Use Of Human Embryonic Stem Cells Research Articles

Reprogramming methods for induced pluripotent stem cells generation

Regenerative medicine expects to replace the function of tissue or organs damaged by disease, trauma, or congenital issues. The tools used to realize these outcomes are tissue engineering and cellular therapies. Cellular therapy is considered a regenerative medicine strategy based on the use of stem cells. Pluripotent stem cells are the hotspots of cellular therapy due to their features that have been showed promising results. Embryonic stem cells (ESCs) are pluripotent, self-renewing cells that are derived from the inner cell mass (ICM) of the developing blastocyst. Pluripotency is the main feature that lead single cell to generate all cell lineages of the developing and adult organism. The use of human ESC (hESC) is ethically controversial, to overcome this problem the induced pluripotent stem cells (iPSCs) were developed. The aim of the present mini-review is to report a comprehensive summary of the different cellular reprogramming techniques from its initial conception to the present day.

Stem cell research and ethics: an update.

Stem cell therapies are not new. Bone marrow stem cell transplants have been performed for decades with much of the general public unaware that this is, in fact, a stem cell therapy. While the use of adult-derived stem cells and storage of cord blood has caused little debate, it is only since 19981 when researchers first learnt how to remove stem cells from human embryos that controversy has ensued. Embryonic stem (ES) cells, being pluripotent, have the potential to form all types of cells, and, therefore, have a huge potential in curing human disease. Research using human ES cells could help to better understand early human development, be used to research possible toxic effects of drugs (drug-screening) and, most importantly, be used in the field of regenerative medicine in the development of cell replacement therapies. However, both political and religious leaders have discussed the moral implications of destroying human embryos. In 2001 in the US there was a restriction on funding for ES cell research by President George Bush. President Bush stated that federal funds could only be used for research on human embryonic stem cell lines that had already been established, preventing researchers from creating more. However, this did not inhibit researchers receiving private funding. President Barack Obama went on to lift the ban in 2009. At the core of the ES cell issue is the question: when does life begin?2 This question closely links to debates over abortion and with the pro-life movement. However, even this debate is not uniform as while some oppose abortion and the use of human ES cells, others oppose abortion but support stem cell research using frozen embryos that remain after a woman or couple has completed infertility treatment, citing the lesser of two evils argument. The debate about stem cells is also a religious one. As Rana Dajani3 explained in her editorial, discussions in Jordan concluded that stem-cell research is permissible in Islam providing it is carried out to improve human health, since Muslim scholars consider life to start 40–120 days after conception. Denominations of the Christian faith, including Roman Catholics and Orthodox Christians, believe that the embryo has a status of a human individual from conception and therefore any decisions/interventions not in favor of the embryo violates the right of the embryo to life.4 Understandably, such conclusions are not easy to reach, and such theological debate is beyond the scope of this article. As a result of the discussions in Jordan, in January 2014 the country passed a law to control research and therapy using human ES cells. The regulation was the first in the Arab and Islamic region. It highlights the recognition that Jordan has for the potential of stem cell therapy and provides a framework for other countries in the region to follow. The law specifically bans private companies from using human ES cells, limiting research or therapies to government, and publically funded organizations/institutions, which have higher levels of transparency and are supervised by the health ministry and a specialized committee. Although there is no published record of ES cell research in Oman, stem cell transplantation (bone marrow, peripheral blood, and cord blood) has been carried out at Sultan Qaboos University Hospital (SQUH) since 1995.5 More recently, researchers at SQUH established the first national voluntary cord blood bank in the country. This caters for parents who have a child with a disorder that would normally be cured by bone marrow transplantation and who wanted their newborn baby’s cord blood to be collected and processed for a possible matched related transplant for their offspring.5 Adult-derived stem cell sources are also available. Adult stem cells are multipotent, meaning that they are capable of producing multiple (but not all) Adult-derived stem cell sources are also available. Adult stem cells are multipotent, meaning that they are capable of producing multiple (but not all) cell types. These are found in a variety of tissues, including the fetus. Initially researchers believed that adult stem cells could only form the cell types of the organ from which they were derived, but the cells have shown more versatility than this.6,7 The first transplanted human organ grown from adult stem cells was performed in 2008 by researchers from the University of Padua, the University of Bristol, and Politecnico di Milano.8 They harvested a section of trachea from a donor and stripped it of all cells, leaving a cartilage scaffold which was seeded with stem cells taken from the recipient patient’s bone marrow. The new section of trachea was then grown in the laboratory over four days and transplanted into the patient. Four months later their research, which was published in The Lancet, reported that the patient’s immune system showed no signs of rejection. Other breakthroughs, such as the ability to induce pluripotency in adult cells (iPS cells) may bring the debate over ES cells to an end. However, it will not remove the ethical concerns over the use of human ES cells. iPS cells are not exactly the same as human ES cells, which will be needed to act as the control for measuring the stemness of other cells.9 As yet, the cells that will be the most useful for cell replacement therapies has not been determined, which warrants the study of all stem cell types. An additional ethical consideration is that iPS cells have the potential to develop into a human embryo, in effect producing a clone of the donor. However, many nations are already prepared for this, having legislation in place that bans human cloning for various purposes, although there is no consensus around the world on these policies.10

A review and update on the current status of stem cell therapy and the retina

Many diseases of the retina result in irreversible visual loss. Stem cell (SC) therapy is a rapidly developing field and represents a novel approach to replace non-functioning neuro-retinal cells. A systematic computerized literature search was conducted on PubMed (http://www.ncbi.nlm.nih.gov/pubmed/). The use of stem cells (SCs) in animal models of retinal diseases has resulted in improvement in visual function and performance. SC therapy represents an exciting prospect in restoring vision. Areas of controversy The use of human embryonic SCs raises ethical concerns. Human trials using SCs in retinal diseases have recently been approved. The success of SCs in retinal therapy depends not only on implanted cell survival, but also on how well SCs migrate, integrate and form synapses. Further research will be needed to overcome these hurdles.

Deciphering the complexities of human diseases and disorders by coupling induced‐pluripotent stem cells and systems genetics

The recent discovery that adult mouse and human somatic cells can be 'reprogrammed' to a state of pluripotency by ectopic expression of only a few transcription factors has already made a major impact on the biomedical community. For the first time, it is possible to study diseases on an individual patient basis, which may eventually lead to the realization of personalized medicine. The utility of induced-pluripotent stem cells (iPSCs) for modeling human diseases has greatly benefitted from established human embryonic stem cell (ESC) differentiation and tissue engineering protocols developed to generate many different cell and tissue types. The limited access to preimplantation genetic tested embryos and the difficulty in gene targeting human ESCs have restricted the use of human ESCs in modeling human disease. Afforded by iPSC technology is the ability to study disease pathogenesis as it unfolds during tissue morphogenesis. The complexities of molecular signaling and interplay with protein transduction during disease progression necessitate a systems approach to studying human diseases, whereby data can be statistically integrated by sorting out the signal to noise issues that arise from global biological changes associated with disease versus experimental noise. Using a systems approach, biomarkers can be identified that define the initiation or progression of disease and likewise can serve as putative therapeutic targets.

Human-induced pluripotent stem cells as a source of hepatocyte-like cells: new kids on the block

One of the most exciting recent discoveries in the field of biology was the demonstration that both mouse and human somatic cells engineered via epigenetic reprogramming for the expression of combinations of few defined transcription factors (including Oct4, KLF4, Sox2, NANOG, LIN28, and c-Myc) can become pluripotent [1–3]. These cells closely resemble embryonic stem (ES) cells for their pluripotency and have been defined as induced pluripotent stem (iPS) cells. These cells have rapidly emerged as very promising tools for the achievement of significant advancement in different fields without the controversies and ethical concerns associated with the use of human ES cells [4]. In particular, the possibility to get iPS cells from readily obtainable somatic cells theoretically opened the way to a number of perspectives and practical applications, including (1) to design and test patient-customized (i.e., autologous) cell therapy without the need for immune suppression and (2) to increase our knowledge on mechanisms of inherited diseases with a realistic opportunity, as recently shown for human iPS-derived hepatocytes [5–7], for modeling inherited metabolic human diseases, understanding disease pathogenesis, and for drug discovery and testing. It should be anticipated, however, that the latter objective (i.e., the use of human iPS cell-derived hepatocytes for drug discovery and testing) can currently be envisaged as the most useful and safe application. Indeed, a number of relevant limitations have progressively emerged in relation to the clinical use of iPS cells and different laboratories have developed strategies in order to at least partially overcome such limitations. The original methods used in order to derive iPS cells from either human or mouse somatic cells employed viral vectors, a strategy leading to the integration of both desired transgenes and vector backbone into the host cell genome [1–3]. Of relevance, the use of these vectors is at risk to produce insertional mutations and genetic alterations able to interfere with normal functions of cells derived from iPS cells and to favor reactivation of reprogramming factors at later stages. Indeed, residual transgene expression has been reported to affect differentiation into specific lineages [2] or even result in tumorigenesis [8–10]. At present, the oncogenic potential of iPS cells represents a major concern for clinical application [9] since these cells, much as ES cells, can readily form teratomas when injected into immunodeficient mice. This may rely, for both iPS and ES cells, on the presence of residual diploid pluripotent cells that have not undergone differentiation in the population of transplanted human ES or iPS cells [9]. Moreover, tumor formation in iPS cell chimeric mice has been attributed to the expression of c-Myc in iPS cell-derived somatic cells, irrespective of the type of the original somatic cell employed [9–13]. However, as well reviewed [9], several other reasons are likely to underlie iPS cell tumorigenicity. In particular, although iPS cells may behave similarly to ES cells, differences between these two kinds of cells indeed exist and this is particularly significant for human cells, with human iPS cells being more tumorigenic than human ES cells because of genetic and epigenetic causes [9]. Along these lines, human iPS cells have been reported to acquire chromosomal alterations and genetic changes (including aneuploidy, gain or loss of small chromosomes, and point mutations) even more readily than ES cells C. Busletta E. Novo M. Parola (&) Department of Experimental Medicine and Oncology, InterUniversity Center for Hepatic Pathophysiology, University of Torino, Corso Raffaello 30, 10125 Turin, Italy e-mail: maurizio.parola@unito.it

Regenerative medicine spoilt for choice

Embryonic stem cells, induced pluripotent cells, transdifferentiated cells – there is now a wide range of options for medical researchers trying to regenerate disease-affected tissues and organs. But which approach will win the day, and can science policy keep up with the bioethical implications of rapid progress? Michael Gross investigates. Embryonic stem cells, induced pluripotent cells, transdifferentiated cells – there is now a wide range of options for medical researchers trying to regenerate disease-affected tissues and organs. But which approach will win the day, and can science policy keep up with the bioethical implications of rapid progress? Michael Gross investigates. Only ten years ago, ‘stem cells’ was a buzz word for all kinds of anticipated miracle cures, following the first production of human embryonic stem (ES) cell lines from blastocysts (early stage embryos) in 1998. As embryonic stem cells are pluripotent, they can generate the specialised cells for any kind of organ or tissue that one may want to create or repair, given the right enticement. While some countries, such as Germany, had qualms about the use of blastocysts discarded after IVF, others, including the UK, took the pragmatic view that these would have been destroyed anyway and took the lead in a rapidly growing and promising field. Then, in 2006, Shinya Yamanaka showed that a cocktail of only four factors suffices to turn back the clock of embryonic development and turn a differentiated somatic cell into a pluripotent one, which came to be known as induced pluripotent stem (iPS) cell. Earlier on, the cloning of Dolly the sheep had demonstrated that the clock can be turned back in a cellular environment, but the finding that researchers can achieve this in vitro with a relatively simple protocol came as a surprise. The bad news was that one of the factors was a known oncogene. While this factor could be eliminated eventually, it drew attention to the more fundamental problem that pluripotency includes the potential for the cells to become malignant. Recently, a third option came onto the scene, when direct conversion of one cell type into another was demonstrated first for mouse and then for human cells. Although the rules are still being established, this so called transdifferentiation appears to be possible even between completely unrelated cell types, without having to go via pluripotent cells. In February 2010, the group of Marius Wernig at Stanford reported the rapid and efficient conversion of mouse fibroblasts (a cell type found in connective tissue, which is also used for the production of iPS cells) into functional neurons in vitro (Nature (2010), 463, 1035–1041). They achieved the conversion by inducing only three transcription factors specific for the targeted cell type. This was the first conversion between unrelated cell lineages – previous efforts had shown that one type of blood cell can be converted into another, and different types of neurons, as well as pancreatic cell types, can be interconverted. Last November, the group of Mickie Bhatia at McMaster University at Hamilton, Ontario, Canada, reported a similar transdifferentiation success with human cells. This group had managed to convert human fibroblasts into blood progenitor cells expressing the marker CD45, shared by all types of leukocytes, without going through a pluripotent stage (Nature (2010), 468, 521–526). These progenitors then differentiated to a range of blood cell lineages, including granulocytic, monocytic, megakaryocytic, and erythroid cells. Earlier this year, the group of Sheng Ding at the Scripps Institute in La Jolla, California, reported the conversion of mouse fibroblasts into functional heart cells or cardiomyocytes (Nat. Cell Biol. (2011), 13, 215–222). By avoiding any pluripotent states with the associated malignancy risk, the transdifferentiation approach appears to be safer than either ES or iPS cells, although this will have to be established in systematic studies. However, this direct route is also forsaking the opportunity to grow stable stem cell lines in culture, and thus to produce large amounts of cells for regeneration. The limited availability of suitable cells for conversion may well limit the usefulness of the transdifferentiation route, so it would be too early to write off pluripotent cells. Medical researchers now have three fundamentally different approaches to choose from, and it is far from clear which will offer the best options. Paul Fairchild, co-director of the Oxford Stem Cell Institute at the Oxford Martin School, comments: “Never has the landscape of regenerative medicine been so promising with advances in the use of adult stem cells and the availability of both embryonic and induced pluripotent stem cells. Nevertheless, the magnitude of the obstacles to be overcome should never be underestimated. The task ahead is, therefore, to begin sifting the chaff from the wheat to determine which approaches might lead to robust clinical applications with manageable levels of risk.” To begin the sifting process, five research groups have recently assessed the quality of iPS cell lines, as summarised in a recent comment in Nature by Martin Pera (Nature (2011), 471, 46–47). The studies find that genetic and epigenetic abnormalities are much more frequent in iPS cell lines than in ES cells. Specific problems of iPS cells include gross chromosomal aberrations, which don't often appear in ES cells, mutation rates 10 times higher than in the fibroblasts from which the iPS cell lines were derived, and unexpected copy number variations (CNVs). At the epigenetic level, the researchers found imperfections in the reprogramming, leaving some characteristic marks of the cells of origin intact. There are indications that some of the faults arise by selection rather than accident, which would make them even harder to eliminate. On the other hand, research with ES cells still faces hurdles at the legal and bioethical front. The European Court of Justice currently deliberates a case that might end up with a complete ban on all patents based on the use of human ES cells. A recent statement from Yves Bot, the European Advocate General, although not legally binding, appeared to point in this direction. While acknowledging that embryonic stem cells are not equivalent to embryos in the eyes of the law, Bot indicated that the fact that embryonic cell lines are originally derived from fertilized human eggs means patents cannot be granted for techniques that involve the use of embryonic stem cells (www.eurostemcell.org). Stem cell researcher Oliver Brüstle from the University of Bonn, whose patent on producing brain cells from ES cells triggered the court case when it was challenged by Greenpeace in 2004, commented: “The Advocate General has taken a more restrictive view than the European Commission or any of the European Member States that have taken a position on this issue. No-one in Great Britain or Sweden would think of questioning a patent of this kind.” Putting the case in the global context, Brüstle said: “One wonders why the EU spends millions of Euros supporting the development of therapies based on embryonic stem cells, if practical progress towards the clinic is to be blocked by patenting restrictions. While stem cell technologies are already beginning to reach patients in the US and Asia, we are still discussing policy issues and wasting Europe's valuable competitive edge.” The decision of the court is expected in around May. Meanwhile, researchers at the medical applications front are already working towards regenerating certain defective cell types from whatever precursor or stem cell is available. The group of John Kessler at the Northwestern University recently attracted media attention with a report of brain cells grown from human ES cells (Stem Cells (2011) DOI: 10.1002/stem.626). The cell type created in Kessler's experiments is one that is affected by the early stages of Alzheimer's disease, so the hope is to be able to treat the disease by regenerating the population of these cells. Another part of the central nervous system that is very popular with experimental medicine is the retina. In the quest for ways to reverse the loss of sight, cell-based regeneration is in direct competition with bio-electronic approaches based on artificial sensors. At a recent meeting, for instance, Mandeep Singh from the Nuffield Department of Ophthalmology at the University of Oxford reported preliminary animal studies suggesting that retinal regeneration can be treated with precursor cells, which, after injection into the collapsed retina started to develop synapses and make contact with bipolar cells. The treatment was also successful in restoring some retina function, even though, intriguingly, there was a placebo effect in the control experiments, due to the body's own cells re-colonising the space created when inactive control material is injected into the collapsed retina. Short of creating replacement tissues, stem cells can be useful in allowing researchers to create a disease model in vitro. This is one of the approaches pursued in the field of Parkinson's disease. Recently, researchers at Stanford University have cultivated brain cells derived from skin cells (via iPS cells) of a patient with Parkinson's disease and have been able to observe the disease symptoms in the cell culture, specifically the accumulation of α-synuclein, an increase of oxidative stress, and an increased sensitivity to a range of stress factors (Cell Stem Cell (2011), 3, 267–280). Having an ‘out of body’ model of the diseased tissue of a living patient should help drug development in general and may also enable the researchers to develop personalised treatment options in the future. One organ that decays dramatically with aging is the thymus. The loss of function starts in early adulthood and leads to changes in the composition of the immune system in later life, as evident from an increased susceptibility to infection and a weakened response to vaccinations. Clare Blackburn from the MRC Centre for Regenerative Medicine, University of Edinburgh, is using ES cells and fetal thymic stem cells in an attempt to bypass the bottleneck of tissue supply for thymus transplants. “We hope one day to be able to boost immune system function by regenerating or replacing the thymus, but to do this we need to understand the fundamental mechanisms which regulate the epithelial cells in the thymus over the lifespan and how these are impacted by aging,” Blackburn explains. “Our research is focused on regulation of thymic cell identity by key transcription factors, and also by the cell's external environment (Nature (2010), 466, 978–982). Work of this type is the essential foundation for controlling tissue stem cells – both in vivo and in vitro – with the degree of precision currently possible only for pluripotent stem cells.” In fast-moving research fields like genomics or stem cells, politics can easily be left behind. As former MP Evan Harris told a stem cell symposium at Oxford in March, this problem gets even worse when the research involves morally complex issues such as dealing with human embryos. “Politicians don't like these issues,” Harris said, “so we don't get timely legislation.” Still, the UK has been relatively lucky due to the fact that the pioneering achievement of the first IVF birth in 1978 shocked politicians into creating the 1990 Human Fertilisation and Embryo Act, which was then cautiously updated in 2008. Harris called the 2008 bill “a missed opportunity” as it tiptoed around the critical issues of current research and anticipated therapies. One important outcome of the 1990 Act was the introduction of the Human Fertilisation and Embryo Authority (HFEA), which not only supervised IVF clinics but also acted as a bioethics council in prickly questions surrounding stem cells, therapeutic cloning, and pre-implantation diagnostics. As the current, Conservative-led government is planning to abolish the HFEA and transfer some of its functions to the Human Tissues Authority (HTA), it is not yet clear what kind of guidance in bioethics questions there will be in the future. If and when human cell lines, be they ES or iPS cells, enter medical practice, the ethical and intellectual property issues surrounding such cell lines will certainly require some work, as a recent report from the Hinxton Group, an international thinktank dealing with stem cell policy and bioethics (www.hinxtongroup.org), points out. At the moment, there are stem cell banks in various places, but there is no central resource where researchers could find information about existing cell lines and the associated intellectual property rights. The Hinxton Group report recommends establishing a central hub for stem cell information and patents, along with improvements to international coordination of research and the sharing of materials and data. At this moment, the future of regenerative medicine appears to be wide open, but it is less than clear whether society will be able to handle what this future may hold.

Mesenchymal Stem Cells

Stem cells have two features: the ability to differentiate along different lineages and the ability of self-renewal. Two major types of stem cells have been described, namely, embryonic stem cells and adult stem cells. Embryonic stem cells (ESC) are obtained from the inner cell mass of the blastocyst and are associated with tumorigenesis, and the use of human ESCs involves ethical and legal considerations. The use of adult mesenchymal stem cells is less problematic with regard to these issues. Mesenchymal stem cells (MSCs) are stromal cells that have the ability to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, such as umbilical cord, endometrial polyps, menses blood, bone marrow, adipose tissue, etc. This is because the ease of harvest and quantity obtained make these sources most practical for experimental and possible clinical applications. Recently, MSCs have been found in new sources, such as menstrual blood and endometrium. There are likely more sources of MSCs waiting to be discovered, and MSCs may be a good candidate for future experimental or clinical applications. One of the major challenges is to elucidate the mechanisms of differentiation, mobilization, and homing of MSCs, which are highly complex. The multipotent properties of MSCs make them an attractive choice for possible development of clinical applications. Future studies should explore the role of MSCs in differentiation, transplantation, and immune response in various diseases.

Potential sources of stem cells as a regenerative therapy for Parkinson's disease.

Stem cells are believed to hold enormous promise as potential replacement therapy in the treatment of neurodegenerative diseases such as Parkinson’s disease (PD). Stem cells were investigated to be the alternative therapeutic source capable of differentiating into dopamine (DA) neurons. Multiple important signaling factors were recorded for the induction of DA neuronal traits from mouse embryonic stem cells (ESCs) such as fibroblast growth factor 8, sonic hedgehog, and Wnt 1. Recent protocols were described for the differentiation of human ESCs into DA neurons, achieving high efficiency of DA neuronal derivation. Despite that, the use of human ESCs is still ethically controversial. The transcription factors necessary for DA neuron development from adult neural stem cells (NSCs), such as Pitx3, Nurr1, En-1, En-2, Lmx1a, Lmx1b, Msx1, and Ngn2, were investigated. In addition to replacement of lost DA neurons, adult NSCs were recorded to provide neuroprotective and neurogenic factors for the mesencephalon. In addition, induced pluripotent stem cells and bone marrow-derived mesenchymal stem cells represent reliable stem cell sources of DA neurons. Future studies are recommended to provide further insight into the regenerative capacity of stem cells needed for the treatment of PD.

Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells

Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12-24 μg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.

Engineered embryonic stem cells for regnerative nedicine

Due to their ability to reproduce the embryological differentiation of nearly all different cellular phenotypes, embryonic stem (ES) cells represent an ideal tool to study processes of embryogenesis under in vitro conditions, in particular the signalling cascades and genes involved in the functional development (functional genomics), as well as to provide a new source for cellular replacement therapy. We cultivated ES cells in three dimensional cell aggregates (embryoid bodies), where they differentiate into derivatives of all three germ layers. To select only one lineage, e.g. the cardiac lineage, and to allow the identification of the transplanted cells, transgenic ES cells were used. They contained a bicistronic vector with two cloning sites for EGFP and an antibiotics resistance for selective selection both under the α-MHC promoter. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild type mice. Immunological cross reactions were avoided by using the same inbred mouse strain. EGFP positive transplanted cardiomyocytes could easily be detected in the native heart at different intervals after operation. The cells were found to engraft and to differentiate into adult-like cardiomyocytes as confirmed by cross striation after immunostaining with a-actinin. These data were corroborated by patch clamp experiments on isolated EGFP positive cardiomyocytes at different time points after operation. The transplanted cells displayed ventricular action potentials and β-adrenergicas well as muscarinic regulation. When survival was investigated in a large colony of mice, the control group had an almost double mortality rate as compared to the transplanted mice. Our data show engraftment and differentiation of embryonic cardiomyocytes after transplantation into cryoinfarcted areas of the heart. Before a clinical use of human ES cells for therapeutic trials in humans two major prerequisites must be fulfilled: (i) they must be safe, i.e. the development of tumours because of the high proliferative potential must be omitted and (ii) the rejection of the transplanted cells must be prevented. For the criterion (i) the technology of “lineage selection” appears optimal, for the criterion (ii) reprogramming techniques are now tested. Although autologous bone marrow cells appear ideal to circumvent rejection, we were not able to demonstrate a transdifferentiate into cardiomyocytes. Hence emphasis is made on reprogrammed human ES cells. The complexity of questions to be answered makes it necessary to establish closer collaborations between various disciplines including cell-, molecular-, developmentalbiology, molecular genetics, biochemistry, immunology, as well as clinical disciplines with medical basic sciences, experimental surgery and bioengineering together with cell culture techniques and material sciences. Cell Research (2008) 18:s111. doi: 10.1038/cr.2008.201; published online 4 August 2008

Genetic Modification of Human Embryonic Stem Cells

Embryonic stem (ES) cells, originated from the inner cell mass (ICM) of a blastocyst embryo, are self-renewable (Evans and Kaufman, 1981; Martin, 1981). The pluoripotent nature of ES cells endows them as a great tool for dissecting cell lineage development in mammals and they can be used as a limitless source for producing specialized cells for potential cell therapy. Application of gene targeting technology in mouse ES cells has allowed the cloning of mice with modified genomes (Frohman and Martin, 1989; Koller et al., 1989), thus significantly widening the use of mice as a vertebrate experimental model in biological research today. Forced overexpression or knockout of critical genes by genetic modification has become a routine technique in mouse ES cells to create transgenic mice for analyzing gene function and genetic pathways in the context of intact animals and in cell lines derived from such animals. Gene targeting in mouse ES cells also allows the creation of mouse models of human diseases (Bedell et al., 1997a; Bedell et al., 1997b), which offers insights into the genetic, biochemical and pathological basis of the diseases and may help developing treatments. Like their murine counterparts, human ES cells (Reubinoff et al., 2000; Thomson et al., 1998) offer invaluable and perhaps the only system for directly studying early human development as manipulation of human embryos is prohibited and information gained from model organisms (Anderson and Ingham, 2003) does not always reflect what is occurring in humans. The ability of human ES cells to produce almost any cell types in our body brings hopes to many incurable diseases affecting the central nervous system (CNS) (Dunnett et al., 2001; Silani et al., 2004), pancreas (Bonner-Weir and Weir, 2005), and heart (Srivastava and Ivey, 2006), etc. The potential use of human ES cells can be further enhanced by genetic modification (Drukker, 2005; Kobayashi et al., 2005). Application of currently available genetic modification techniques in human ES cells will provide researchers with even more versatile tools to study early human development and to expand the potential use in medicine. Basic mechanisms underlying early human development can be dissected through interfering with specific signaling pathways by forced gene overexperssion or silencing. Genetic defects may be corrected in human ES cells by site directed gene modifications, which may lead to the development of treatment for many inheritable diseases (Chang et al., 2006; Rideout et al., 2002). By labeling cells with tissue specific markers, genetic modification may help us obtain pure functional cells of desired types after differentiation, which can be used as more effective transplant medicine. Unlike their murine counterparts, genetic modification in human ES cells has been technically challenging. Standard chemical or mechanical methods for gene delivery exhibit very low efficiency in human ES cells, with the most effective approaches yielding only approximately one stable transfectant per 105 transfected cells (Eiges et al., 2001; Zwaka and Thomson, 2003). After transfection, transgene expression appears to be suppressed more severely in human ES cells than in mouse ES cells (Xia, 2006). Thus successful examples are still rare. In this review, we will summarize the current methodologies for genetic modification of human ES cells from very limited literatures and propose future directions to overcome the present problems in genetic manipulation of human ES cells.

Stem cells with potential to generate insulin producing cells in man.

Replacement of insulin-producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans--although successful in experienced centres--is limited by the lack of donor organs. Generation of insulin-producing cells from stem cells represents an attractive alternative. Stem cells with the potential to differentiate into insulin-producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, central nervous system, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns and the inability to create patient specific ESC with therapeutic cloning. Among adult stem cells mesenchymal stem cells appear to have a particular developmental plasticity ex vivo that include their ability to adopt a pancreatic endocrine phenotype. The present review summarises the current knowledge on the development of insulin-producing cells from stem cells with special emphasis on human mesenchymal stem cells isolated from the pancreas and adipose tissue.

Human Embryonic Stem Cells as a Powerful Tool for Studying Human Embryogenesis

Human embryonic stem cells (HESC) are pluripotent stem cell lines derived from the inner cell mass (ICM) of human blastocyst-stage embryos. They are characterized by their unlimited capacity to self-renew in culture. In addition, they have a broad developmental potential, as demonstrated by their ability to form practically any cell type in vivo and in vitro. These two features have made HESC extremely important in basic and applied research. In addition, they may serve as a powerful tool for studying human development. HESC can recapitulate embryogenesis by expressing developmentally regulated genes and by activating molecular pathways as they occur in vivo. Moreover, they can be used to analyze the effect of specific mutations on particular developmental events and may enable us to identify critical factors that play a role in the processes of cell commitment, differentiation, and adult cell reprogramming. Thus, modeling human embryogenesis by the use of HESC may allow new insights into developmental processes, which would otherwise be inaccessible for research.

Différenciation neurale des cellules souches embryonnaires

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.

Mouse and human embryonic stem cell models of hematopoiesis: past, present, and future

Human embryonic stem (ES) cells provide a unique model and an important resource to analyze early hematopoietic development. Other systems to study mammalian hematopoiesis include mouse ES cells, dissection of timed mouse embryos, or use of human postnatal hematopoietic tissue typically isolated from bone marrow or umbilical cord blood. All these models have particular strengths and weaknesses. The extensive studies on murine hematopoiesis provide a basis for work on the human developmental system. Since there are likely some important species differences, use of human ES cells now provides an optimal means to evaluate basic cellular and molecular mechanisms that regulate the beginning stages of human blood development, prior to derivation of hematopoietic stem cells (HSCs). Eventually, research on human ES cells may provide an alternative source of HSCs and other blood products for hematopoietic cell transplantation or other cellular therapies.

The ultimate source of human hematopoietic stem cells: thinking outside the marrow.

The ability to reconstitute cellular components of the hematopoietic system has immense utility in several areas of clinical medicine. These include replacement of cells responsible for innate and acquired immunity, providing red cells for oxygen transport, and ultimately the ability to recover hematopoietic function by repopulating all lineages comprising the entire blood system. This latter property functionally defines the mammalian hematopoietic stem cell (HSC). Recently, human embryonic stem cells (ESCs) have been suggested to be a viable source of transplantable hematopoietic cells. Although the number of human ESCs is virtually unlimited, the ability to efficiently differentiate adequate numbers of cells that possess hematopoietic repopulating ability remains to be determined. Achieving this goal is confounded by the difficulty of experimentally generating murine hematopoietic cell types capable of in vivo reconstitution from mouse ESC, suggesting that similar limitations may arise using human counterparts. Although the use of human ESCs and adult somatic HSCs have their independent merits, a direct comparison between HSCs derived from each source using similar assays will ultimately be required to determine the best source for clinical use. Here we will summarize the results from efforts to differentiate and assay primitive hematopoietic cells derived from ESCs, and compare these findings to similar parameters using putative mammalian HSCs harvested from the adult.

Stem cells: you can't tell a cell by its cover.

Embryonic stem (ES) cells are capable of unlimited self-renewal and have the ability to give rise to all tissue types in the body. The use of human ES cells for tissue and cell therapeutics has been suggested, but is limited by ethical concerns as these cells are derived from the inner cell mass of human embryos. In addition, the need for HLA matching of ES cell-derived tissues for allogeneic transplantation would require a bank of several thousand ES cell lines to make tissue therapeutics practical. Recently, adult stem cells-of which those in bone marrow are the best studied-have been shown to be capable of multilineage differentiation into cells of various non-blood tissues. Umbilical cord blood (UCB) haematopoietic stem cells have been shown to be equivalent to bone marrow stem cells for reconstitution of the haematopoietic system. Preliminary studies have also demonstrated that UCB haematopoietic stem cells are multipotent and capable of differentiating into non-blood cell types. This observation raises the exciting possibility of replacing human ES cells for tissue and cell therapeutics with UCB blood haematopoietic stem cells that are normally discarded with the placenta after delivery.

Stem cell research in Germany: ethics of healing vs. human dignity.

On 25 April 2002, the German Parliament has passed a strict new law referring to stem cell research. This law took effect on July 1, 2002. The so-called embryonic Stem Cell Act ("Stammzellgesetz-StZG") permits the import of embryonic stem (ES) cells isolated from surplus lvF-embryos for research reasons. The production itself of ES cells from human blastocysts has been prohibited by the German Embryo Protection Act of 1990, with the exception of the use of ES cells which exist already. The debate on the legitimate use of ES cells escalated, after the main German research funding agency, the Deutsche Forschungsgemeinschaft (DFG), unexpectedly published new guidelines recommending a restricted use of human ES cells for research. Meanwhile, the debate has ethically divided society, political parties, government and church members into a group supporting and a group rejecting ES cell research. The arguments in favour of such a research can be summarized as arguments derived from a new "ethics of healing" calling for a therapeutic imperative, whereas the arguments against can be summarized as arguments violating the fundamental principle of human dignity as they imply the destruction of human embryos. This article will try to present and evaluate various ethical arguments founded on the latest biological and medical data on the potential use of stem cell technologies. It will finally come to the conclusion that ES cell research is opposed to human dignity, since the procedures of isolating ES cells require the destruction and instrumentalization of human embryos. Human embryos are human beings at a very early stage of their development, fully possessing the ability of completing their development. At this very early stage, human embryos are extremely dependent and fragile, and thus vulnerable corporealities. Vulnerability and human dignity demand the protection of the embryo's corporeal integrity. Hence, this essay will try to propagate research with adult stem (AS) cells, a procedure which does not require the destruction of human embryos; with regard to the necessary plasticity, it should be emphasized that AS cells very much resemble ES cells.

Human embryo research in France.

The French law on bioethics, voted upon in July 1994, is going to be revised. This is the occasion for France to reconsider its position concerning research on human embryos, which is currently prohibited in France, as it is in Germany, Switzerland and Austria. However, such research is authorised in other European countries such as the UK, Spain, Belgium, Italy and The Netherlands. The establishment of human embryonic stem (ES) cells has reopened the debate in France because of their potential in human therapy. Indeed, ES cells, derived from early embryos (5-6 days old), preserve in vitro a pluripotent character, and they could provide an infinite source of different tissues that could be used in replacement therapy. This consists of ES cells differentiated in vitro into the desired tissues or cell types and grafted into the patient. The use of human ES cells in replacement therapy raises the major problem of graft rejection. One of the proposed solutions would be to carry out a 'therapeutic cloning' and to derive ES cells from the embryos obtained in this way. We do consider that, for the moment, the interest of the cloning study lies mainly in the understanding of the mechanisms responsible for reprogramming the nuclei. This research can be performed first on animal models. France is now thinking to allow human embryo research. We present here the French law proposed on human embryo research. French government is proposing to allow research exclusively on frozen supernumerary embryos, which no longer have any parental or adoption potential. Creation of human embryos for research purposes will still be prohibited. However, allowance of studies on human cloning in order to realise therapeutic cloning is mentioned in the proposal. We think that allowing research in humans on therapeutic cloning is premature and contradicts the prohibition of the creation of human embryos for research.

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.