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.     

胚胎干细胞专利相关信息分析

目的对1985年至2004年12月的胚胎干细胞相关专利信息进行详细阅渎和分析,了解国内、外胚胎干细胞相关专利的情况。方法以干细胞及胚胎干细胞为主题词,以德文特(Der- went)数据库为背景,对多个专利数据库进行检索、筛查和分类,建立胚胎干细胞专利数据库。从专利的角度入手,对数据库中的630篇国际及44篇国内胚胎干细胞专利文献进行分类分析。结果胚胎干细胞研究领域的专利申请情况总体呈上升趋势;申请范围主要涉及动物细胞或组织、修饰的细胞、脊椎动物新品种及未分化的人类/动物细胞等方面,国际(13.4%)和国内(20.9%)申请均以涉及动物细胞或组织的占据首位;国内申请中涉及未分化的人/动物细胞的专利较多(19.8%);转基因动物、细胞建系及核移植技术相关的专利申请是胚胎干细胞相关专利中的重要组成部分。结论国内外胚胎干细胞相关信息的分析表明,目前我国在胚胎干细胞研究申请专利方面相对比较宽松,我们应该在涉及胚胎干细胞的分离、建系、在基因工程及细胞组织工程学方面的用途和方法以及胚胎干细胞在药物筛选过程中的用途等方面集中投入力量,争取有新的突破。     

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.     

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.     

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.     

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.     

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.     

Herzgewebe aus embryonalen Stammzellen

Embryonic stem cells can give rise to all somatic cells, making them an attractive cell source for tissue engineering applications. The propensity of cells to form tissue-like structures in a culture dish has been well documented. We and others made use of this intrinsic property to generate bioartificial heart muscle. First proof-of-concept studies involved immature heart cells mainly from fetal chicken, neonatal rats and mice. They eventually provided evidence that force-generating heart muscle can be engineered in vitro. Recently, the focus shifted to the application of stem cells to eventually enable the generation of human heart muscle and reach following long-term goals: (1) development of a simplified in vitro model of heart muscle development; (2) generation of a human test-bed for drug screening and development; (3) allocation of surrogate heart tissue to myocardial repair applications. This overview will provide the background for cell-based myocardial repair, introduce the main myocardial tissue engineering concepts, discuss the use of embryonic and non-embryonic stem cells, and lays out the potential direct and indirect therapeutic use of human tissue engineered myocardium.     

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.     

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.     

Derivation of human embryonic stem cell lines, towards clinical quality

Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells.     

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.     

冷冻胚胎经序贯共培养用于建立人胚胎干细胞系

目的:利用长期冷冻人卵裂期胚胎经序贯共培养形成的囊胚建立人类胚胎干细胞系。方法:将冷冻≥10年的卵裂期胚胎复苏后,采用单层卵丘细胞序贯共培养至囊胚期,经辅助孵出,置鼠胚胎成纤维细胞(MEF)饲养层全胚培养,原代培养6d后加入20%MEF条件培养基培养至形成原代克隆。观察人胚胎干细胞集落的生长状态并通过碱性磷酸酶染色、Oct-4基因表达、核型分析及体外分化实验等方法进行生物学鉴定。结果:9枚冷冻卵裂期胚胎经序贯共培养,有6枚发育到囊胚期,最终获得1株胚胎干细胞系。经鉴定该细胞系具有碱性磷酸酶活性、表达Oct-4胚胎干细胞特异标记、形成拟胚体、在体外形成具有自动节律性的心肌细胞、并具有46,XY核型等5种特性。结论:冷冻胚胎经序贯共培养能够改善胚胎发育潜能,有助于建立人胚胎干细胞系。     

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.     

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。     

昆明鼠胚胎干细胞系建立中原代克隆生长及其传代方法的研究

目的:探讨昆明鼠胚胎内细胞团分离方法、原代干细胞克隆分离时机及干细胞传代方法对干细胞建系效率的影响。方法:取昆明鼠3.5d囊胚建胚胎干细胞系,比较全胚培养法与免疫外科法两种内细胞团分离方法对胚胎干细胞建系效率的影响,观察原代克隆的分离时机,机械法传代和酶消化传代对胚胎干细胞建系效率的影响。结果:全胚培养法30个囊胚建系3个,免疫外科法32个囊胚建系4个,两组均可以有效地形成胚胎干细胞系;昆明鼠原代干细胞克隆分离最佳时机是增殖4～6d;5代以前的干细胞以机械传代方法较好,5代后用酶消化法传代效果较好。结论:全胚培养法和免疫外科法均能有效建立昆明鼠胚胎干细胞系,采用机械化与酶消化法相结合传代更适合昆明鼠干细胞建系。     

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.     

人胚胎干细胞建系研究中的伦理管理

目的：建立国人胚胎干细胞系递交国际干细胞库,并在此基础上建立既符合中国国情又得到国际认可的相关伦理管理体系。方法：在比尔盖茨基金会的资助下,与北京大学生命科学院再生生物学实验室合作,募集胚胎建立人胚胎干细胞系,在此过程中探讨可行的符合国际伦理原则的相关伦理管理机制。结果：成功建立了国人胚胎干细胞系及相关伦理管理体系。结论：进行干细胞研究时应充分重视伦理问题,国际干细胞伦理管理与中国相关伦理原则是可以有机结合的。     

人胚胎干细胞建系研究中的伦理管理

目的：建立国人胚胎干细胞系递交国际干细胞库,并在此基础上建立既符合中国国情又得到国际认可的相关伦理管理体系。方法：在比尔盖茨基金会的资助下,与北京大学生命科学院再生生物学实验室合作,募集胚胎建立人胚胎干细胞系,在此过程中探讨可行的符合国际伦理原则的相关伦理管理机制。结果：成功建立了国人胚胎干细胞系及相关伦理管理体系。结论：进行干细胞研究时应充分重视伦理问题,国际干细胞伦理管理与中国相关伦理原则是可以有机结合的。