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 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.     

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.     

干细胞向雌性生殖细胞分化的研究进展

干细胞是一类具有自我更新和多向分化潜能的细胞。根据其来源不同,干细胞可分为胚胎干细胞、成体干细胞以及诱导性多能干细胞。生殖细胞系负责跨代传递遗传和表观遗传信息,以确保新的正常个体产生。人类辅助生殖技术(ART)虽可解决部分难治性不孕不育患者的生育问题,但尚不能解决由于卵巢早衰生殖细胞缺乏导致的不孕,如果在体外可以将干细胞定向诱导分化为生殖细胞,则可能通过ART帮助卵巢早衰患者获得健康后代。雌性配子发育经历了多个严格、复杂的过程,包括原始生殖细胞(PGC)特化、增殖、迁移到生殖嵴并最终分化为成熟的卵母细胞。然而具体过程尚不明确。近年来学者已建立了干细胞向雌性生殖细胞分化的体外模型,并取得了长足进步。     

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.     

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.     

Maintenance of pluripotential embryonic stem cells by stem cell selection

As gastrulation proceeds, pluripotential stem cells with the capacity to contribute to all primary germ layers disappear from the mammalian embryo. The extinction of pluripotency also occurs during the formation of embryoid bodies from embryonic stem (ES) cells. In this report we show that if the initial differentiated progeny are removed from ES cell aggregates, further differentiation does not proceed and the stem cell population persists and expands. Significantly, the presence of even minor populations of differentiated cells lead to the complete loss of stem cells from the cultures. This finding implies that the normal elimination of pluripotent cells is dictated by inductive signals provided by differentiated progeny. We have exploited this observation to develop a strategy for the isolation of pluripotential cells. This approach, termed stem cell selection, may have widespread applicability to the derivation and propagation of stem cells.     

Germ cells and pluripotent stem cells in the mouse

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivo in chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.     

Embryonic stem cell therapy in experimental stroke: host-dependent malignant transformation

INTRODUCTION: Therapeutic application of embryonic stem cells in neurodegenerative disorders like stroke is widely investigated in preclinical animal models. AIM: The authors studied the therapeutic potential of murine embryonic stem cells in two rodent models of stroke. METHODS: Undifferentiated and predifferentiated stem cells were implanted into the non-ischemic hemisphere of mice and rats following focal brain ischemia. The brains were analysed by immunohistochemistry and histology. The in vitro differentiation of the cells was checked by immunocytochemistry and Western-blot. RESULTS: After xenotransplantation in rats undifferentiated cells migrated along the corpus callosum towards the ischemic injury. Later stem cells differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. These experiments demonstrated a hitherto unrecognized adverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells. CONCLUSION: This observation raises serious concerns about safety provisions when the therapeutic potential of human embryonic stem cells is tested in preclinical animal models. The clinical trials are based on the positive outcome of the xenologous experiments.     

Male germ line stem cells: from cell biology to cell therapy

Research using stem cells has several applications in basic biology and clinical medicine. Recent advances in the establishment of male germ line stem cells provided researchers with the ability to identify, isolate, maintain, expand and differentiate the spermatogonia, the primitive male germ cells, as cell lines under in vitro conditions. The ability to culture and manipulate stem cell lines from male germ cells has gradually facilitated research into spermatogenesis and male infertility, to an extent beyond that facilitated by the use of somatic stem cells. After the introduction of exogenous genes, the spermatogonial cells can be transplanted into the seminiferous tubules of recipients, where the transplanted cells can contribute to the offspring. The present review concentrates on the origin, life cycle and establishment of stem cell lines from male germ cells, as well as the current status of transplantation techniques and the application of spermatogonial stem cell lines.     

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。     

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.     

两株人胚胎干细胞的无饲养层扩增

目的:建立两株中国人胚胎干细胞(hESCs)的无饲养层的培养体系,观察条件培养基所需的饲养层密度,并探索最佳的传代方式。方法:两株人胚胎干细胞hES-846XX和hES-1846XY分别使用不同密度(3×105/mL、6×105/mL和1.2×106/mL)获得的条件培养基(CM)在无饲养层培养体系培养12代以上,用机械分离法和Ⅳ型胶原酶法进行传代,观察比较结果并对所得hESCs鉴定。结果:6×105/mL～1.2×106/mL之间的密度都是合适密度;Ⅳ型胶原酶在长期传代中优于机械法传代;所得细胞维持干细胞未分化状态和全能性。结论:无饲养层的培养体系可以用于中国胚胎干细胞培养,两株中国人胚胎干细胞(hESCs)的倍增周期、所需饲养层密度及分化率等方面与国外hESCs有明显差别。     

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.     

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。     

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.     

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.     

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。