Isolation,characterization and differentiation of cells expressing pluripotent/multipotent markers from adult human ovaries

Pluripotent stem cells are still generally accepted not to exist in adult human ovaries, although increasing studies confirm the presence of pluripotent/multipotent stem cells in adult mammalian ovaries, including those of humans. The aim of this study is to isolate, characterize and differentiate in vitro stem cells that originate from the adult human ovarian cortex and that express markers of pluripotency/multipotency. After enzymatic degradation of small ovarian cortex biopsies retrieved from 18 women, ovarian cell cultures were successfully established from 17 and the formation of cell colonies was observed. The presence of cells/colonies expressing some markers of pluripotency (alkaline phosphatase, surface antigen SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLA), germinal lineage (DDX4/VASA) and multipotency (M-CAM/CD146, Thy-1/CD90, STRO-1) was confirmed by various methods. Stem cells from the cultures, including small round SSEA-4-positive cells with diameters of up to 4 μm, showed a relatively high degree of plasticity. We were able to differentiate them in vitro into various types of somatic cells of all three germ layers. However, these cells did not form teratoma when injected into immunodeficient mice. Our results thus show that ovarian tissue is a potential source of stem cells with a pluripotent/multipotent character for safe application in regenerative medicine.     

Can ovarian infertility be treated with bone marrow- or ovary-derived germ cells?

A year ago, reproductive biologists and general public were astonished with evidence reported by Johnson et al. in Nature 428:145 that mammalian ovaries possess persisting large germline stem cells, which allegedly enable follicular renewal in adult females. Recently, the same research group declared such view obscure, and reported that mammalian oocytes originate from putative germ cells in bone marrow and are distributed by peripheral blood to the ovaries (Cell 122:303). While neglecting available data on the germ cell origin from the ovarian surface epithelium (OSE) in adult mouse and human females and complexity of follicular renewal in humans, the authors widely extrapolated their observations on formation of allogeneic oocytes after bone marrow (or blood) transplantation in ovaries of adult mice treated with cytostatics to clinical implications in the public media. Yet, the resulting outcome that such allogeneic oocytes may enable the propagation of ovarian cycles is a poor alleviation for the women with ovarian infertility. Women lacking primary follicles, or carrying follicles with low quality eggs persisting in aging ovaries, are not concerned about the lack of menstrual cycles or ovarian steroids, but about virtually no chance of having genetically related children. Johnson et al. also reported that the germ cell formation in bone marrow disappears in ovariectomized mice. Such observation, however, raises solid doubts on the bone marrow origin of oocytes. Since germ cells developing from the OSE cells of adult human ovaries during periodical follicular renewal are known to enter blood vessels in order to enable formation of primary follicles at distant ovarian sites, they also contaminate peripheral blood and hence bone marrow. Better knowledge on the complexity of follicular renewal in humans and exploration of a potential of human OSE cells to produce new oocytes in vitro are essential for novel approaches to the autologous treatment of premature ovarian failure and age induced ovarian infertility.     

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.     

Potential applications of germline cell-derived pluripotent stem cells in organ regeneration

Impressive progress has been made since the turn of the century in the field of stem cells. Different types of stem cells have now been isolated from different types of tissues. Pluripotent stem cells are the most promising cell source for organ regeneration. One such cell type is the germline cell-derived pluripotent cell, which is derived from adult spermatogonial stem cells. The germline cell-derived pluripotent stem cells have been obtained from both human and mouse and, importantly, are adult stem cells with embryonic stem cell-like properties that do not require specific manipulations for pluripotency acquisition, hence bypassing problems related to induced pluripotent stem cells and embryonic stem cells. The germline cell-derived pluripotent stem cells have been induced to differentiate into cells deriving from the three germ layers and shown to be functional in vitro. This review will discuss the plasticity of the germline cell-derived pluripotent stem cells and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.     

Origin of germ cells and formation of new primary follicles in adult human ovaries

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles.     

The role of pluripotent embryonic-like stem cells residing in adult tissues in regeneration and longevity

From the point of view of regenerative potential, the most important cells are pluripotent stem cells (PSCs). Such cells must fulfill certain in vitro as well as in vivo criteria that have been established by work with PSCs isolated from embryos, which are known as embryonic stem cells (ESCs). According to these criteria, pluripotent stem cells should: (i) give rise to cells from all three germ layers, (ii) complete blastocyst development, and (iii) form teratomas after inoculation into experimental animals. Unfortunately, in contrast to immortalized embryonic ESC lines or induced PSCs (iPSCs), these last two criteria have thus far not been obtained in a reproducible manner for any potential PSC candidates isolated from adult tissues. There are two possible explanations for this failure. The first is that PSCs isolated from adult tissues are not fully pluripotent; the second is that there are some physiological mechanisms involved in keeping these cells quiescent in adult tissues that preclude their "unleashed proliferation", thereby avoiding the risk of teratoma formation. In this review we present an evidence that adult tissues contain remnants from development; a population of PSCs that is deposited in various organs as a backup for primitive stem cells, plays a role in rejuvenation of the pool of more differentiated tissue-committed stem cells (TCSCs), and is involved in organ regeneration. These cells share several markers with epiblast/germ line cells and have been named very small embryonic-like stem cells (VSELs). We suggest that, on one hand, VSELs maintain mammalian life span but, on the other hand, they may give rise to several malignancies if they mutate. We provide an evidence that the quiescent state of these cells in adult tissues, which prevents teratoma formation, is the result of epigenetic changes in some of the imprinted genes.     

Ovarian stem cells: From basic to clinical applications

The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004, an article published in nature challenged this central dogma of mammalian reproductive biology. Tilly’s group reported the existence of ovarian germline stem cells (GSCs) in postnatal ovaries of mice and suggested that the bone marrow could be an extragonadal source of ovarian GSCs. These findings were strongly criticized; however, several independent groups have since successfully isolated and characterized ovarian GSCs in postnatal mice. The ovarian GSCs are located in the ovarian surface epithelium and express markers of undifferentiated GSCs. When transplanted into mouse ovaries, mouse ovarian GSCs could differentiate and produce embryos and offspring. Similarly, in a recent study, ovarian GSCs were found to be present in the ovaries of women of reproductive age. Conversely, there is increasing evidence that stem cells responsible for maintaining a healthy state in normal tissue may be a source of some cancers, including ovarian cancer. Cancer stem cells (CSCs) have been found in many tissues, including ovaries. Some researchers have suggested that ovarian cancer may be a result of the transformation and dysfunction of ovarian GSCs with self-renewal properties. Drug resistant and metastasis-generating CSCs are responsible for many important problems affecting ovarian cancer patients. Therefore, the identification of CSCs will provide opportunities for the development of new therapeutic strategies for treatments for infertility and ovarian cancer. In this article, we summarize the current understanding of ovarian GSCs in adult mammals, and we also discuss whether there is a relationship between GSCs and CSCs.     

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

We reported earlier that occasional neurons evolve in human cultures of pluripotent ovarian epithelial stem cells. In subsequent experiments, frequent transdifferentiation into neural stem cells (NSC) and differentiating neurons was observed in human ovarian epithelial stem cells and porcine granulosa cells after exposure to certain combinations of sex steroids. Testosterone (TS), progesterone (PG) or estradiol (E2) alone do not increase the emergence of neurons. However, a mixture of TS+PG after E2 pretreatment converted a majority of ovarian epithelial stem cells or porcine granulosa cells into NSC and differentiating neuronal cells within one to three hours. Cultured neurons manifested an interconnectivity resembling primitive neuronal pathways in culture. These converted cells expressed the cell markers SSEA-1, SSEA-4, NCAM, and Thy-1 glycoconjugates of NSC and neurons, and differentiating cells showed characteristic neuronal morphology. Emergence of NSC and neuronal cells was associated with significant cellular depletion of L-glutamic acid (glutamate), which serves as the major excitatory neurotransmitter in the vertebrate CNS and its fast removal is essential for preventing glutamate excitotoxicity. These observations suggest that certain sequential systemic treatment with common sex steroids and their mixture might be effective in the treatment or prevention of degenerative CNS disorders. The ovarian stem cell cultures readily obtainable from human ovaries regardless of the woman's age have the potential to produce NSC for autologous regenerative treatment of neurologic diseases in aging women. Finally, the proper combination of sex steroids could possibly be employed for transdifferentiation of adult bone marrow stem cells or mobilized peripheral blood cells into autologous NSC and stimulate their neuronal differentiation after homing in the CNS.     

X chromosome inactivation in human and mouse pluripotent stem cells

Since the groundbreaking hypothesis of X chromosome inactivation (XCI) proposed by Mary Lyon over 50 years ago, a great amount of knowledge has been gained regarding this essential dosage compensation mechanism in female cells. For the mammalian system, most of the mechanistic studies of XCI have so far been investigated in the mouse model system, but recently, a number of interesting XCI studies have been extended to human pluripotent stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Emerging data indicate that XCI in hESCs and hiPSCs is much more complicated than that of their mouse counterparts. XCI in human pluripotent stem cells is not as stable and is subject to environmental influences and epigenetic regulation in vitro. This mini-review highlights the key differences in XCI between mouse and human stem cells with a greater emphasis placed on the understanding of the epigenetic regulation of XCI in human stem cells.     

Myogenic potential of mouse primordial germ cells

Primordial germ cells are the only stem cells that retain true developmental totipotency after gastrulation, express markers typical of totipotent/pluripotent status and are able both in vivo and in vitro to give rise to pluripotent stem cells as EC and EG cells. We have therefore explored the possibility of the trans-differentiation of mouse PGCs to a myogenic lineage by transplanting them directly or after in vitro culture into a regenerating muscle and by culturing them on monolayers of differentianting muscle cells. The results obtained suggest that mouse PGCs may trans-differentiate into myogenic cells, provided that their somatic environment is preserved. This occurs at an estimated frequency of 0.01%, which is no higher than that reported for stem cells of adult tissues.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Oogenesis in cultures derived from adult human ovaries

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.     

Tracking the progression of the human inner cell mass during embryonic stem cell derivation

The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.     

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).     

Energy metabolism in human pluripotent stem cells and their differentiated counterparts

BACKGROUND: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. METHODOLOGY/PRINCIPAL FINDINGS: We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. CONCLUSIONS/FINDINGS: Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH).