The next (re)generation of ovarian biology and fertility in women: is current science tomorrow's practice?

Stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women. However, utilization of embryonic stem cells or induced pluripotent stem cells to produce oocytes has had limited success in vitro. A recent report of the isolation and characterization of endogenous oocyte-producing or oogonial stem cells (OSCs) from ovaries of reproductive age women describes the first stable and pure human female germ cell culture model in which a subset of cells appear to initiate and complete meiosis. In addition, purified human OSCs introduced into adult human ovarian cortical tissue generate oocytes that arrest at the diplotene stage of meiosis and successfully recruit granulosa cells to form new primordial follicles. This overview examines the current landscape of in vitro and in vivo gametogenesis from stem cells, with emphasis on generation of human oocytes. Future research objectives for this area of work, as well as potential clinical applications involving the use of human OSCs, are discussed.     

Ovarian stem cells—resolving controversies

A recent review on ovarian stem cells by Horan and Williams entitled “Oocyte Stem Cells: Fact or Fantasy?” suggests that the debate on ovarian stem cells (OSCs) is still not over. They did not even discuss the presence of two distinct populations of stem cells in the ovary in their review. OSCs are located in the ovary surface epithelium and Tilly’s group reported them in the size range of 5–8 μm whereas Virant-Klun’s group has reported pluripotent, 2–4 μm OSCs. Our group reported OSCs of two distinct sizes including pluripotent very small embryonic-like stem cells (VSELs) which are smaller in size than RBCs (similar to those reported by Virant-Klun’s group) and slightly bigger (similar to those reported by Tilly’s group) tissue committed progenitors (OSCs) that presumably differentiate from VSELs. These stem/progenitor cells express receptors for follicle stimulating hormone (FSH) and are activated by FSH. Our opinion article provides explanation to several open-ended questions raised in the review on OSCs by Horan and Williams. VSELs survive chemotherapy; maintain life-long homeostasis; loss of their function due to a compromised niche results in age-related senescence and presence of overlapping pluripotent markers suggest that they may also be implicated in epithelial ovarian cancers.     

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary

Background

Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies.

Results

Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02?±?0.008, 0.03?±?0.017 and 0.08?±?0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary.

Conclusions

VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

     

干细胞诱导分化为生殖细胞的研究进展

干细胞具有多系谱分化的可塑性,是再生医学的研究热点。通过各种调控、诱导手段从干细胞获得生殖细胞,为生殖细胞发生与发育障碍导致的不孕不育患者带来希望。本文将探讨从各种干细胞[胚胎干细胞(embryonic stem cells,ESCs)、诱导多能干细胞(induced pluripotent stem cells,i PSCs)、原始生殖细胞(primordial germ cells,PGCs)、精原干细胞(spermatogonial stem cells,SSCs)和卵巢干细胞(ovarian stem cells,OSCs)]获得生殖系细胞的研究进展并提出其临床应用前景与挑战。     

The potential of stem cells

Although stem cells have held the fascination of scientists for years, the attention of the general public has recently been captured by the derivation of human embryonic stem cells. In this review we describe the historical experiments leading up to the isolation of human embryonic stem cells and discuss recent advances in our understanding of both embryonic and somatic stem cells. Select examples are used to illustrate the potential of stem cells, both in the sense of their ability to differentiate into specific cell types and in the sense of their power to treat various diseases and conditions. Also discussed are recent studies describing current progress toward the treatment of Parkinson disease, spinal cord injuries, diabetes, and cardiac disease. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians LEARNING OBJECTIVES: After completion of this article, the reader will be able to describe the various types of stem cells, outline potential clinical uses of stem cells, and summarize the somatic cell transdifferentiation debate.     

Attributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary

Epithelial ovarian cancer (EOC) remains the most lethal of all the gynaecological malignancies with drug resistance and recurrence remaining the major therapeutic barrier in the management of the disease. Although several studies have been undertaken to understand the mechanisms responsible for chemoresistance and subsequent recurrence in EOC, the exact mechanisms associated with chemoresistance/recurrence continue to remain elusive. Recent studies have shown that the parallel characteristics commonly seen between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) are also shared by a relatively rare population of cells within tumors that display stem cell-like features. These cells, termed ‘cancer initiating cells’ or ‘cancer stem cells (CSCs)’ have been shown not only to display increased self renewal and pluripotent abilities as seen in ESCs and iPSCs, but are also highly tumorigenic in in vivo mouse models. Additionally, these CSCs have been implicated in tumor recurrence and chemoresistance, and when isolated have consistently shown to express the master pluripotency and embryonic stem cell regulating gene Oct4. This article reviews the involvement of Oct4 in cancer progression and chemoresistance, with emphasis on ovarian cancer. Overall, we highlight why ovarian cancer patients, who initially respond to conventional chemotherapy subsequently relapse with recurrent chemoresistant disease that is essentially incurable.     

人乳干细胞研究进展

近年研究证实,母乳中存在不同分化阶段的干细胞,包括多能干细胞、造血干细胞和间充质干细胞等。人乳干细胞组成存在个体差异,且受分娩孕周、泌乳阶段等多因素影响。通过母乳喂养进入子代的母乳干细胞也许可以促进婴儿的生长发育。同时,人乳干细胞具有多向分化潜能,未来可能作为干细胞替代治疗的新来源,具有广泛临床应用前景。现就上述几个方面的内容展开综述。     

卵巢生殖干细胞与卵巢表面生发上皮关系的研究进展

雌性哺乳动物体内是否存在分裂活跃的生殖干细胞(GSCs),依然存在争议。近年来研究证实,在人类和小鼠卵巢中分离得到了具有分裂活性的GSCs样细胞。假定的雌性哺乳动物GSCs可能定位于卵巢表面生发上皮中。卵巢微环境对GSCs池的维持和分化至关重要,而年龄增加和免疫系统退变可以通过改变卵巢微环境使GSCs无法获得足够的支持来形成新的卵子和卵泡。雌性GSCs的存在,不仅可以为干细胞领域的研究提供新的来源,对辅助生殖技术的未来发展也有着重要意义。     

Will stem cells in cord blood, amniotic fluid, bone marrow and peripheral blood soon be unnecessary in transplantation?

There are now various sources of stem cells. Those derived from blastocysts, named embryo stem (ES) cells, have attracted most attention and are highly multipotent. Human cord blood became widely used as a source of stem cells with differing properties to ES cells and their therapeutic application has grown steadily as they are stored in increasing numbers of stem cell banks. Other sources of human stem cells are derived from peripheral blood and amniotic fluid. They may arise from a common origin in epiblast. This review stresses the use of cord blood stem cells, but describes new approaches which may supersede the use of most stem cells. The advantages and disadvantages of these various classes are described in relation to potential methods involving gene conversion to change somatic cells to ES cells.     

Identification of stem cell marker-positive cells by immunofluorescence in term human amnion

The placenta contains different populations of stem/progenitor cells such as mesenchymal, hematopoietic, trophoblastic and pluripotent stem cells. Although some tissue-specific stem cells are restricted to particular parts of the placenta, the localization of embryonic stem cell-like cells in term human placenta has not been determined. We have used immunofluorescence staining techniques with antibodies to pluripotent stem cell antigens, SSEA-3, SSEA-4, TRA 1-60 and TRA 1-81, and confocal microscopic analysis to identify and localize stem cells within the placenta. Stem cell marker-positive cells were found in amnion but not in choriodecidua, tissues known to contain hematopoietic and trophoblastic stem cells. Amniotic mesenchymal cells did not react with these pluripotent stem cell markers, while all amniotic epithelial cells reacted with at least one antibody. The TRA 1-60 and TRA 1-81 positive cells were solitary and present throughout the surface of amniotic membrane without a specific pattern of distribution, whereas SSEA-3 was negative and SSEA-4 was weakly positive on all amniotic epithelial cells. These data suggest that the human amnion contains stem cell-like cells at different states of differentiation. Human term amnion may be useful source of pluripotent stem cells for regenerative medicine.     

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。     

Derivation of germ cells from mouse embryonic stem cells.

Deriving oocytes from mouse embryonic stem (ES) cells in the laboratory would be a major advance in therapeutic cloning from today's insufficient process. The first step in this attempt is to study the precursors - primordial germ cells (PGCs), including their emergence, presentation, migration and differentiation. By comparing various reported antigens of germ cells, we found stage-specific embryonic antigen-1 (SSEA-1) and Octamer-4 (Oct-4) are the most useful markers to verify the growth and maturation of germ cell culture from mice. Transgenic mice with specific antigens such as SSEA-1 or Oct-4 are helpful to pick up colonies and follow PGC differentiation. We also delineate the physiological structure for germ cell development by reviewing important studies which employed re-aggregation methods to make germ cells more mature and ready for clinical use. Although some mysteries about reprogramming and intragonadal signal interactions still remain unsolved, each step in uncovering these mechanisms will bring us closer to establishing an unlimited source of germ cells from ES cells.     

Pregnancy-associated progenitor cells: an under-recognized potential source of stem cells in maternal lung

Novel therapies are needed for the treatment of acute and chronic lung diseases, many of which are incurable. The use of exogenous stem cells has shown promise in both animal models and clinical trials. However, to date, the stem cell literature has under-recognized naturally acquired pregnancy-associated progenitor cells (PAPCs). These cells are found at sites of injury or disease in female tissues. They persist for decades after parturition in maternal blood and organs, with the largest number being found in the maternal lungs. Their presence there may be one explanation for the sex differences observed in the prevalence and prognosis of some lung diseases. Although the clinical significance of these cells is as yet unknown, the literature suggests that some of the PAPCs are stem cells or have stem cell-like properties. PAPCs harvested from the blood or organs of parous women could potentially be used as an alternate source of cells with regenerative properties for the woman herself or her children. Because PAPCs preferentially traffic to the maternal lung they may play a significant role in recovery or protection from lung disease. In this review article, we discuss ongoing research investigating the administration of both adult and placenta-derived stem cells to treat lung disease, and how PAPCs may also play an important future therapeutic role.     

Establishment and characterization of new human embryonic stem cell lines

Human embryonic stem cells (hESC), with their ability to differentiate into all cell types in the human body, are likely to play a very important therapeutic role in a variety of neurodegenerative and life-threatening disorders in the near future. Although more than 120 different human embryonic stem cell lines have been reported worldwide, only a handful are currently available for researchers, which limits the number of studies that can be performed. This study reports the isolation, establishment and characterization of new human embryonic stem cell lines, as well as their differentiation potential into variety of somatic cell types. Blastocyst-stage embryos donated for research after assisted reproductive techniques were used for embryonic stem cell isolation. A total of 31 blastocysts were processed either for immunosurgery or direct culture methods for inner cell mass isolation. A total of nine primary stem cell colonies were isolated and of these, seven cell lines were further expanded and passaged. Established lines were characterized by their cellular and colony morphology, karyotypes and immunocytochemical properties. They were also successfully cryopreserved/thawed and showed similar growth and cellular properties upon thawing. When induced to differentiate in vitro, these cells formed a variety of somatic cell lineages including cells of endoderm, ectoderm and mesoderm origin. There is now an exponentially growing interest in stem cell biology as well as its therapeutic applications for life-threatening human diseases. However, limited availability of stem cell lines as well as financial or ethical limitations restrict the number of research projects. The establishment of new hESC lines may create additional potential sources for further worldwide and nationwide research on stem cells.     

Amniotic fluid and placental stem cells

The amniotic fluid and the placenta are unique sources of different populations of stem cells--mesenchymal, hematopoietic, trophoblastic--and, possibly, of more primitive stem cells. Although much of the amniotic cavity/fluid and the placenta share a common embryonic origin, the specific origins of the stem cells found in these two compartments remain to be determined. Accordingly, it is not yet known whether all or part of these two stem-cell subsets are actually the same. The multilineage potential of the different stem cell populations from these two sources has begun to be described but still much remains to be learned. Thus, it is not surprising that clinical applications related to the use of these cells have yet to be reported. Nevertheless, fertile experimental work from many different groups has introduced a number of promising novel therapeutic concepts utilizing these cells, such as in tissue engineering, cell transplantation, and gene therapy.     

Embryonic stem cells: advances toward potential therapeutic use

Established lines of human pluripotent stem cells provide a convenient tool for investigating cell differentiation in a way that is pertinent to human embryonic development, providing insights into the causes of birth defects and diseases such as cancer that involve aberrant cell proliferation and differentiation. In principle, human pluripotent stem cells, including embryonic stem and embryonic germ cells, are capable of differentiating into all of the cell types that are present in the adult human. They therefore have the potential to provide a source of tissues for replacement in diseases in which native cell types are inactivated or destroyed. Intense media and public interest has surrounded the announcement of human pluripotent stem cells derivation, focusing on the ethical implications of embryo-related work and on the prospects of an unlimited source of tissues for transplantation-based treatments. Recent studies have focused on identifying method for culture of these cells and inducing their differentiation into specific cell types.     

卵巢癌干细胞与上皮-间质转化的研究进展

近年有研究指出肿瘤可能是肿瘤干细胞性疾病。随着各种肿瘤干细胞不断被分离与鉴定出来,卵巢癌干细胞的分离与鉴定也取得了进展。卵巢癌干细胞是卵巢癌中具有自我更新和多向分化能力的细胞。越来越多的证据表明,肿瘤干细胞可能是介导肿瘤转移和耐药的关键细胞。同时,研究发现上皮-间质转化（EMT）在肿瘤转移中发挥着重要作用,且EMT可能介导肿瘤细胞获得干细胞特性。综述卵巢癌干细胞与EMT之间的关系,进一步探讨卵巢癌的潜在治疗策略。