A European discussion about stem cells for therapeutic use

Stem cells as a source material for growing cellular transplants to repair dysfunctional organs appear to be a new challenge for medical science. Though stem cells are also present in foetal and adult organs, embryonic stem cells from the pre-implantation embryo in particular have the potency to proliferate easily in vitro and the capacity to differentiate into all the body's organ-specific cells. Therefore, these are the ideal cells for developing new cell transplantation therapies for diseases such as Parkinson's disease, diabetes mellitus and heart failure. The use of spare in vitro fertilization (IVF) embryos or pre-implantation embryos specially created to harvest human embryonic stem cells is, however, controversial and an ethical problem. In a European discussion platform organised by the European Commission Research Directorate-General, the status quo of the progress was presented and subsequently commented upon and discussed in terms of medical-ethical, social, industrial and patient interests. The expectations of this new medical technology were high, but clinical trials seem only acceptable once the in vitro differentiation of stem cells can be adequately controlled and once it is known how in vitro prepared stem cells behave after implantation. The ethical justification of the use of in vitro pre-implantation embryos remains controversial. The prevailing view is that the interests of severely ill patients for whom no adequate therapy exists, surmounts the interest of protection of a human in vitro pre-implantation embryo, regardless of whether it was the result of IVF or of transplantation of a somatic cell nucleus of the patient in an enucleated donor egg cell (therapeutic cloning).     

干细胞研究与女性生殖

成年卵巢可能通过干细胞而再生卵母细胞,体外培养胚胎干细胞可形成卵母细胞,为治疗性克隆提供可能的卵母细胞来源,对提高和保存女性生育力有重要意义。近年妊娠相关的干/祖细胞参与母体组织修复也引起关注。随着干细胞和组织工程研究技术的发展,用不同的方法深入研究,希望能阐明女性生殖的发育生物学机制及生殖系统疾病的发病机制,为临床治疗提供新的切入点。     

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.     

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.     

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 in debate]

Human embryos can be conceived by cell nuclear transfer in order to isolate human embryonic stem cells (hES cells) for research into autologous cell therapy (therapeutic cloning). However, this technique broaches the major ethical problem concerning the instrumental use of human preimplantation embryos. From the viewpoint of subsidiarity, it is argued that various potential alternatives for therapeutic cloning should first be investigated further. The question as to whether therapeutic cloning should be allowed only becomes apparent when research with surplus embryos obtained in the course of in-vitro fertilization suggests that usable transplants can be obtained in vitro from hES cells, and when the potential alternatives for therapeutic cloning are either less promising or need more time for development than is currently expected.     

Novel method for demonstrating nuclear contribution in mouse nuclear transfer

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.     

The mitochondrial contribution to stem cell biology

The distribution and functions of mitochondria in stem cells have not been examined, yet the contributions of these organelles to stem cell viability and differentiation must be vitally important in view of their critical roles in all other cell types. A key role for mitochondria in stem cells is indicated by reports that they translocate in the oocyte during fertilisation to cluster around the pronuclei and can remain in a perinuclear pattern during embryo development. This clustering appears to be essential for normal embryonic development. Because embryonic stem cells are derived from fertilised oocytes, and eventually can differentiate into 'adult' stem cells, it was hypothesised that mitochondrial perinuclear clustering persists through preimplantation embryo development into the stem cells, and that this localisation is indicative of stem cell pluripotency. Further, it was predicted that mitochondrial activity, as measured by respiration and adenosine triphosphate (ATP) content, would correlate with the degree of perinuclear clustering. It was also predicted that these morphological and metabolic measurements could serve as indicators of 'stemness.' This article reviews the distribution and metabolism of mitochondria in a model stem cell line and how this information is related to passage number, differentiation and/or senescence. In addition, it describes mitochondrial DNA deletions in oocytes and embryos that could adversely affect stem cell performance.     

Sucrose-exposed chemically enucleated mouse oocytes support blastocyst development of reconstituted embryos

This study was carried out to test the ability of sucrose-exposed chemically enucleated mouse oocytes to support the development of reconstituted embryos in vitro. Cumulus-enclosed germinal-vesicle-stage mouse oocytes were matured in vitro to metaphase I stage and were chemically enucleated with 50 microg mL(-1) etoposide in tissue culture medium 199. The chemically enucleated oocytes were grouped into two groups. Group I was exposed to 0.75 M sucrose and group II was not exposed to sucrose. The zonae pellucidae of the chemically enucleated oocytes were removed with acid Tyrode's solution (pH 2.7). They were then aggregated into couplets with karyoplasts from pronuclear-stage embryos using phytohemagglutinin-P. The couplets were electrically fused to form reconstituted embryos. The reconstituted embryos were activated with 7% ethanol and cultured in vitro in simplex optimisation medium to test their developmental ability to the blastocyst stage. Some of the reconstituted embryos that developed to the blastocyst stage were used for chromosome counts to test their ploidy. The results of the present study showed that chemically enucleated oocytes exposed to sucrose supported the development of reconstituted embryos to the blastocyst stage (21.5%), whereas those not exposed to sucrose did not. The chromosome counts showed that the reconstituted embryos had normal ploidy (40 chromosomes). It is concluded that sucrose exposure improves the quality of chemically enucleated mouse oocytes. Thus they can be used as recipients for mouse embryo cloning and nucleocytoplasmic interaction studies.     

Therapeutic and reproductive cloning: a critique

This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.     

Cellular therapy--the possible future of regenerative medicine

Stem cells are a unique cell population capable of self-renewal and differentiation into different cell lines. There are two main types of stem cells: embryonic stem cells (pluripotent) and somatic/adult stem cells (multipotent cells differentiated into the specific types of the tissue they originate from). Scientists are now interested in finding the sources of cells that can be used for therapeutic cloning as a method of saving human life and a new trend in regenerative medicine. Reproductive cloning, which aims at creating genetically identical human beings, is prohibited and is subject to national legislation in each country. Mesenchymal stem cells, with their capability to elude detection by the host's immune system and their relative ease of expansion in culture, are a very promising source of stem cells for regenerative medicine. This is the vast potential of cellular therapy for treating damaged and degenerating tissues.     

Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V, Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL(-1) in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9-10 x 10(6) spermatozoa mL(-1) in Brackett and Oliphant medium and the cleavage rate was recorded 40-42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P < 0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL(-1). It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.     

Embryonic Stem Cell Patents and Human Dignity

This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells.     

Dry storage of sperm: applications in primates and domestic animals

Cryopreservation of spermatozoa, oocytes and embryos, as well as somatic cells or cell lines for cloning from cells, are all options for the long-term storage of unique genotypes and endangered species. Spermatozoal cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration-based methods for long- or short-term storage, which requires routine maintenance and extensive space requirements. The preservation of stem cells also has strict requirements for long-term storage to maintain genetic integrity. Dessicated (lyopreserved) sperm and stem cells will provide an unprecedented type of long-term storage without the need for expensive and burdensome cryogenic conditions. Experiments were conducted to determine an effective intracellular concentration of the lyoprotectant trehalose. High-pressure liquid chromatography studies revealed that trehalose can be incorporated into mature sperm cells as well as spermatogonial stem cells from rhesus monkeys. In addition, using fourier transform infrared spectroscopy, we determined that thermotropic phase transitions for fresh ejaculates from rhesus monkey and stallion sperm occurred at 10-15, 33-37 and 55-59 degrees C. Preliminary studies in our laboratory have indicated that spermatogonial stem cells can be dried to <3 g g(-1) water and maintain viability following rehydration. Studies in our laboratory have provided preliminary results suggesting that the desiccated storage of sperm and spermatogonial stem cells may be a viable alternative to conventional cryopreservation.     

Commercial applications of nuclear transfer cloning: three examples

Potential applications of cloning go well beyond the popularly envisioned replication of valuable animals. This is because targeted genetic modifications can be made in donor cells before nuclear transfer. Applications that are currently being pursued include therapeutic protein production in the milk and blood of transgenic cloned animals, the use of cells, tissues and organs from gene-modified animals for transplantation into humans and genetically modified livestock that produce healthier and safer products in an environmentally friendly manner. Commercial and social acceptance of one or more of these early cloning applications will lead to yet unimagined applications of nuclear transfer technology. The present paper summarises progress on three additional applications of nuclear transfer, namely the development of male livestock that produce single-sex sperm, the transfer of immune responses from animals to their clones to permit the production of unlimited supplies of unique polyclonal antibodies, and the generation of genetically modified animals that accurately mimic human diseases for the purpose of developing new therapies. However, the myriad applications of cloning will require appropriate safeguards to ensure safe, humane and responsible outcomes of the technology.     

Circulating stem cells and tissue repair

Stem cells are defined as cells that have clonogenic, self-renewing capacities and the capability to differentiate into multiple cell lineages. Whereas embryonic stem cells are derived from mammalian embryos in the blastocyst stage and can generate terminally differentiated cells of all 3 embryonic germ layers, adult human stem cells are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiologic cell turnover or tissue injury. The traditional idea of organ-restricted stem-cell differentiation is now being challenged by the suggestion that adult stem cells retain developmental plasticity. Preclinical and clinical studies described in this review provide evidence that within the blood circulate not only progenitor cells that differentiate into hematopoietic cells, but also stem/progenitor cells which can participate in the homeostasis, repair and replacement of solid organ tissues. In addition to the occurrence of cell fusion, there are 4 suggested mechanisms of adult stem cell differentiation into solid organ cells. Preclinical data support these models particularly that of transdifferentiation as the most likely model, allowing stem/progenitor cells to differentiate across lineage, tissue, and germ layer boundaries. There is increasing evidence that we can manipulate in vivo circulating adult stem cells to repair or regenerate solid organ tissue, which offers potential clinical benefit in the treatment of many hereditary and acquired diseases.     

Stem cell research: licit or complicit? Is a medical breakthrough based on embryonic and fetal tissue compatible with Catholic teaching?

In November 1998 biologists announced that they had discovered a way to isolate and preserve human stem cells. Since stem cells are capable of developing into any kind of human tissue or organ, this was a great scientific coup. Researchers envision using the cells to replace damaged organs and to restore tissue destroyed by, for example, Parkinson's disease, diabetes, or even Alzheimer's. But, since stem cells are taken from aborted embryonic and fetal tissue or "leftover" in vitro embryos, their use raises large ethical issues. The National Institutes of Health (NIH) recently decided to fund research employing, not stem cells, but "cell lines" derived from them. The NIH has essentially made an ethical determination, finding sufficient "distance" between cell lines and abortion. Can Catholic universities sponsoring biological research agree with this finding? Probably not. In Catholic teaching, the concept of "complicity" would likely preclude such research. However, Catholic teaching would probably allow research done with stem cells obtained from postpartum placental tissue and from adult bone marrow and tissue. These cells, which lack the pluripotency of embryonic and fetal stem cells, are nevertheless scientifically promising and do not involve the destruction of human life.     

Somatische Stammzellen des Pankreas Perspektiven für die Transplantationsmedizin

Insulin-dependent Diabetes mellitus is a devastating disease and affected individuals depend on exogenous insulin to maintain glucose homeostasis. Furthermore, the disease invariably results in a variety of debilitating complications that threaten the quality of life and the life expectancy of these patients. Pancreas transplantation displays the most logical approach to restore glucose homeostasis. Recent advances include novel, glucocorticoid-free immunosuppression and improved islet isolation for transplantation. This will augment current indications for transplantation and will increase the demand for the appropriate tissues. However, transplantation is restricted by the shortage of donor tissue. Consequently, protocols need to be established to obtain insulin-secreting cells from alternative sources. Pluripotent pancreatic stem cells, stimulated to develop into islet cells offer the possibility of a renewable source of tissue to treat Diabetes. In recent years promising progress has been made in this field and Diabetes mellitus may be one of the first disorders treated by stem cell therapy.     

人类成熟卵母细胞玻璃化冷冻研究

目的:研究人类成熟卵母细胞玻璃化的方法和冷冻液,探讨人类卵母细胞玻璃化冷冻保存的方法及临床应用价值。方法:运用cryoleaf进行人类成熟卵母细胞玻璃化冷冻保存,复苏后行胞浆内单精子注射-胚胎移植或培养至囊胚形成,观察复苏后卵母细胞受精及发育能力。根据冷冻方法分为程序化冷冻组和玻璃化冷冻组,又将玻璃化冷冻组分为自配液冷冻组和成品液冷冻组,比较各组存活率、受精率、卵裂率及囊胚形成率。结果:程序化冷冻组和玻璃化冷冻组间存活率、卵裂率、囊胚形成率均无显著性差异(P>0.05),但是受精率存在显著性差异(P<0.05);程序化组与自配液组、成品液组间存活率、卵裂率均无显著性差异(P>0.05),但受精率存在显著性差异(P<0.05)。成品液组与自配液组、程序化组间囊胚形成率存在显著性差异(P<0.05)。程序化组移植4例,获得临床妊娠并分娩1例,玻璃化组移植7例,获得1例生化妊娠。结论:程序化冷冻与玻璃化冷冻法均可应用于人类成熟卵母细胞,冻融后卵母细胞具备正常受精能力及卵裂能力。玻璃化冻存人类卵母细胞具有更大发展趋势。