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.     

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.     

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.     

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.     

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.     

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.     

Genomic imprinting in primate embryos and embryonic stem cells

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.     

Durchbruch in der Stammzellforschung?

Embryonic stem (ES) cells are capable of generating all cell types and tissues of the body. As such they represent an attractive source for therapeutic approaches. However, transplanted cells may be rejected by the immune system. One way to address this problem is to generate patient-specific ES cells. This, however, requires the transformation of the genetic program of somatic cells back to that of an early embryonic state. The field of stem cell research and reprogramming is rapidly evolving. This article aims at providing background information to understand some of the most exciting recent developments. Subsequently, the different existing strategies of converting somatic cells into ES-like cells are reviewed and evaluated.     

Shifting paradigms? Reflections on regenerative medicine,embryonic stem cells and pharmaceuticals

Will human embryonic stem (hES) cells lead to a revolutionary new regenerative medicine? We begin to answer this question by drawing on interviews with scientists and clinicians from leading labs and clinics in the UK and the USA, exploring their views on the bench‐bedside interface in the fields of hES cells, neuroscience and diabetes. We employ Bourdieu's concepts of field, habitus and capital in order to understand stem cell science and cell transplantation. We also build on research on the sociology of expectations, and explore expectations of pharmaceutical approaches in hES research through our concept of ‘expectational capital’. In the process we discuss emerging expectations within stem cell research, most especially the ‘disease in a dish’ approach, where hES cells will be used as tools for unravelling the mechanisms of disease to enable the development of new drugs. We argue that experts’ persuasive promises advance their interests in the uncertain stem cell field, and explore how this performative strategy might stabilise the emerging ‘disease in a dish’ model of translational research.     

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.     

生物技术与医学模式变革:迈向再生医学时代

基因工程、细胞工程、组织工程和整体动物工程等新的生物技术使医学模式发生变革,从以化学药物加手术刀为主要治疗手段的传统医学模式,迈向以基因治疗、细胞移植或生物人工组织器官移植为主要的治疗手段的"再生医学"模式。使用基因工程技术生产各种重组蛋白药物或疫苗越来越广泛地应用于临床。人类基因组计划(HGP)将在2003年完成全部高质量的基因组序列,HGP所开发的资源将对未来的20年的医学领域药物开发、疾病诊断、疾病易感性预测、个体化疾病预防及治疗方案设计等方面都产生巨大的影响。基因治疗从简单的单基因形式走向一整套基因的替代的复杂形式。DNA微阵列和蛋白组微阵列等科学研究技术将应用于临床诊断。核移植技术正在用于培养通用干细胞,用于移植以治疗艾滋病、糖尿病和帕金森氏病等难治性疾病。通过干细胞移植可以再生受损的组织器官。在体外构建可供移植的生物人工组织器官以取代丧失功能的组织器官,这正是组织工程的目标。再生医学存在不少科学技术困难和伦理问题。新的生物技术不仅给我们带来希望,而且还带我们带来不可预测危险。     

脐带间充质干细胞与脐血造血干细胞临床应用的研究进展

人胚胎干细胞(HESC)系及生殖细胞系的建立,被美国时代周刊(Times)列为20世纪世界十大科技成就之首。脐带间充质干细胞(UC-MSC)和脐血造血干细胞(UCB-HSC)的研究作为其中的一部分,具有广阔的临床应用前景。UC-MSC和UCB-HSC是具有自我更新、分化能力和产生多系或单系特异细胞功能的细胞,位于细胞发育谱系的顶端。笔者拟对UC-MSC和UCB-HSC的来源、生物学特点及其临床应用等方面的进展,综述如下。     

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.     

Stem cell sources for cardiac regeneration

Cell-based cardiac repair has the ambitious aim to replace the malfunctioning cardiac muscle developed after myocardial infarction, with new contractile cardiomyocytes and vessels. Different stem cell populations have been intensively studied in the last decade as a potential source of new cardiomyocytes to ameliorate the injured myocardium, compensate for the loss of ventricular mass and contractility and eventually restore cardiac function. An array of cell types has been explored in this respect, including skeletal muscle, bone marrow derived stem cells, embryonic stem cells (ESC) and more recently cardiac progenitor cells. The best-studied cell types are mouse and human ESC cells, which have undisputedly been demonstrated to differentiate into cardiomyocyte and vascular lineages and have been of great help to understand the differentiation process of pluripotent cells. However, due to their immunogenicity, risk of tumor development and the ethical challenge arising from their embryonic origin, they do not provide a suitable cell source for a regenerative therapy approach. A better option, overcoming ethical and allogenicity problems, seems to be provided by bone marrow derived cells and by the recently identified cardiac precursors. This report will overview current knowledge on these different cell types and their application in cardiac regeneration and address issues like implementation of delivery methods, including tissue engineering approaches that need to be developed alongside.     

胚胎干细胞及诱导多能干细胞在胚胎毒性研究中的应用

药物对于生殖细胞或者早期胚胎的影响将会引起不孕或者种植前胚胎的发育异常,进而引起胚胎毒性或者是后代的畸形,因此药物的临床应用需要有可靠的实验数据证明其对胚胎的影响,而胚胎干细胞(embryonic stem cell,ESC)由于其无限增殖及多向分化的潜能而作为研究药物胚胎毒性的细胞模型得到广泛应用,以ESC为基础的胚胎干细胞实验(embryonic stem cell test,EST)是获得国际认可的胚胎毒性评价的体外替代方法,但是该实验方法的快速性和准确性存在一定的局限性,目前该细胞模型的研究主要集中于快速性和准确性的优化。新兴的诱导多能干细胞(induced pluripotent stem cells,i PSC),由于具有与ESC相似的增殖和分化特性,目前也被逐步应用于药物胚胎毒性的研究。