The derivation and potential use of human embryonic stem cells

Human embryonic stem cells lines can be derived from human blastocysts at high efficiency (>50%) by immunosurgical isolation of the inner cell mass and culture on embryonic fibroblast cell lines. These cells will spontaneously differentiate into all the primary embryonic lineages in vitro and in vivo, but they are unable to form an integrated embryo or body plan by themselves or when combined with trophectoderm cells. They may be directed into a number of specific cell types and this enrichment process requires specific growth factors, cell-surface molecules, matrix molecules and secreted products of other cell types. Embryonic stem (ES) cells are immortal and represent a major potential for cell therapies for regenerative medicine. Their use in transplantation may depend on the formation of a large bank of suitable human leucocyte antigen (HLA) types or the genetic erasure of their HLA expression. Successful transplantation may also require induction of tolerance in recipients and ongoing immune suppression. Although it is possible to customize ES cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that these strategies will be used extensively for producing ES cells compatible for transplantation. Embryonic stem cell research may deliver a new pathway for regenerative medicine.     

Human embryonic stem cells: challenges and opportunities

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.     

Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures

Current knowledge on the biology of mammalian embryonic stem cells (ESC) is stunningly sparse in light of their potential value in studies of development, functional genomics, generation of transgenic animals and human medicine. Despite many efforts to derive ESC from other mammalian species, ESC that retain their capacity for germ line transmission have only been verified in the mouse. However, the criterion of germ line transmission may not need to be fulfilled for exploitation of other abilities of these cells. Promising results with human ESC-like cells and adult stem cells have nourished great expectations for their potential use in regenerative medicine. However, such an application is far from reality and substantial research is required to elucidate aspects of the basic biology of pluripotent cells, as well as safety issues associated with the use of such cells in therapy. In this context, methods for the derivation, propagation and differentiation of ESC-like cultures from domestic animals would be highly desirable as biologically relevant models. Here, we review previously published efforts to establish bovine ESC-like cells and describe a procedure used in attempts to derive similar cells from bovine Day 12 embryos.     

Embryonale Stammzellen der Maus Eigenschaften, Potenzial und Verwendung

During the last few years research on embryonic stem cells has received much public attention due to the fact that these cells are able to differentiate in vitro into many specialized cells and thus may serve as a source for a variety of tissues. The following article focuses on mouse embryonic stem cells (murine ES cells), because research on these cells has given insight into the potential of embryonic stem cells. Murine ES cells are permanent cell lines established from the inner cell mass (ICM) of early embryos (blastocysts). ES cells are undifferentiated pluripotent cells that are able to undergo an unlimited number of cell divisions without loosing the undifferentiated phenotype. The same is true for mouse primordial germ cell lines (murine EG cell lines), that where established from the fetal progenitor cells of primordial germ cells. Mouse embryonic stem cells are used for different purposes. In basic research they are used to study the consequences of mutations within genes that control embryonic development and/or the development of diseases. Because of their ability to differentiate into a variety of specialized cell types, murine ES cells also serve as model systems to establish specific differentiation protocols. In the last few years protocols were established for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiac, skeletal muscle, neural, adipogenic, haematopoietic, endothelial, chondrogenic or vascular smooth muscle cells. Last but not least, studies on mouse ES cells have demonstrated that embryonic cells and their differentiated derivatives can be used to analyse the effects of toxic substances or of pharmaceutical drugs.     

Enrichment of blood from embryonic stem cells in vitro

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.     

干细胞治疗薄型子宫内膜的研究进展

在辅助生殖技术中,子宫内膜厚度可以反映内膜功能状态,薄型子宫内膜是指子宫内膜厚度不足以获得胚胎着床及临床妊娠。虽然临床对薄型子宫内膜治疗的方式较多,但总体治疗效果欠佳。干细胞因具有多向分化和自我更新的潜能而展现出广阔的发展前景,包括胚胎干细胞、成体干细胞和诱导多能干细胞,随着对干细胞研究的深入,目前有研究诱导干细胞向子宫内膜细胞定向分化并增生,以促进子宫内膜生长,进而用于治疗薄型子宫内膜。现主要就骨髓间质干细胞（BMSCs）、子宫内膜干细胞（EDSCs）、人胚胎干细胞（hESCs）及人脐带华通胶间充质干细胞（WJ-MSCs）在薄型子宫内膜治疗的研究进展以及这4种细胞用于临床治疗的优缺点进行综述。     

Somatische Stammzellen Grundlagen, Konzepte und Herausforderungen

Adult stem cells are multipotent cells that have the ability to self-renew and to differentiate into highly specialized cells. Differentiation of specific cells is not limited to early embryonic development. It also takes place within the adult organism. Adult stem are found in tissues and organs showing a high turn-over rate such as blood, skin or intestine. Moreover, tissues like liver or skin are able to regenerate after injury, indicating the presence of stem cells within these organs. Whereas blood stem cells are already used in transplantation medicine, the biology and therapeutic potential of stem cells isolated from other tissues are still under investigation. Currently, research is focused on the identification and analysis of the factors regulating stem cell self-renewal and differentiation. A better understanding of stem cell biology certainly will lead to new therapeutic concepts, i.e. the use of stem cells and/or their derivatives as replacement cells to treat diseases including for example diabetes, Parkinson's disease, spinal cord injury, stroke, burns, rheumatoid arthritis.     

LIF: a molecule with divergent actions on myeloid leukaemic cells and embryonic stem cells

We have previously characterized, purified and cloned a novel murine and human regulator [leukaemia inhibitory factor, LIF] which induces the differentiation of certain murine and human myeloid leukaemic cells. Recently we have shown that there are specific LIF receptors on murine embryonic stem [ES] and embryonal carcinoma [EC] cells and that purified recombinant LIF can substitute for feeder cells and crude sources of differentiation inhibiting activity [DIA] [such as BRL-cell-conditioned medium] in the maintenance of ES cells in a pluripotential state in vitro. Furthermore, ES cells maintained in culture in recombinant LIF for a prolonged period can give rise to germline chimaeric mice. Thus, based on a number of biochemical and biological similarities, it is likely that LIF and DIA are the same molecule. The identification of LIF as a molecule, necessary and sufficient for the maintenance of ES cells in culture, should have a profound impact on the use of these cells for genetic manipulations.     

The Commercialization of Human Stem Cells: Ethical and Policy Issues

The first stage of the human embryonic stem(ES) cell research debate revolved aroundfundamental questions, such as whether theresearch should be done at all, what types ofresearch may be done, who should do theresearch, and how the research should befunded. Now that some of these questions arebeing answered, we are beginning to see thenext stage of the debate: the battle forproperty rights relating to human ES cells. The reason why property rights will be a keyissue in this debate is simple and easy tounderstand: it costs a great deal of money todo this research, to develop new products, andto implement therapies; and private companies,researchers, and health professionals requirereturns on investments and reimbursements forgoods and services. This paper considersarguments for and against property rightsrelating to ES cells defends the followingpoints: (1) It should be legal to buy and sellES cells and products. (2) It should be legalto patent ES cells, products, and relatedtechnologies. (3) It should not be legal tobuy, sell, or patent human embryos. (4) Patentson ES cells, products, and related technologiesshould not be excessively broad. (5) Patents onES cells, products, and related technologiesshould be granted only when applicants statedefinite, plausible uses for their inventions. (6) There should be a research exemption in EScell patenting to allow academic scientists toconduct research in regenerative medicine. (7)It may be appropriate to take steps to preventcompanies from using patents in ES cells,products, and related technologies only toblock competitors. (8) As the field ofregenerative medicine continues to develop,societies should revisit issues relating toproperty rights on a continuing basis in orderto develop policies and develop regulations tomaximize the social, medical, economic, andscientific benefits of ES cell research andproduct development.     

Genetic modification of somatic cells for producing animal models and for cellular transplantation

Great progress has been made in two technologies related to biomedical research: (1) manipulating the genomes of cells; and (2) inducing stem cells in culture to differentiate into potentially useful cell types. These technologies can be used to create animal models of human disease and to provide cells for transplantation to ameliorate human disease. Both embryonic stem cells and adult stem cells have been studied for these purposes. Genetically modified somatic cells provide another source of cells for creating animal models and for cellular transplantation.     

Embryonic stem cells in companion animals (horses, dogs and cats): present status and future prospects

Reproductive technologies have made impressive advances since the 1950s owing to the development of new and innovative technologies. Most of these advances were driven largely by commercial opportunities and the potential improvement of farm livestock production and human health. Companion animals live long and healthy lives and the greatest expense for pet owners are services related to veterinary care and healthcare products. The recent development of embryonic stem cell and nuclear transfer technology in primates and mice has enabled the production of individual specific embryonic stem cell lines in a number of species for potential cell-replacement therapy. Stem cell technology is a fast-developing area in companion animals because many of the diseases and musculoskeletal injuries of cats, dogs and horses are similar to those in humans. Nuclear transfer-derived stem cells may also be selected and directed into differentiation pathways leading to the production of specific cell types, tissues and, eventually, even organs for research and transplantaton. Furthermore, investigations into the treatment of inherited or acquired pathologies have been performed mainly in mice. However, mouse models do not always faithfully represent the human disease. Naturally occurring diseases in companion animals can be more ideal as disease models of human genetic and acquired diseases and could help to define the potential therapeutic efficiency and safety of stem cell therapies. In the present review, we focus on the economic implications of companion animals in society, as well as recent biotechnological progress that has been made in horse, dog and cat embryonic stem cell derivation.     

Male germ cell transplantation: promise and problems

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.     

Stem cells and lineage development in the mammalian blastocyst

The mammalian blastocyst is the source of the most pluripotent stem cells known: embryonic stem (ES) cells. However, ES cells are not totipotent; in mouse chimeras, they do not contribute to extra-embryonic cell types of the trophectoderm (TE) and primitive endoderm (PrE) lineages. Understanding the genetic pathways that control pluripotency v. extra-embryonic lineage restriction is key to understanding not only normal embryonic development, but also how to reprogramme adult cells to pluripotency. The trophectoderm and primitive endoderm lineages also provide the first signals that drive patterned differentiation of the pluripotent epiblast cells of the embryo. My laboratory has produced permanent mouse cell lines from both the TE and the PrE, termed trophoblast stem (TS) and eXtra-embryonic ENdoderm (XEN) cells. We have used these cells to explore the genetic and molecular hierarchy of lineage restriction and identify the key factors that distinguish the ES cell v. the TS or XEN cell fate. The major molecular pathways of lineage commitment defined in mouse embryos and stem cells are probably conserved across mammalian species, but more comparative studies of lineage development in embryos of non-rodent mammals will likely yield interesting differences in terms of timing and details.     

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 IvF-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 are stricted use of human ES cells for research. Meanwhile, the debate has ethically dividedsociety, 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 ofhealing 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 willtry 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.This revised version was published online in October 2005 with corrections to the Cover Date.     

Inositol transport in mouse embryonic stem cells

The uptake of myo-inositol by mouse embryonic stem (ES) cells was measured using [2-(3)H]myo-inositol. Uptake of myo-inositol by ES cells occurred in a mainly saturable, sodium-, time- and temperature-dependent manner, which was inhibited by glucose, phloridzin and ouabain. Self inhibition by inositol was much greater than inhibition by glucose indicating that transport was not occurring via a sodium-dependent glucose transporter. Uptake rate was much greater than efflux rate indicating a mainly unidirectional transport mechanism. Estimated kinetics parameters for sodium-dependent inositol uptake were a K m of 65.1 +/- 11.8 micromol L(-1) and a V max of 5.0 +/- 0.59 pmol microg protein(-1) h(-1). Inositol uptake was also sensitive to osmolality; uptake increased in response to incubation in hypertonic medium indicating a possible role for inositol as an osmolyte in ES cells. These characteristics indicate that myo-inositol transport in mouse ES cells occurs by a sodium-dependent myo-inositol transporter protein.     

Derivation characteristics and perspectives for mammalian pluripotential stem cells

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.     

Therapeutic cloning: far from application at this stage]

Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.     

Ethical Aspects of the Use of Stem Cell Derived Gametes for Reproduction

A lot of interest has been generated by the possibility of deriving gametes from embryonic stem cells and bone marrow stem cells. These stem cell derived gametes may become useful for research and for the treatment of infertility. In this article we consider prospectively the ethical issues that will arise if stem cell derived gametes are used in the clinic, making a distinction between concerns that only apply to embryonic stem cell derived gametes and concerns that are also relevant for gametes derived from adult stem cells. At present, it appears preferable to use non-embryonic stem cells for the derivation of gametes. Adult stem cell derived gametes do not present any problems with regard to the moral status of the human embryo, bypass the safety risks linked to SCNT and do not present any ambiguity or novel problems with regard to informed consent, psychological consequences for the child or genetic parenthood. A remaining ethical concern, however, regards the safety of the procedure in terms of the welfare of the resulting children. This should spark a thorough reflection on how far one must go to accommodate a person’s wish to have a genetically related child.     

Stem cell therapy for diabetes mellitus: progress, prospects and challenges

Curative therapy for diabetes mellitus mainly implies replacement of missing insulin-producing pancreatic beta cells, with pancreas or islet-cell transplants. The limited supply currently available from cadaveric donor islets for transplantation, however, determines that researchers must explore alternative sources of graft material. Stem cells represent a promising solution to this problem, and current research is being aimed at the creation of islet-endocrine tissue from these undifferentiated cells. Both embryonic stem cells (derived from the inner cell mass of a blastocyst) and adult tissue stem cells (found in the postnatal organism) have been used to generate surrogate beta cells or otherwise restore beta cell functioning. Nevertheless, cell replacement therapies that are stem cell based will remain fiction rather than fact until we can efficiently and reproducibly ensure that stable, fully functional cells can be generated in vitro. It is also critical to ensure that any surrogate or regenerated beta cells have perfectly regulated insulin production, which is essential for physiological glucose homeostasis. As in every emerging field in biology, early reports seem confusing and conflicting. Therefore, discrepancies between different results need to be reconciled. In addition, encouraging studies in rodent models may ultimately set the stage for large-animal studies. In this review, the authors provide insight into research efforts to overcome existing hurdles for this promising therapy.