人卵母细胞样细胞体内分化模型的建立

干细胞通过诱导培养,在体外能够分化为卵母细胞样细胞 (Oocyte-like cell, OLC),将其置于体内环境能够有效地改善OLC的质量和发育能力。通过检测猪卵泡液中激素和Bmp 15蛋白的含量,选取了中等卵泡的卵泡液对人羊水干细胞进行体外诱导培养,10 d后,经实时定量PCR检测发现,早期生殖细胞样细胞团高表达生殖基因oct4和重新甲基化转移酶基因dnmt3b。将这些细胞团用猪卵泡膜包裹后形成移植物,移植到小鼠肾被膜下。1个月后取出移植物,发现移植物内的细胞在形态上,不仅与正常的卵母细胞极为相似,而且还表达生殖细胞和卵母细胞特异标记基因 (oct4、nanog、stella、ifitm3、dazl、nanos3、bmp15和gdf9)。证明技术体系能够有效改善OLC的形成和发育能力。     

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.     

Primordial Germ Cell-Like Cells Differentiated In Vitro from Skin-Derived Stem Cells

Background

We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.

Methodology/Principal Findings

When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.

Significance

The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.     

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20–30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.     

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.     

Germ cell differentiation and synaptonemal complex formation are disrupted in CPEB knockout mice.

CPEB is a sequence-specific RNA binding protein that regulates translation during vertebrate oocyte maturation. Adult female CPEB knockout mice contained vestigial ovaries that were devoid of oocytes; ovaries from mid-gestation embryos contained oocytes that were arrested at the pachytene stage. Male CPEB null mice also contained germ cells arrested at pachytene. The germ cells from the knockout mice harbored fragmented chromatin, suggesting a possible defect in homologous chromosome adhesion or synapsis. Two CPE-containing synaptonemal complex protein mRNAs, which interact with CPEB in vitro and in vivo, contained shortened poly(A) tails and mostly failed to sediment with polysomes in the null mice. Synaptonemal complexes were not detected in these animals. CPEB therefore controls germ cell differentiation by regulating the formation of the synaptonemal complex.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.     

Skin-Derived Mesenchymal Stem Cells Help Restore Function to Ovaries in a Premature Ovarian Failure Mouse Model

Skin-derived mesenchymal stem cells (SMSCs) can differentiate into the three embryonic germ layers. For this reason, they are considered a powerful tool for therapeutic cloning and offer new possibilities for tissue therapy. Recent studies showed that skin-derived stem cells can differentiate into cells expressing germ-cell specific markers in vitro and form oocytes in vivo. The idea that SMSCs may be suitable for the treatment of intractable diseases or traumatic tissue damage has attracted attention. To determine the ability of SMSCs to reactivate injured ovaries, a mouse model with ovaries damaged by busulfan and cyclophosphamide was developed and is described here. Female skin-derived mesenchymal stem cells (F-SMSCs) and male skin-derived mesenchymal stem cells (M-SMSCs) from red fluorescence protein (RFP) transgenic adult mice were used to investigate the restorative effects of SMSCs on ovarian function. Significant increases in total body weight and the weight of reproductive organs were observed in the treated animals. Both F-SMSCs and M-SMSCs were shown to be capable of partially restoring fertility in chemotherapy-treated females. Immunostaining with RFP and anti-Müllerian hormone (AMH) antibodies demonstrated that the grafted SMSCs survived, migrated to the recipient ovaries. After SMSCs were administered to the treated mice, real-time PCR showed that the expression levels of pro-inflammatory cytokines TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFNγ were significantly lower in the ovaries than in the untreated controls. Consistent with this observation, expression of oogenesis marker genes Nobox, Nanos3, and Lhx8 increased in ovaries of SMSCs-treated mice. These findings suggest that SMSCs may play a role within the ovarian follicle microenvironment in restoring the function of damaged ovaries and could be useful in reproductive health.     

Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.     

Männliche Keimzellen aus embryonalen Stammzellen

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.     

Reconstitution of Gametogenesis In Vitro:Meiosis Is the Biggest Obstacle

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.     

人卵母细胞样细胞体内分化模型的建立

赋予抗TNF-α 单链抗体片段 (TNF-scFv) 对炎症组织的特异性,用一段来自人清蛋白 (HSA) 的柔性连接肽在基因水平上连接TNF-scFv和抗B型纤维连接蛋白 (B-FN) 的额外域B (ED-B) 的scFv L19,构建了抗TNF-α/抗ED-B单链双特异抗体BsDb,其中B-FN为炎症组织中特异表达的抗原。BsDb在毕赤酵母中获得了分泌表达,表达产物经鉴定和纯化制备后,进行了功能分析。结果表明,BsDb保留了其亲本抗体TNF-scFv和L19对抗原的免疫反应性,能够同时结合TNF-α和ED-B,并中和TNF-α的生理作用。而且,BsDb对抗原的亲和力及中和能力与大肠杆菌包涵体来源的亲本抗体相比显著增强。在小鼠佐剂型关节炎 (AIA) 模型中,BsDb能选择性地积累和保留于小鼠的炎症关节,并快速从血浆中清除。说明BsDb兼备炎症组织的特异性和正常组织的低毒性,在类风湿关节炎及其他慢性炎症性疾病的治疗上具有较大潜力。     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

IL-4 induces differentiation and expansion of Th2 cytokine-producing eosinophils

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.     

Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.