. For those who believe that life begins at conception, the blastocyst is a human life and to destroy it is unacceptable and immoral.

This seems to be the only controversial issue standing in the way of stem cell research in North America.

Where It Stands

In the summer of 2006 President Bush stood his ground on the issue of stem cell research and vetoed a bill passed by the Senate that would have expanded federal funding of embryonic stem cell research.

Currently, American federal funding can only go to research on stem cells from existing (already destroyed) embryos. Similarly, in Canada, as of 2002, scientists cannot create or clone embryos for research but must used existing embryos discarded by couples. The UK allows embryonic stem cell cloning.

Use of stem cell lines from alternative non-embryonic sources has received more attention in recent years and has already been demonstrated as a successful option for treatment of certain diseases. For example, adult stem cells can be used to replace blood-cell-forming cells killed during chemotherapy in bone marrow transplant patients. Biotech companies such as Revivicor and ACT are researching techniques for cellular reprogramming of adult cells, use of amnionic fluid, or stem cell extraction techniques that do not damage the embryo, that also provide alternatives for obtaining viable stem cell lines.

Out of necessity, the research on these alternatives is catching up with embryonic stem cell research and, with sufficient funding, other solutions might be found that are acceptable to everyone.

On March 9, 2009, President Obama overturned Bush's ruling, allowing US Federal funding to go to embryonic stem cell research. However, the stipulation applies that normal NIH policies on data sharing must be followed. Despite the progress being made in other areas of stem cell research, using pluripotent cells from other sources, many American scientists were putting pressure on the government to allow their participation and compete with the Europeans. However, many people are still strongly opposed.

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Embryonic Stem Cells and the Germ Cell Lineage | InTechOpen

2.1. Primordial germ cell specification and candidate genes controlling germline establishment

The establishment of early germ cells and their successful maturation are complex processes, and require frequent changes in physiology, location and transcriptional profile of involved cells. Germline establishment in mammals occurs via inductive signaling, in contrast to lower organisms such as flies and worms where the germ cell identity is transmitted via the inheritance of germ plasm (McLaren, 1999, 2000; Saitou et al., 2002). In rodents and humans, the first glimpses of primordial germ cell (PGC) formation are observed in the embryo after implantation and gastrulation, when the epiblast, endoderm, mesoderm and ectoderm are first established (Matsui & Okamura, 2005). At this time, in response to molecular cues including Bone Morphogenetic Proteins (BMP4 and BMP8a) from the yolk sac, a population of pluripotent stem cells is segregated from the ICM and set physically apart from the extra-embryonic ectoderm or yolk sac of the embryo (Figure 1) (Lawson et al., 1999; Ying 2000, 2001). While BMPs provide the inductive signal to epiblast stem cells to supply PGCs, it is not clear what signals control the size of the PGC founder population or what molecules signal the termination of PGC specification. BMPs are crucial to specification of PGCs due to their activation of the ALK2 receptor and Smad-1/5 signaling pathways, as evidenced in mice (Hayashi et al., 2002). In the human embryo, the effects of BMPs on germline specification are unclear because there is very limited access to early embryonic samples. From work in our laboratory, it appears that similar BMP protein pathways are activated in the human embryo during gastrulation that direct PGC specification (Clark & Reijo Pera, 2006, Kee et al, 2006).

Additional mechanisms that may assist germline specification are the activation of pathways that either promote PGC survival and/or inhibit molecules that promote somatic differentiation of PGCs (Ewen & Koopman, 2010). As such, mouse and human PGCs retain expression of several biomarkers of pluripotency, including Oct4, Nanog and Sox2 which underlies their close resemblance to other pluripotent stem cells (Clark & Reijo Pera, 2006; Medrano et al., 2010; Nicholas et al., 2009). Interestingly, the transcription factor Sox2 is expressed on both mouse and human embryonic stem cells but unlike in the mouse, human Sox2 expression is diminished when PGCs migrate to the fetal gonad (Perrett et al., 2008). Coincident with the timing of germline specification in the mouse (E7.5), PGCs near the extraembryonic ectoderm begin to express germ-cell specific markers such as Blimp-1 (Prdm1 in human), Stella (Dppa3 in human), E-Cadherin, and Dazl and harbor alkaline phosphatase activity. Blimp-1/Prdm-1 is a transcriptional repressor whose activity is restricted to the germ lineage and appears to be critical for maintaining a PGC fate. There is strong evidence to suggest that in the mouse, Blimp-1 actively represses the somatic fate of PGCs by inhibiting expression of key somatic regulators during development (Ohinata et al., 2005; Hayashi et al., 2007). It is unclear whether Prdm-1 carries out a similar function in human germline formation. Once cell-cell communication has been established, the Fragilis (IFITIM-1 in human) and Stella/ Dppa3 genes promote further development of PGCs and may do so in a similar fashion as Blimp-1 in mice (Saitou et al., 2002).

Migration of germ cells to the gonad begins at E8.5 in the mouse and during weeks 4 to 6 of human gestation (first trimester). At this stage, PGCs accumulating at the base of the allantois exit the extraembryonic ectoderm and begin migration to the developing gonads, also known as the genital ridge. During migration, PGCs also proliferate by undergoing mitosis and express a new set of biomarkers, including the CXCR4 receptor in mice and the proto-oncogene c-KIT and its ligand, KIT in both mice and humans (Molyneaux & Wylie, 2004; Gomperts et al., 1994). The DAZ gene homologue, DAZL is also expressed on migrating PGCs. In the mouse, migratory PGCs display pseudopodia that may assist in movement through the hindgut and it is plausible that human germ cells behave similarly since they are also observed in the hindgut during migration. Various somatic tissues interact with PGCs during the migratory path to the gonad. It is likely therefore, that these tissues express molecules and factors that guide or cue the PGCs and help maintain their survival. In the mouse, several candidate molecules have been identified, including receptors such as -1 Integrin and extracellular matrix components such as Collagen I (Chuva de Sousa Lopes et al., 2005; De Felici et al., 2005). Although migration of PGCs is less well understood in human development, germ cells have been histologically observed during the late first trimester, when they undergo migration to the hindgut (Fujimoto et al., 1977; Gaskell et al., 2004; Goto et al., 2004). Male and female PGCs have been isolated from

Developmental Cycle of Mammalian Germ Cells. Life cycle of the mouse and human embryo following fertilization, progressing through gastrulation and producing the germline. The germline develops in the gonads and transmits genetic information to the next generation, thus completing the cycle. Fertilization of oocytes by sperm promotes the formation of a 1-cell zygote that undergoes cell division and cleavage to form a blastocyst. The outer layer of blastocyst gives rise to the trophectoderm while the inner cell mass (ICM) contains embryonic stem cells (ESCs). During gastrulation (E7.5 in mouse; Day 15+ in human), the blastocyst cavitates and develops the three germ layers and the epiblast. The primordial germ cells (PGCs) are specified and localize near the extra-embryonic ectoderm, at the base of the allantois. Once PGCs are specified, they migrate to the fetal gonads and undergo sex-specific developmental to male and female gonocytes. Subsequently, male gonocytes undergo spermatogenesis while female gonocytes enter meiotic prophase I and begin oogenesis. Adapted from Schuh-Huerta et al., 2011.

10-week old fetuses and were observed to express Alkaline Phosphatase (AP), a marker of PGCs (Goto et al., 2004). The morphology of human PGCs also resemble the rounded shape of mouse PGCs. Female-specific germ cells have also been visualized at the ultrastructural level during gonadal development in human fetuses (Motta et al., 1997). Finally, recent studies by Kerr et al. with human fetal gonads (testis and ovary) provide a detailed analysis of pluripotency and germ cell-specific markers. The germ cells of the fetal testis are Oct4 +/Nanog +/ c-Kit+ from week 7 to 15 after which these cells become localized to the testis periphery. Meanwhile, the presumptive gonocytes in the week 15 testis show strong expression of Pumilio2 (PUM2), VASA and DAZL and express low to no pluripotency markers (Oct4, Nanog, c-Kit, Tra1-60, Tra1-81) (Kerr et al., 2007). In the human fetal ovary, as in the testis, the expression of pluripotency markers peaks by week 8 and then declines after week 9, as oocytes enter meiosis (Kerr et al., 2008). Interestingly, the cell surface markers SSEA-1 and SSEA-4 are co-expressed on the female germ cells from week 5 onwards although only SSEA-1 is restricted to the germ cell lineage.

Upon arrival at the genital ridge, germ cells express another germ-cell specific marker, VASA, a cytoplasmic protein that is implicated in translational regulation. The gene encoding VASA expression, DDX4 (Mvh in mouse) is highly conserved among species and is expressed exclusively in both male and female pre-meiotic germ cells (Gustafson & Wessel, 2010). This finding underlines the importance of the VASA protein in germline function and makes it an attractive candidate for further study. Along with VASA, other factors produced are germ cell nuclear antigen-1 (GCNA-1) and E-cadherin. The sex-specific character of the developing gonad is controlled by the chromosomal constitution of gonadal somatic cells. In particular, the SRY gene expressed on the Y chromosome in mammals is thought to be an essential regulator of various downstream targets including the Sox9 gene that controls male gonadal development (McLaren, 1995, 2003). Once within the gonad, germ cells associate with Sertoli cells to form testis cords and this interaction induces the expression of VASA in post-migratory PGCs. VASA expression is induced in both male and female PGCs and persists until these cells enter meiosis and after which its levels diminish (Castrillon et al., 2000; Toyooka et al., 2000).

During germline development, an extensive remodeling of the epigenetic landscape occurs. This takes place during embryogenesis and during PGC migration to the gonad (Nicholas et al, 2009). The first wave of epigenetic remodeling occurs during implantation of the blastocyst and involves the erasure of all DNA methylation at CpG islands except those at imprinted gene loci. This transition is observed in all cells of the embryo, including the primitive germline. In female PGCs, there is another level of epigenetic change in the form of random X inactivation wherein one copy of the X chromosome pair is silenced. The second wave of epigenetic remodeling occurs when PGCs migrate to the primitive gonads and their paternal or maternal imprinted loci undergo a gradual process of erasure (Hajkova et al., 2002; Yamazaki et al., 2003). This phase occurs only in germ cells and may help to prime them for sex-specific DNA remethylation, when their developmental programs are established (Durcova-Hills et al., 2006). In the mouse, the re-establishment of imprints takes place prior to birth in the male prospermatogonia (E15) and only after birth in oocytes (Lucifero et al., 2002). In addition to DNA methylation changes, male and female germ cells also undergo post-translational histone modifications and RNA-mediated silencing (Reviewed in Tasler, 2009 & Nicolas et al., 2009).

The successful passage of germ cells through meiosis is a unique and highly rigorous process. However, between male and female embryos, the timing of meiosis is different. Male germ cells are restricted from entering meiosis while female germ cells enter meiosis within the embryo. Although the mechanisms for these contrasting behaviors are unclear, it appears that the gonadal cells provide the signal for (or against) meiotic entry (Brennan & Capel, 2004; Ewen & Koopman, 2010). One signal could be retinoic acid (RA) produced in the fetal ovary, which in turn induces Stra8, a key regulator of meiotic entry. In female germ cells destined to become oocytes, mitotic divisions cease and meiotic prophase begins with the correct stimuli (Borum, 1961); eventually oocytes arrest during Meiosis I prior to fetal birth and will only resume meiosis upon receiving hormonal signals during adulthood (Peters, 1970). Meanwhile, male germ cells transition from primordial status to the gonocyte stage, stop proliferating and remain quiescent in the fetal seminiferous tubules until after birth. The post-natal gonocytes then commit to a spermatogonial stem cell (SSC) fate and amplify through self-renewal or enter meiosis to initiate spermatogenesis. In both male and female germ cells, the synaptonemal complex proteins (SCPs) SCP-1, SCP-2 and SCP-3 are critical components of the meiotic machinery during chromosomal segregation (Chuma et al., 2001; Parra et al., 2004; Yuan et al., 2000). The completion of meiosis signals that germ cells have matured into haploid male and female gametes. At this stage, oocytes exclusively express GDF9 and spermatocytes express TEKT1. However, one feature that distinguishes human and mouse germline differentiation is the synchronization of meiotic entry, as in human fetal gonads, one can observe both pre-meiotic and meiotic germ cells in close proximity (Anderson, 2007).

Significant efforts have been made to culture mouse and human PGCs and gonocyes in vitro. In the case of mouse germ cells, the addition of endogenous factors known to affect germ cell development such as BMPs, RA, LIF and Forskolin have produced mixed results in maintaining PGCs in culture. For example, adding LIF enhanced PGC survival but the observations with the use of other factors not as clear. PGCs may also behave erratically in culture (showing low survival rates and non sex-specific behavior) because of the lack of a normal somatic environment (Childs et al., 2008). Studies of PGCs by Shamblott et al., Turnpenny et al. and Tu et al. with fetal human gonocytes resulted in a mixture of cellular phenotypes. Some gonocytes appeared rounded while others took on an elongated or spindly appearance. In addition, they appeared to have different proliferation rates (Shamblott et al., 1998; Turnpenny et al., 2003; Tu et al., 2007). Currently, it is not at all clear that these cells resemble their germ cell counterparts in vivo, but improvements in culture conditions and the cellular microenvironment will certainly help in this regard.

As delineated earlier, mammalian germ cells populate the testis and ovary during development in a incredibly dynamic manner. During early prenatal mice and human embryo development, PGCs migrate to the primitive gonad (gonadal ridge) and associate with Sertoli cells to form primitive testicular cords (Brennan et al., 2004). Within the testicular cords, the primitive germline stem cells (now termed gonocytes) remain in the testis as the gonad differentiates. Eventually, Sertoli cells, peritubular myoid cells and gonocytes form more compact structures known as the seminiferous tubules. When the gonocytes migrate to the periphery of the tubules, they transform into prospermatogonia and then into spermatogonia (Gondos & Hobel, 1971). In the fetal testis, prospermatogonia enter mitotic arrest, a feature observed at E12.5 to E14.5 in the mouse. At the molecular level, during entry into meiosis, both male and female human gonocytes express DAZL proteins and Vasa transcripts and downregulate OCT3/4 expression (Anderson et al., 2007). Interestingly, it is the early migrating germ cells that share similar properties with embryonic stem cells and testicular germ cell tumors (Ezeh et al., 2005). From what is known, the development of gonocytes in the fetal ovary follows a similar path in that OCT3/4 expression is reduced while VASA, Germ Cell Nuclear Antigen (GCNA) and DAZL are expressed (McLaren, 2003). In contrast to the male gonocytes, the female gonocytes receive sex-specific signals from the fetal gonad to enter meiotic prophase. After initiating meiosis, the female gonocytes will develop into primordial follicles and subsequently into primary follicles at puberty. A key difference between mouse and human systems is the timing of primary follicle formation: the mouse achieves this stage at birth while in the human ovary, this occurs at puberty (Bukovsky et al., 2005). There is some speculation whether this difference in follicle development is due to autocrine signals produced from the oocyte itself or from the ovarian environment (Hutt & Albertini, 2007). A plausible hypothesis is that the immediate environment of early germ cells determines whether they are commit to spermatogenesis or oogenesis. The most obvious source of signals are the mesonephros, primitive Sertoli cells in the testis, and primitive Granulosa cells in the ovary.

Timeline of Germline Specification and Germ Cell Marker Expression. A temporal representation of the stages of human and mouse germline differentiation in vivo. At each cellular stage, important molecular and somatic signals controlling that stage are indicated above the diagram. Specific germ cell molecular markers are indicated on left with arrows depicting the duration of development during which they expression has been observed. Genes that are italicized are present in both mouse and human germ cells. At the bottom, an approximate timing of each stage during mouse or human germline development is indicated. Adapted from Schuh-Huerta & Reijo Pera, 2011.

The seminiferous tubule serves as the sperm production center, where approximately 123 x 106 spermatozoa are produced from germ cells daily, or about 1000 sperm/second (Amann et al., 1980; de Rooij, 2009). Developing germ cells are arranged along the basement membrane in a highly ordered sequence and extend into the lumen of the tubule. At the most basal portion of the tubules lies the spermatogonial stem cell (SSC) population, closely associated with the adjacent Sertoli cells. Morphologic analysis of the various germ cells reveals at least 13 recognizable germ cell types in the human testis (Heller & Clermont, 1963, 1964). Each cell type is thought to represent a different step in spermatogenesis. From the least to the most differentiated, they have been named dark type A spermatogonia (Ad); pale type A spermatogonia (Ap); type B spermatogonia (B); preleptotene (R), leptotene (L), zygotene (z) and pachytene primary spermatocytes (p); secondary spermatocytes (II); and Sa, Sb, Sc, Sd1, and Sd2 spermatids (Figure 3). The early, type A spermatogonia are the most interesting germ cell type from a stem cell point of view (de Rooij, 2009). In fact, time-course studies using GFP-based reporters with early type A, type Ad and type Ap spermatogonia in the mouse revealed that the early type A cell has the ability to divide, self-renew, and give rise to the Ad and Ap sun-populations (Nakagawa T. et al., 2007). These observations provide evidence for the existence of SSCs in the testis and their clonal behavior is protypical of other adult stem cells.

It is currently thought that pale type A (Ap) spermatogonia in the basal, stem cell niche of the seminiferous tubule divide at 16-day intervals and differentiate to type B spermatogonia, which then become spermatocytes (Clermont, 1972). The ability of SSCs within the testis stem cell niche to undergo stem cell renewal is governed by several known factors. The growth factor-receptor kit ligand/c-kit receptor system and the niche factor glial cell line-derived neurotrophic factor (GDNF) are important in this process (Oatley & Brinster, 2008). In fact, spermatogenesis in the rat is dependent on c-Kit receptor activity, whereas spermatogonial stem cell renewal may be c-kit independent (Dym, 1994). GDNF appears to provide a significant stimulus to self-renewal of SSCs through receptors for GDNF on SSCs such as c-Ret and GFR-1(Meng et al., 2000). Despite this, our knowledge of other receptor-ligand systems that control human SSC renewal is limited at this point. During spermatogenesis, the cytoplasm between spermatogonial daughter cells remains conjoined after mitosis, forming cytoplasmic bridges between adjacent cells (Ewing et al., 1980). Cytoplasmic bridges are thought to be important for synchronized cellular proliferation, differentiation, and possibly regulation of gene expression. Thus, SSCs and early spermatogonia in the adult testis are critical for germ cell renewal and differentiation into sperm and raise important questions about the source of proliferative and differentiation signals for spermatogenesis. The majority of stages in mouse spermatogenesis delineated above are translatable to the human germ cell development pathway except for differences in the timing of each stage during development.

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Cloning/Embryonic Stem Cells – National Human Genome Research …

Cloning/Embryonic Stem Cells

The term cloning is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases, isolated genes or cells are duplicated for scientific study, and no new animal results. The experiment that led to the cloning of Dolly the sheep in 1997 was different: It used a cloning technique called somatic cell nuclear transfer and resulted in an animal that was a genetic twin -- although delayed in time -- of an adult sheep. This technique can also be used to produce an embryo from which cells called embryonic stem (ES) cells could be extracted to use in research into potential therapies for a wide variety of diseases.

Thus, in the past five years, much of the scientific and ethical debate about somatic cell nuclear transfer has focused on its two potential applications: 1) for reproductive purposes, i.e., to produce a child, or 2) for producing a source of ES cells for research.

The technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. In the simplest terms, the technique used to produce Dolly the sheep - somatic cell nuclear transplantation cloning - involves removing the nucleus of an egg and replacing it with the diploid nucleus of a somatic cell. Unlike sexual reproduction, during which a new organism is formed when the genetic material of the egg and sperm fuse, in nuclear transplantation cloning there is a single genetic "parent." This technique also differs from previous cloning techniques because it does not involve an existing embryo. Dolly is different because she is not genetically unique; when born she was genetically identical to an existing six-year-old ewe. Although the birth of Dolly was lauded as a success, in fact, the procedure has not been perfected and it is not yet clear whether Dolly will remain healthy or whether she is already experiencing subtle problems that might lead to serious diseases. Thus, the prospect of applying this technique in humans is troubling for scientific and safety reasons in addition to a variety of ethical reasons related to our ideas about the natural ordering of family and successive generations.

Several important concerns remain about the science and safety of nuclear transfer cloning using adult cells as the source of nuclei. To date, five mammalian species -- sheep, cattle, pigs, goats, and mice -- have been used extensively in reproductive cloning studies. Data from these experiments illustrate the problems involved. Typically, very few cloning attempts are successful. Many cloned animals die in utero, even at late stages or soon after birth, and those that survive frequently exhibit severe birth defects. In addition, female animals carrying cloned fetuses may face serious risks, including death from cloning-related complications.

An additional concern focuses on whether cellular aging will affect the ability of somatic cell nuclei to program normal development. As somatic cells divide they progressively age, and there is normally a defined number of cell divisions that can occur before senescence. Thus, the health effects for the resulting liveborn, having been created with an "aged" nucleus, are unknown. Recently it was reported that Dolly has arthritis, although it is not yet clear whether the five-and-a-half-year-old sheep is suffering from the condition as a result of the cloning process. And, scientists in Tokyo have shown that cloned mice die significantly earlier than those that are naturally conceived, raising an additional concern that the mutations that accumulate in somatic cells might affect nuclear transfer efficiency and lead to cancer and other diseases in offspring. Researchers working with clones of a Holstein cow say genetic programming errors may explain why so many cloned animals die, either as fetuses or newborns.

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered on the misperception that a child or many children could be produced who would be identical to an already existing person. This fear is based on the idea of "genetic determinism" -- that genes alone determine all aspects of an individual -- and reflects the belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which he or she develops. Nonetheless, many of the concerns about cloning have focused on issues related to "playing God," interfering with the natural order of life, and somehow robbing a future individual of the right to a unique identity.

Several groups have concluded that reproductive cloning of human beings creates ethical and scientific risks that society should not tolerate. In 1997, the National Bioethics Advisory Commission recommended that it was morally unacceptable to attempt to create a child using somatic cell nuclear transfer cloning and suggested that a moratorium be imposed until safety of this technique could be assessed. The commission also cautioned against preempting the use of cloning technology for purposes unrelated to producing a liveborn child.

Similarly, in 2001 the National Academy of Sciences issued a report stating that the United States should ban human reproductive cloning aimed at creating a child because experience with reproductive cloning in animals suggests that the process would be dangerous for the woman, the fetus, and the newborn, and would likely fail. The report recommended that the proposed ban on human cloning should be reviewed within five years, but that it should be reconsidered "only if a new scientific review indicates that the procedures are likely to be safe and effective, and if a broad national dialogue on societal, religious and ethical issues suggests that reconsideration is warranted." The panel concluded that the scientific and medical considerations that justify a ban on human reproductive cloning at this time do not apply to nuclear transplantation to produce stem cells. Several other scientific and medical groups also have stated their opposition to the use of cloning for the purpose of producing a child.

The cloning debate was reopened with a new twist late in 1998, when two scientific reports were published regarding the successful isolation of human stem cells. Stem cells are unique and essential cells found in animals that are capable of continually reproducing themselves and renewing tissue throughout an individual organism's life. ES cells are the most versatile of all stem cells because they are less differentiated, or committed, to a particular function than adult stem cells. These cells have offered hope of new cures to debilitating and even fatal illness. Recent studies in mice and other animals have shown that ES cells can reduce symptoms of Parkinson's disease in mouse models, and work in other animal models and disease areas seems promising.

In the 1998 reports, ES cells were derived from in vitro embryos six to seven days old destined to be discarded by couples undergoing infertility treatments, and embryonic germ (EG) cells were obtained from cadaveric fetal tissue following elective abortion. A third report, appearing in the New York Times, claimed that a Massachusetts biotechnology company had fused a human cell with an enucleated cow egg, creating a hybrid clone that failed to progress beyond an early stage of development. This announcement served as a reminder that ES cells also could be derived from embryos created through somatic cell nuclear transfer, or cloning. In fact, several scientists believed that deriving ES cells in this manner is the most promising approach to developing treatments because the condition of in vitro fertilization (IVF) embryos stored over time is questionable and this type of cloning could overcome graft-host responses if resulting therapies were developed from the recipient's own DNA.

For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical.

Since 1996, Congress has prohibited researchers from using federal funds for human embryo research. In 1999, DHHS announced that it intended to fund research on human ES cells derived from embryos remaining after infertility treatments. This decision was based on an interpretation "that human embryonic stem cells are not a human embryo within the statutory definition" because "the cells do not have the capacity to develop into a human being even if transferred to the uterus, thus their destruction in the course of research would not constitute the destruction of an embryo." DHHS did not intend to fund research using stem cells derived from embryos created through cloning, although such efforts would be legal in the private sector.

In July 2001, the House of Representatives voted 265 to 162 to make any human cloning a criminal offense, including cloning to create an embryo for derivation of stem cells rather than to produce a child. In August 2002, President Bush, contending with a DHHS decision made during the Clinton administration, stated in a prime-time television address that federal support would be provided for research using a limited number of stem cell colonies already in existence (derived from leftover IVF embryos). Current bills before Congress would ban all forms of cloning outright, prohibit cloning for reproductive purposes, and impose a moratorium on cloning to derive stem cells for research, or prohibit cloning for reproductive purposes while allowing cloning for therapeutic purposes to go forward. As of late June, the Senate has taken no action. President Bush's Bioethics Council is expected to recommend the prohibition of reproductive cloning and a moratorium on therapeutic cloning later this summer.

Prepared by Kathi E. Hanna, M.S., Ph.D., Science and Health Policy Consultant

Last Reviewed: April 2006

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Cloning/Embryonic Stem Cells - National Human Genome Research ...

Stem Cell Basics I. | stemcells.nih.gov

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos more than 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures. When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later section of this document.

Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, the inner cells give rise to the entire body of the organism, including all of the many specialized cell types and organs such as the heart, lungs, skin, sperm, eggs and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.

Given their unique regenerative abilities, stem cells offer new potentials for treating diseases such as diabetes, and heart disease. However, much work remains to be done in the laboratory and the clinic to understand how to use these cells for cell-based therapies to treat disease, which is also referred to as regenerative or reparative medicine.

Laboratory studies of stem cells enable scientists to learn about the cells essential properties and what makes them different from specialized cell types. Scientists are already using stem cells in the laboratory to screen new drugs and to develop model systems to study normal growth and identify the causes of birth defects.

Research on stem cells continues to advance knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. Stem cell research is one of the most fascinating areas of contemporary biology, but, as with many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries.

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Stem Cell Basics I. | stemcells.nih.gov