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.     

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.     

生殖干细胞的研究进展

卵巢中始基卵泡是女性的基本生殖单位，也是卵细胞储备的唯一形式。传统观点认为，出生后卵母细胞数量不再增加，随着卵泡的发育、成熟和闭锁，卵母细胞不断耗竭，卵巢功能衰退，从而进入绝经期。随着人类在干细胞研究领域不断深入，应用干细胞治疗相关疾病已成为现实。在临床应用中，骨髓造血干细胞移植成为白血病等血液系统疾病患者的重要治疗方式。近年来，研究人员在成年小鼠卵巢内发现进行有丝分裂的生殖细胞，可能参与卵母细胞再生，这种细胞很可能就是生殖干细胞（GSCs）。GSCs的存在为卵巢功能不全、卵巢早衰和恶性肿瘤患者生育力保存及绝经后期女性健康维护带来希望。现就GSCs的研究进展进行综述。     

Germ cell transplantation for the propagation of companion animals, non-domestic and endangered species

The transplantation of spermatogonial stem cells between males results in a recipient animal producing spermatozoa carrying a donor's haplotype. First pioneered in rodents, this technique has now been used in several animal species. Importantly, germ cell transplantation was successful between unrelated, immuno-competent large animals, whereas efficient donor-derived spermatogenesis in rodents requires syngeneic or immuno-compromised recipients. Transplantation requires four steps: recipient preparation, donor cell isolation, transplantation and identifying donor-derived spermatozoa. There are two main applications for this technology. First, genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in production of transgenic spermatozoa. Transgenesis through the male germ line has tremendous potential in species in which embryonic stem cells are not available and somatic cell nuclear transfer and reprogramming pose several problems. Second, spermatogonial stem cell transplantation within or between species offers a means of preserving the reproductive potential of genetically valuable individuals. This might have significance in the captive propagation of non-domestic animals of high conservation value. Transplantation of germ cells is a uniquely valuable approach for the study, preservation and manipulation of male fertility in mammalian species.     

Blutstammzelltransplantation Ein Beispiel praktizierter Stammzelltherapie

Hematopoietic stem cells (HSC) generate all blood cell lineages during ontogeny, and their continuous function is required for the maintenance of blood cells and immune tissues throughout life. HSC are characterized by certain key properties (multipotency, clonogenicity, self-renewal). These stem cell criteria also apply to non-hematopoietic stem cells. The capacity of HSC to self-renew is the basis for experimental and clinical bone marrow transplantation in which host HSC can engraft in donor bone marrow at long term (engraftment). HSC can also be used as cellular vectors for gene transfers in vivo. Most recently, several reports have suggested the exciting possibility that HSC can, in addition to blood cells, also generate non-blood cell types such as heart muscle or neuronal tissues. Is this potential for transdifferentiation a physiological HSC function? Are transdifferentiating HSC recruited to sites of tissue damage in vivo, and, if so, can this HSC property be used to “repair” adult tissues from bone marrow stem cells? The answers are, at present, highly speculative. In the present review we will thus focus primarily on the basic biology of HSC, and on some aspects of the clinical use of HSC in bone marrow transplantation.     

[Long-term bone marrow culture]

Long-term bone marrow culture is an experimental in vitro model of hematopoiesis imitating conditions in vivo. It contains hematopoietic elements at various stages of differentiation as well as a supportive stromal microenvironment. Primitive hematopoietic stem cells of mesenchymal origin, the long-term culture initiating cells proliferate and differentiate into different cell types, giving rise to the adherent stromal layer and to various hematopoietic elements attached to it or floating freely in the supernatant medium. The stromal layer keeps the hematopoietic cells aggregated, helps their mitosis, differentiation and maturation by cell-to-cell contact, produces hematopoietic growth factors (cytokines), and forms the extracellular matrix required for cell attachment. Hematopoiesis occurs without exogenous growth factors. The appearance and development of the stroma, the proliferation and differentiation of hematopoietic progenitor cells, and the production of cytokines differ in long-term cultures of the normal and of pathologically altered bone marrow. Long-term bone marrow culture is applied in fundamental studies of normal and pathologically altered hematopoiesis, in pharmacological research, in the purging of residual leukemia cells from bone marrow autotransplants, and in the gene transfer. It is also suitable for testing carcinogenic and toxic chemicals causing hematopoietic damage through occupational or habitual exposure.     

Regeneration of the immune system after bone marrow transplantation

After haematopoietic stem cell transplantation, reconstitution of bone marrow consists of two distinct phenomena, numerical recovery of bone marrow cellular elements on the one hand and functional recovery of cellular interactions on the other. Immune reactivity during the first month postgrafting is extremely low. Cytotoxic and phagocytic functions usually recover by day 100, while more specialized and cooperative functions of T and B cells remain impaired up to one year or more postgrafting. Regeneration of total CD4+ T cell number in adult (and especially in elderly) transplant recipients is severely limited and occurs largely by peripheral expansion of mature CD4+ T cells. While restoration of total CD8+ T cell number is commonly seen in adults, potentially important alterations in the subset composition of CD8+ populations remain. Contracted T cell repertoires for CD4+ and CD8+ T cells are consistently found in adults after T cell regeneration. This suggests that thymic function is frequently limiting in adults and that thymic-independent pathways are insufficient for restoring host immunocompetence. Although there are similarities in immune reconstitution after alllo- and autologous haematopoietic stem cell transplantations, allogeneic transplantation involves graft versus host disease and the use of immunosuppressive therapy to control it, both of which further interfere in the early developmental stages of immune reconstitution.     

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.     

骨髓基质细胞诱导神经干细胞移植治疗颞叶癫痫幼年大鼠学习记忆障碍的实验研究

目的:探讨骨髓基质细胞诱导神经干细胞移植至KA大鼠海马后对学习记忆障碍的改善作用,从而为干细胞移植治疗癫痫提供理论依据。方法:首先分离幼年大鼠骨髓基质细胞,并在特定的条件下培养、诱导分化为神经干细胞。然后用KA建立大鼠癫痫模型,之后把神经干细胞移植至KA致痫鼠的海马内,用Morris水迷宫测试大鼠的学习记忆能力。结果:癫痫移植组与癫痫未移植组相比幼年大鼠空间学习记忆能力有显著性差异(P<0.05)。结论:骨髓基质细胞诱导神经干细胞移植至KA幼年大鼠海马后对学习记忆障碍有明显的治疗作用,但其具体的作用机理有待进一步研究。     

体外诱导胚胎干细胞分化为神经细胞特性的实验研究

目的研究体外利用全反视黄酸(RA)诱导小鼠胚胎干细胞(mESC)分化为神经细胞的培养方法。方法悬滴3天转悬浮1天两者结合的培养方法得到胚体(EBs),再利用RA对其处理4天后,进行免疫组织化学、RT-PCR以及兴奋性功能的检测,观察神经细胞获得的效率及其生理特性。结果利用RA诱导方法得到的神经细胞,免疫组化表明nestin蛋白表达的阳性率为(53.49±6.02)%,且RT-PCR法分析其能表达nestin、glutaminase和Brn-3等神经细胞特异基因,同时在Glu刺激下具有与脑来源神经元相似的特性和功能。结论利用本实验诱导方法,获得神经细胞的比例较高,且具有与神经元相似的生物学特性。     

Bone marrow transplantation in adolescents

This paper reviews bone marrow transplantation in adolescents. The primary indications for bone marrow transplantation are malignancies, usually relapsed lymphomas or acute/chronic leukemias. Autologous bone marrow transplantation is used as a high-dose consolidation therapy in some solid tumor patients with varied success. Peripheral blood stem cells are a feasible source of autologous stem cells in adolescents. The process of stem cell transplantation and the complications are the same in adolescents as in younger children and adults. Adolescents face the same biologic barriers to allogeneic transplant (minimal residual disease, availability of donor), but may also face more problems with their insurance status. The psychological and social aspects of bone marrow transplantation during adolescence are unique to their developmental stage. With appropriate medical, nursing, and psychosocial support, bone marrow transplantation offers cure for the adolescent with high-risk disease.     

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.     

Stem cell transplantation in the treatment of gastrointestinal diseases

The stem cells with self-renewal ability are capable to form one or more cell types. They will be in the target of cell and gene therapy because of their multipotency and easy retrieval. Application of adult mesenchymal, neuronal, epidermal and haematopoietic stem cell can be favourable in the treatment of cardiac (myocardial infarction), bone (osteoarthritis), neurological (Parkinson's, Alzheimer's) and hematological (hemophilia, thrombocytopenia) disorders. Authors summarize the knowledge in connection with their application in the therapy of gastrointestinal diseases. Haematopoietic stem cell transplantation has been successful for the treatment of refractory Crohn's disease, as well as in selected group of celiac patients. Mesenchymal stem cell transplantation has been proved beneficial in the prevention of liver fibrotic process. It will gain more grounds in the treatment of autoimmune liver diseases: autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis in the future. Well-designed, multicentric, prospective studies are needed to confirm the results of case reports and clinical studies with small group of patients.     

Male germ cell transplantation in livestock

Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production.     

Estimation of the energy fraction emitted by alpha particles in the mineralized part of trabecular bone which is absorbed in the bone marrow

The cells at particular carcinogenic risk in the skeleton are haematopoietic stem cells of the marrow, which are predominantly distributed throughout the haematopoietic marrow within the trabecular bone. The Monte Carlo method for estimating the fraction of the energy of alpha particles emitted in a volume of the mineral part of the bone which is deposited in the marrow is described. The relationship between the absorbed fraction (AF) of the alpha particles and their energy was found to be linear. AF (Red Marrow----Trabecular Bone) is calculated to be 0.016 for the radionuclides emitting 3 MeV alpha particles and 0.080 for those emitting 8 MeV alpha particles. As radionuclides are for purposes of bone dosimetry dichotomously classified as surface and volume seekers, the estimation of energy deposition in the skeletal target organs can be highly dependent on this classification because of complicated geometric relationships between the source and target regions.     

Stem cells: hype and hope

There is increasing interest in the use of stem cells for therapeutic purposes. The use of embryonic stem cells carries ethical and legal restrictions that limit their role in tissue regeneration. These restrictions do not apply to somatic stem cells, such as haematopoietic stem cells, which normally reside in the bone marrow. Preclinical studies have produced very promising results using these cells in experimental models of myocardial infarction. Bone-marrow cells have also been used to generate several different types of tissue. However, experimental data suggest that bone marrow also houses other non-haematopoietic stem cells, which could account for the alleged plasticity of haematopoietic stem cells. So far, the results of randomised clinical trials in patients with myocardial infarction or heart failure have been disappointing. It is clear that further research in this field is needed.     

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.     

Studies with 1,2-dithiole-3-thione as a chemoprotector of hydroquinone-induced toxicity to DBA/2-derived bone marrow stromal cells.

Stromal cells from DBA/2 mouse bone marrow have been shown to be susceptible to cytotoxicity induced by several redox-active metabolites of benzene, including hydroquinone (HQ). Treatment with HQ also alters the composition of stromal cell populations by preferentially killing stromal macrophages compared to stromal fibroblasts. This cytotoxicity can be prevented by 1,2-dithiole-3-thione (DTT) as a result of the induction of quinone reductase (QR), a quinone-processing enzyme, and glutathione. The inductive activities of DTT protected stromal cells against HQ-induced cytotoxicity and against HQ-induced impairment of stromal cell ability to support myelopoiesis. In vivo feeding of DTT to DBA/2 mice increased QR activity within the bone marrow compartment and protected bone marrow stromal cells isolated from the DTT-fed animals from ex vivo HQ challenge. Thus, the inducibility of cellular defense mechanisms and xenobiotic-processing enzymes by chemoprotective agents such as DTT may be a useful strategy for protecting against chemically induced bone marrow toxicities.