小鼠胚胎干细胞体外诱导分化为精原干细胞的研究

目的:初步探讨在体外条件下诱导小鼠胚胎干细胞(EGFP-mESC)分化为精原干细胞(SSC)的条件,建立有效的技术平台。方法:采用部分模拟胚胎早期发育的方法,将EGFP-mESC(XY)经体外悬滴培养,制备拟胚体(EB);使用免疫磁珠分选法从EB中分离出表达原始生殖细胞(PGC)表面标志SSEA-1的细胞;获得的细胞经2μmol/L维甲酸(RA)诱导增殖,使之向雄性生殖细胞分化。对诱导的细胞采用RT-PCR及免疫荧光检测特异性基因及蛋白的碱性磷酸酶。结果:在诱导EGFP-mESC向雄性生殖细胞分化的过程中,采用RT-PCR方法检测到了雄性生殖细胞特异表达基因Sry、fragilis、stella、Tex14等mRNA的表达;利用激光共聚焦方法检测到SSC表面标志蛋白Stra8、integrin-α6及Hsp90α的表达。结论:采用RA体外诱导EGFP-mESC定向分化为SSC是可行的。     

小鼠胚胎干细胞分化为精子细胞的研究进展

胚胎干细胞(ESCs)是一种具有分化发育为三个胚层组织细胞潜能的全能性细胞,哺乳动物的精子起源于原始生殖细胞(PGCs),ESCs可分化为PGCs,并进一步分化为精子细胞。通过在培养基中添加诱导分化因子(如维甲酸等)或与希望诱导分化的目的细胞(如Sertoli细胞等)共培养,并通过鉴别ESCs分化为生殖细胞的各阶段特异性基因标志物c-kit、VASA、DAZL、fragilis、miwi、mil1和mil2等,获取不同阶段的生殖细胞。鼠的ESCs已诱导出了不成熟的精子细胞,但到目前为止尚无成熟精子培养成功,且诱导分化的效率很低。     

小鼠胚胎干细胞在遗传和发育毒理学中的应用

小鼠胚胎干细胞是具有无限自我更新能力的未分化细胞系,在体外可以分化形成内胚层、中胚层和外胚层的所有细胞,能再现体内胚胎发育的全过程和基因表达模式。包括3种类型:①小鼠胚胎干细胞(embryonic stem cells, ES cells),来源于受精卵发育至囊胚阶段的内细胞团(inner cell mass, ICM);②胚胎生殖细胞(embryonic germ cells, EG cells),来源于9.5-12.5日龄胚胎生殖脊的原始生殖细胞(primordial germ cells, PGC);③胚胎癌细胞(embryonic carcinoma cells, EC cells),来源于恶性畸胎瘤的干细胞群。小鼠胚胎干细胞是唯一可以同时研究致突变物对未分化胚胎细胞和分化体细胞致突变性的细胞株,其对致突变物的敏感性因检测终点的不同而有所不同。在胚胎毒性检测方面,由于胚胎干细胞可以同时检测化学物对细胞增殖和分化的影响,从而大大提高了体外替代实验的预测符合率,有望成为化学物致畸试验的体外替代实验模型。同时,转基因技术的广泛应用和人源胚胎干细胞的建系将更有助于提高化学物胚胎毒性和致畸性体外预测符合率,但同时也存在较大的伦理学争议。     

胚胎干细胞体外分化为生殖细胞的研究进展

鼠胚胎干细胞体外已成功诱导分化为生殖细胞和配子,人胚胎干细胞也表现出相似的分化能力。本文重点综述了原始生殖细胞(PGC)的起源和发育、PGC发育的相关因素、体外胚胎干细胞分化为原始生殖细胞和配子的过程及其影响因素、多能成体干细胞分化为生殖细胞等方面。     

胚胎干细胞与雌性生殖细胞互相转化的研究进展

胚胎干细胞是全能性的干细胞,可以向3个胚层的细胞转化;雌性生殖细胞也被认为是一种"干细胞",因为其将遗传信息从一代传至下一代。两者具有相同和不同的特殊标志物,并且可以通过不同的方法互相转化,为"胚胎干细胞-卵母细胞环路"提出设想。     

如何高效制备生殖系传递的嵌合体小鼠

目前,嵌合体技术已成为胚胎干细胞(ES细胞)和诱导多能性干细胞(iPS细胞)研究不可或缺的一部分,嵌合体小鼠模型主要用于基因功能、调控机制、致病机理等各方面的研究。要安全可靠地进行ES细胞和iPS细胞的各种应用性研究,首先需要验证细胞在体内向各种组织定向分化的能力,特别是生殖系传递的能力。而高效制备多能性干细胞生殖系传递的嵌合体小鼠,需要在分子水平、细胞水平、动物个体水平以及制备方法等各方面全面综合地分析考虑,才能达到实验设计的最优化。制备嵌合体小鼠主要有注射法和聚集法2种方法,各有利弊,在整个制备过程中,需要考虑不同发育阶段以及不同小鼠品系胚胎的分化潜能、选用远交系和杂交系的胚胎作为受体,同时要考虑ES细胞的遗传背景,选择低代次的远交系和杂交系的ES细胞或者iPS细胞,以及优选KSR培养体系。对于操作者,需根据实际情况,全方位地综合考虑,从而制定出合理的操作方案。     

人胚胎干细胞建系的研究进展

人胚胎干细胞(human embryonic stem cells,hES细胞)体外成功建系,对人类胚胎发生和发育生物学、新药物开发、细胞和组织的移植等研究领域具有重大意义和深远影响。近年干细胞的研究发展很快,已成为生物学领域研究的热点之一。现对人类胚胎干细胞建系的胚胎来源、内细胞团分离、ES细胞培养体系、伦理问题、研究中存在的问题以及应用前景等作一综述。     

成年干细胞分化为卵母细胞样细胞的研究进展

近年研究表明不仅胚胎干细胞、胚胎性腺和皮肤细胞能分化产生卵母细胞,而且成年骨髓干细胞能表达生殖干细胞的标志基因、移植雌鼠骨髓细胞可使不育的受体卵巢中重新出现卵母细胞和卵泡,卵巢表面上皮细胞能形成生殖细胞样细胞,并分化为透明带蛋白阳性的卵母细胞和组装形成新的初级卵泡,以及胰腺干细胞也能诱导出现干细胞标志物、生殖细胞标志物以及减数分裂标志基因表达,产生卵母细胞样细胞,但是这些新产生的卵母细胞是否有正常功能受到质疑,卵母细胞生成调控的分子基础正在进一步探索之中。     

胚胎干细胞研究进展

胚胎干细胞来源于着床前的囊胚内细胞团或早期胚胎的原始生殖细胞,是一类未分化的全能性(多能性)干细胞,具有无限增殖和分化潜能.人类胚胎干细胞系的成功建立以及其体外诱导分化为特定细胞、组织甚至器官的研究进展,使其在临床应用中显示出诱人的前景.就胚胎干细胞系的建立、定向分化及临床应用等研究现状作一综述.     

干细胞在妇产科领域的研究和应用前景

干细胞研究，已取得了举世瞩目的成就。干细胞及其衍生组织器官的临床应用，必将导致一次医学革命，产生一种全新的治疗技术。干细胞是一类具有自我更新和增殖分化能力的细胞，可以产生出与细胞自身完全相同的子细胞，同时还可以分化为仍保持干细胞特性的祖细胞。按分化阶段不同，干细胞可分为胚胎干细胞(embryonic stem cell，ESC)     

Induced pluripotent stem cells in reproductive medicine

Despite recent advances in reproductive medicine, there are still no effective treatments for severe infertility caused by congenital absence of germ cells or gonadotoxic treatments during prepubertal childhood. However, the development of technologies for germ cell formation from stem cells in vitro, induction of pluripotency from somatic cells, and production of patient-specific pluripotent stem cells may provide new solutions for treating these severe fertility problems. It may be possible to produce germ cells in vitro from our own somatic cells that can be used to restore fertility. In addition, these technologies may also bring about novel therapies by helping to elucidate the mechanisms of human germ cell development. In this review, we describe the current approaches for obtaining germ cells from pluripotent stem cells, and provide basic information about induction of pluripotency and germ cell development.     

Trial evaluation of bone marrow derived mesenchymal stem cells (MSCs) transplantation in revival of spermatogenesis in testicular torsion

Defective spermatogenesis due to the failure in germ cell proliferation and differentiation is the major sign of male infertility pathogenesis; and male factor is involved in up to half of all infertile couples. Testicular torsion is an acute vascular event causing the rotation of the vascular pedicle of the testis, thereby impeding the blood flow to the testis and the scrotal contents. Azoospermia caused by torsion in testis is a common source of impotency, which has not been touched by this approach, yet. Here, we use the capacity of mesenchymal stem cells (MSCs), as multipotent adult stem cells, to revive spermatogenesis in torsion-induced azoospermia. For this purpose, MSCs were extracted from rat bone marrow, cultured and transplanted into torsioned testis. Germ cell specific markers (Oct4, Vasa and c-Kit) were monitored for the differentiation of MSCs after transplantation into the torsion azoospermed testis. This study is a trial evaluation of mesenchymal stem cells in rat torsion testis to follow up the regenerative capacity of stem cells in spermatogenesis revival. These approaches can provide the powerful tools to investigate the basic biology of stem cells for reproductive engineering and infertility treatment.     

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

Background

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans.

Methods

Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized.

Results

In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species‐specific requirements for growth factors and mechanisms supporting the self‐renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential.

Conclusion

Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.     

Long-term survival of human spermatogonial stem cells in mouse testes

OBJECTIVE: To evaluate colonizing ability of human spermatogonial stem cells in mouse testes. DESIGN: Transplantation of human testis cells into the seminiferous tubules of immunodeficient mice. SETTING: University hospital and academic laboratory. PATIENT(S): Men with obstructive azoospermia or maturation arrest of spermatogenesis.Analyzed up to 6 months after transplantation. Also analyzed: cryopreservation of donor cells, donor cell concentrations, and leuprolide treatment of recipients. MAIN OUTCOME MEASURE(S): Detection of human donor cells in recipient testes using whole-mount immunohistochemistry with antibodies that react with human germ cells. RESULT(S): Mouse testes were colonized by human testis cells obtained from each of 6 patients; overall, human spermatogonia were found in 16 of 22 (73%) recipient testes. Human spermatogonial stem cells survived in mouse testes for at least 6 months and proliferated during the first month after transplantation. No human-differentiating spermatogonia were identified, and meiotic differentiation did not occur in mouse testes. In this initial study, human stem cell colonization was not influenced by cryopreservation of donor cells, donor cell concentration, or leuprolide treatment of recipient mice. CONCLUSION(S): Xenogeneic transplantation of human germ cells using mice as recipients is feasible and could be used as a biological assay system to further characterize human spermatogonial stem cells. This study might provide a mechanism to evaluate the status of the stem cell population in selected infertile male patients.     

Stem cells in gynaecology

Currently, there is enormous interest in stem cells as a new treatment modality for regenerative medicine, commencing when human embryonic stem (hES) cells were first cultured from spare in vitro fertilisation-derived embryos. Emerging evidence also suggests that somatic stem cells may have greater differentiation potential. Stem cell research is now in an exciting phase of development and has the potential to dramatically influence therapeutics as hES cell derivatives and/or adult stem cells are applied to regenerative medicine or to deliver gene therapy. Human ES cells show apparently limitless proliferative potential and differentiation capacity into all tissue types. Adult stem cells are rare cells, which maintain the tissue in which they reside. The challenges facing the use of hES cells and adult stem cells in medicine are highlighted and examples of their use in laboratory studies and the clinic are given. Adult stem cells have been identified in diverse tissues, including human bone marrow, breast, prostate, brain and liver. We hypothesised that adult stem cells reside in the endometrium, a highly proliferative, cyclically regenerating tissue. Our research has demonstrated, for the first time, that human endometrium contains a small population of epithelial cells (0.22%) and stromal cells (1.25%) that exhibit stem/progenitor cell behaviour in vitro; clonogenicity. The progeny in these colonies have been characterised and growth factors supporting clonogenicity identified. The goal is to examine the role of putative endometrial stem/progenitor cells in proliferative disorders of human endometrium, such as endometriosis, adenomyosis, endometrial hyperplasia and endometrial cancer, and the action of hormone-replacement therapy on the post-menopausal endometrium.     

Germ cell specification and pluripotency in mammals: a perspective from early embryogenesis

Germ cells are unique cell types that generate a totipotent zygote upon fertilization, giving rise to the next generation in mammals and many other multicellular organisms. How germ cells acquire this ability has been of considerable interest. In mammals, primordial germ cells (PGCs), the precursors of sperm and oocytes, are specified around the time of gastrulation. PGCs are induced by signals from the surrounding extra-embryonic tissues to the equipotent epiblast cells that give rise to all cell types. Currently, the mechanism of PGC specification in mammals is best understood from studies in mice. Following implantation, the epiblast cells develop as an egg cylinder while the extra-embryonic ectoderm cells which are the source of important signals for PGC specification are located over the egg cylinder. However, in most cases, including humans, the epiblast cells develop as a planar disc, which alters the organization and the source of the signaling for cell fates. This, in turn, might have an effect on the precise mechanism of PGC specification in vivo as well as in vitro using pluripotent embryonic stem cells. Here, we discuss how the key early embryonic differences between rodents and other mammals may affect the establishment of the pluripotency network in vivo and in vitro, and consequently the basis for PGC specification, particularly from pluripotent embryonic stem cells in vitro.     

The promise of human embryonic stem cells

Pluripotency refers to the ability of a cell to give rise to cells that originate from all three germ layers. Among the available human pluripotent cells, human embryonic stem cells (hESCs) are considered to have the greatest probability for practical clinical application because of their simple propagation and stability in culture. Since their first derivation, issues concerning hESC maintenance and self-renewal have been widely addressed. The first part of this review presents the accumulated knowledge concerning the self-renewal of hESCs and discusses recent genetic profile data, which seem to shed light on hESC self-renewal and pluripotency mechanism. The second part deals with the regenerative potential of hESCs. Available lineage-specific differentiations of hESCs are presented, with detailed data on the ability of hESCs to differentiate into trophoblast cells, an observation that might broaden the definition of their developmental potential. Specific focus is given to vascular cell differentiation, including endothelial and smooth muscle cells. Transplantation limitations as well as current steps taken toward resolution conclude the review.     

Immunosuppression by placental indoleamine 2,3-dioxygenase: a role for mesenchymal stem cells

BACKGROUND/OBJECTIVES: Mesenchymal stem cells (MSC) can be isolated from human placenta and have the potential to contribute to the immunosuppressive properties of placental tissue. The objectives of this study were to investigate the phenotype and differentiation characteristics of MSC derived from human placenta and evaluate the role of the tryptophan degrading enzyme, indoleamine 2,3 dioxygenase (IDO), in mediating their immunosuppressive affect. METHODS: MSC obtained from placental tissue (pMSC) were characterised using flow cytometry and tested for multipotency by determining differentiation into all mesenchymal lineages. The immunosuppressive properties of pMSC were tested in allogeneic mixed lymphocyte reactions and IDO expression and activity were measured by semi-quantitative real-time PCR and HPLC respectively. RESULTS: Multipotent stem cells were isolated from placenta and displayed chondrogenic, osteogenic and limited adipogenic differentiation. Cell surface antigen expression of pMSC was similar to bone marrow MSC (bMSC) with lack of the haematopoietic and common leukocyte markers (CD34, CD45), and expression of adhesion (CD29, CD166, CD44) and stem cell (CD 90, CD105, CD73) markers. Placental MSC were suppressive of allogeneic T-cell proliferation, an effect which was intensified following IDO induction by IFN-gamma. Replenishment of tryptophan or treatment with the IDO-blocker, 1-methyl-tryptophan (1-MT), attenuated the immunosuppressive action of pMSC. CONCLUSIONS: These results suggest that placental tissue contains MSC, which are phenotypically and functionally similar to bMSC, and that IDO is a key mediator of their immunosuppressive effect. Further investigation is needed to determine if pMSC function effects pregnancy outcome.