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1.
Infertile couples including cancer survivors stand to benefit from gametes differentiated from embryonic or induced pluripotent stem (ES/iPS) cells. It remains challenging to convert human ES/iPS cells into primordial germ-like cells (PGCLCs) en route to obtaining gametes. Considerable success was achieved in 2016 to obtain fertile offspring starting with mouse ES/iPS cells, however the specification of human ES/iPS cells into PGCLCs in vitro is still not achieved. Human ES cells will not yield patient-specific gametes unless and until hES cells are derived by somatic cell nuclear transfer (therapeutic cloning) whereas iPS cells retain the residual epigenetic memory of the somatic cells from which they are derived and also harbor genomic and mitochondrial DNA mutations. Thus, they may not be ideal starting material to produce autologus gametes, especially for aged couples. Pluripotent, very small embryonic-like stem cells (VSELs) have been reported in adult tissues including gonads, are relatively quiescent in nature, survive oncotherapy and can be detected in aged, non-functional gonads. Being developmentally equivalent to PGCs (natural precursors to gametes), VSELs spontaneously differentiate into gametes in vitro. It is also being understood that gonadal stem cells niche is compromised by oncotherapy and with age. Improving the gonadal somatic niche could regenerate non-functional gonads from endogenous VSELs to restore fertility. Niche cells (Sertoli/mesenchymal cells) can be directly transplanted and restore gonadal function by providing paracrine support to endogenous VSELs. This strategy has been successful in several mice studies already and resulted in live birth in a woman with pre-mature ovarian failure.  相似文献   

2.
The basis for the lifelong differentiation of male gametes are the spermatogonial stem cells (SSCs) in the testis (0.03% of all testicular cells). We and others have succeeded in the generation of SSCs and haploid male germ cells from mouse embryonic stem cells (ES cells). We injected these artificial spermatozoa into unfertilized oocytes, transferred the resulting two-cell embryos into the uterus of pseudopregnant female mice, and viable, fertile mice were born. Our approach provides an in vitro model for the molecular and biochemical analyses of male gametogenesis, especially meiosis and haploidisation.  相似文献   

3.
Embryonic stem cells (ESC) are self-renewal and pluripotent cells that are able to differentiate in vitro into several cell types in favourable conditions. Technical protocols for in vitro gametes production have been developed in mice and human species. The functionality of such differentiated cells is not always analysed and an early meiotic arrest is a current observation. These kinds of experimentations have also been tested from human induced pluripotent stem cells (IPSC). However, differentiation ends shortly at the primordial germ cell stage.  相似文献   

4.
5.
Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.  相似文献   

6.
离体受精作为技术平台在被子植物有性生殖研究中的应用   总被引:3,自引:1,他引:2  
被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质:研究合子细胞被激活的机理:用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。  相似文献   

7.
Despite numerous elegant transgenic mice experiments, the absence of an appropriate in vitro model system has hampered the study of the early events responsible for epidermal and dermal commitments. Embryonic stem (ES) cells are derived from the pluripotent cells of the early mouse embryo. They can be expanded infinitely in vitro while maintaining their potential to spontaneously differentiate into any cell type of the three germ layers, including epidermal cells. We recently reported that ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, which are characteristic of embryonic skin formation. Derivation of epidermal cells from murine ES cells has been successfully established by exposing the cells to precisely controlled instructive influences normally found in the body, including extracellular matrix and the morphogen BMP-4. These differentiated ES cells are able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment similar to native skin. This bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling skin development and epidermal stem cell properties.  相似文献   

8.
The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.  相似文献   

9.
Germ-line cells are responsible for transmitting genetic and epigenetic information across generations, and ensuring the creation of new individuals from one generation to the next. Gametogenesis process requires several rigorous steps, including primordial germ cell (PGC) specification, proliferation, migration to the gonadal ridges and differentiation into mature gametes such as sperms and oocytes. But this process is not clearly explored because a small number of PGCs are deeply embedded in the developing embryo. In the attempt to establish an in vitro model for understanding gametogenesis process well, several groups have made considerable progress in differen- tiating embryonic stem cells (ESCs) and adult stem cells (ASCs) into germ-like cells over the past ten years. These stem cell-derived germ cells appear to he capable of undergoing meiosis and generating both male and female gametes. But most of gametes turn out to be not fully functional due to their abnormal meiosis process compared to endogenous germ cells. Therefore, a robust system of differentiating stem cells into germ cells would enable us to investigate the genetic, epigenetic and environmental factors associated with germ cell development. Here, we review the stem cell-derived germ cell development, and discuss the potential and challenges in the differentiation of functional germ cells from stem cells.  相似文献   

10.
We have devised a general strategy for producing female mice from 39,X0 embryonic stem (ES) cells derived from male cell lines carrying a targeted mutation of interest. We show that the Y chromosome is lost in 2% of subclones from 40,XY ES cell lines, making the identification of targeted 39,X0 subclones a routine procedure. After gene targeting, male and female mice carrying the mutation can be generated by tetraploid embryo complementation from the 40,XY ES cell line and its 39,X0 derivatives. A single intercross then produces homozygous mutant offspring. Because this strategy avoids outcrossing and therefore segregation of mutant alleles introduced into the ES cells, the time and expense required for production of experimental mutant animals from a targeted ES cell clone are substantially reduced. Our data also indicate that ES cells have inherently unstable karyotypes, but this instability does not interfere with production of adult ES cell tetraploid mice.  相似文献   

11.
Potential of embryonic stem cells   总被引:29,自引:0,他引:29  
Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.  相似文献   

12.
高等植物离体受精研究进展   总被引:12,自引:1,他引:11  
高等植物的卵细胞深藏在子房内的胚珠体细胞组织中,形成了对高等植物受精过程研究的技术障碍。以前采用超微结构观察研究受精过程已取得了一定的结果,但用固定切片技术研究受精机理需将卵细胞杀死,并且不能进行定点追踪观察。将高等植物的精、卵细胞分离出来在体外诱导其融合的离体受精技术可在很大程度上克服这些技术障碍,对雌、雄配子的识别和融合,合子开始胚胎发生等一系列的受精和胚胎发生机理进行研究。分离的雌、雄配子及合子使应用分子生物学方法研究这些细胞的结构和功能成为可能。将合子的二倍性和胚胎发生特性与外源DNA转入技术结合起来可使转基因植物研究的后期工作简单化。另外,异种植物离体精、卵细胞融合和杂种合子的培养也是进行远缘杂交的一条有潜力的途径。  相似文献   

13.
Previous reports have shown that embryonic stem (ES) cells, derived from the inner cell mass of mouse or human blastocysts, could differentiate in vitro into female and male germ cells as well as into the cell types of all three germ layers. While in one case, the ES cell‐derived germ cells have been reported to give birth to live offspring in the mouse, these cells differ in fertilization capacity from the sperm and oocytes produced in vivo as they cannot complete meiosis under in vitro conditions. The efficiency of functional germ cell isolation from ES cells is also low. According to published reports, factors such as the proper selection of feeder cells, including ovarian granulosa cells and those which could secrete bone morphogenic protein‐4 (BMP4), and the addition of retinoic acid into culture medium, could to some extent establish and improve the microenvironment ES cells rely on for differentiation into germ cells. This review briefly describes the progress of deriving germ cells from ES cells and discusses possible factors that could improve in vitro gamete production. Mol. Reprod. Dev. 77: 586–594, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

14.
15.
In sexually reproducing species, fertilization brings together in the zygote the genomes of the female and male gametes. In several animal species, female gametes are able to initiate embryogenesis in the absence of fertilization, a process referred to as parthenogenesis. Parthenogenesis has been engineered in mice by tampering with expression of loci under epigenetic controls [1]. In plants, embryo development in the absence of fertilization has been reported in cases in which meiosis is bypassed leading to apomictic development, and parthenogenetic development from a reduced egg cell has been only reported in rare accidental cases [2]. We report that single mutations in the gene MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) are able to initiate parthenogenetic development of the embryo in Arabidopsis thaliana from eggs cells produced by meiosis. The WD40 repeat protein MSI1 is part of the evolutionarily conserved Polycomb group (PcG) chromatin-remodeling complexes [3] and is homologous to the Retinoblastoma binding proteins P55 in Drosophila and RbAp48 in mammals [4]. Nonviable haploid parthenogenetic msi1 embryos express molecular markers and polarity similar to diploid wild-type (wt) embryos produced by fertilization, indicating a maternal contribution to early patterning of the Arabidopsis embryo.  相似文献   

16.
17.
Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.  相似文献   

18.
Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.  相似文献   

19.
Mouse pluripotent embryonal stem ( ES ) cell lines hitherto have been conventionally isolated from the 'inner cell mass' of mouse blastocysts. In this report, I describe a new and simplified method for establishing pluripotent cell lines from mouse morulae of the 16- to 20-cell stage, which were disaggregated by the use of EDTA. From 17 cell lines established in such a way, 7 were characterized with respect to their differentiation potential:
(i) When injected into syngeneic mice, the cells gave rise to solid, fully differentiated teratomas representing derivatives of all three germ layers. (ii) When cultured in suspension in vitro, the cells were able to differentiate into complex organized 'embryoid bodies' analogous to mouse early postimplantation embryos. These results strongly imply that embryonal stem cell lines isolated from mouse morulae are highly homologous to conventionally isolated ES cells.
In addition, my results indicate that murine pluripotent embryonal stem ( ES ) cell lines can be derived with more ease and higher efficiency from disaggregated morulae than from the 'inner cell mass' of blastocysts.  相似文献   

20.
The making of "transgenic spermatozoa"   总被引:8,自引:0,他引:8  
The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.  相似文献   

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