In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes

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.     

Stemming the Tide of Normalisation: An Expanded Feminist Analysis of the Ethics and Social Impact of Embryonic Stem Cell Research

Feminists have indicated the inadequacies of bioethical debates about human embryonic stem cell research, which have for the most part revolved around concerns about the moral status of the human embryo. Feminists have argued, for instance, that inquiry concerning the ethics and politics of human embryonic stem cell research should consider the relations of social power in which the research is embedded. My argument is that this feminist work on stem cells is itself inadequate, however, insofar as it has not incorporated an analysis of disability into its considerations of the ethical and political issues that surround the phenomena. Thus, I consider claims that disability theorists and anti-disability activists have made about the research. I conclude by indicating that stem cell research must be situated within a cultural matrix that operates in the service of normalisation.     

Stem cells in diabetes: what has been achieved

Beta-cell replacement therapy via islet transplantation has received renewed interest due to the recent improved success. In order to make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must be readily available. The most promising sources are stem cells, with efforts of deriving new beta-cells from both embryonic and adult stem cells. Several groups have reported generating insulin-producing cells from mouse embryonic stem cells. The strategies in the first two acclaimed reports were very different. One strategy, used by Soria's group, is gene trapping in which an introduced antibiotic resistance under the control of the insulin promoter allowed the selection of insulin-expressing cells that had spontaneously differentiated within embryoid bodies. Another strategy, used by McKay's group, manipulated culture conditions in a multistep protocol used for generating neural cells but with changed final conditions. Since these reports, there have been modifications of the protocols in efforts to improve the yields and maturity of the resulting cells. While it is unclear if the insulin-producing cells in any of these studies are truly mature beta-cells, these studies show the clear potential of embryonic stem cells and support optimism that similar results will be possible with human embryonic stem cells. We know that new beta-cells are generated throughout adult life, but the identity of adult pancreatic stem cells has been elusive. The potential for expansion and differentiation of pluripotent adult stem cells, whether from bone marrow or as non-pancreas tissue resident SP cells, is being explored but has not yet yielded insulin-producing tissue. In contrast, insulin-producing cells have been generated in vitro from adult pancreatic tissues. We have been examining the hypothesis that the functional source for new beta-cells in the adult pancreas are mature duct epithelial cells that have regressed or lost their mature phenotype after replication. Others have isolated putative stem cells from islets and ducts. For adult cells the issue of expansion as well as of differentiation is a question. The field of generating new beta-cells from stem cells, either embryonic or adult, is still in its infancy. Each new report has been met with a mixture of excitement and skepticism. With continued efforts and rigorous assessments, hopefully the potential of generating enough new beta-cells from stem cells will be realized.     

Targeting frequency for deletion vectors in embryonic stem cells.

We analyzed the gene targeting frequencies and recombination products generated by a series of replacement deletion vectors which target the hprt (hypoxanthine phosphoribosyltransferase) locus in mouse embryonic stem cells. We found that the targeting frequency of a 19.2-kb deletion was comparable to that of a 3-kb deletion or a conventional replacement event in which a 1.7-kb fragment was inserted into the locus. We also observed different integration patterns for these deletion vectors. A result of this finding is that a wide range of genomic deletions in embryonic stem cells is feasible.     

Cancer genes hypermethylated in human embryonic stem cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.     

Low microRNA-199a expression in human amniotic epithelial cell feeder layers maintains human-induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression

Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells, and may be generated from patient- or disease-specific sources, which makes them attractive for personalized medicine, drug screens, or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, as they express endogenous leukemia inhibitory factor (LIF) at high levels. Here, we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs, and in turn on human iPS cell pluripotency. We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels, allowing iPS to maintain a high level of alkaline phosphatase activity in long-term culture and form teratomas in severe combined immunodeficient mice. The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant, compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts. Taken together, these results suggested that LIF expression might be regulated by microRNA-199a, and LIF was a crucial component in feeder cells, and also was required for maintenance of human iPS cells in an undifferentiated, proliferative state capable of self-renewal.     

The tumorigenicity of diploid and aneuploid human pluripotent stem cells

Human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) offer an immense potential as a source of cells for regenerative medicine. However, the ability of undifferentiated HESCs to produce tumors in vivo presents a major obstacle for the translation of this potential into clinical reality. Therefore, characterizing the nature of HESC-derived tumors, especially their malignant potential, is extremely important in order to evaluate the risk involved in their clinical use. Here we review recent observations on the tumorigenicity of human pluripotent stem cells. We argue that diploid, early passage, HESCs produce benign teratomas without undergoing genetic modifications. Conversely, HESCs that acquired genetic or epigenetic changes upon adaptation to in vitro culture can produce malignant teratocarcinomas. We discuss the molecular mechanisms of HESC tumorigenicity and suggest approaches to prevent tumor formation from these cells. We also discuss the differences in the tumorigenicity between mouse embryonic stem cells (MESCs) and HESCs, and suggest methodologies that may help to identify cellular markers for culture adapted HESCs.     

Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

Identification of spectral modifications occurring during reprogramming of somatic cells

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.     

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.     

Putative "stemness" gene jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells

Many genes have been identified that are specifically expressed in multiple types of stem cells in their undifferentiated state. It is generally assumed that at least some of these putative "stemness" genes are involved in maintaining properties that are common to all stem cells. We compared gene expression profiles between undifferentiated and differentiated embryonic stem cells (ESCs) using DNA microarrays. We identified several genes with much greater signal in undifferentiated ESCs than in their differentiated derivatives, among them the putative stemness gene encoding junctional adhesion molecule B (Jam-B gene). However, in spite of the specific expression in undifferentiated ESCs, Jam-B mutant ESCs had normal morphology and pluripotency. Furthermore, Jam-B homozygous mutant mice are fertile and have no overt developmental defects. Moreover, we found that neural and hematopoietic stem cells recovered from Jam-B mutant mice are not impaired in their ability to self-renew and differentiate. These results demonstrate that Jam-B is dispensable for normal mouse development and stem cell identity in embryonic, neural, and hematopoietic stem cells.     

Mouse embryonic stem cells exhibit indefinite proliferative potential

The proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life-span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of "crisis" or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ-line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.     

GP130 stimulation and the maintenance of stem cells

Recent advantages with the cultivation of adult and embryonic stem cells have raised hopes for therapeutic applications of such cells in the treatment of neurodegenerative diseases and cancer. Cultivation of stem cells on feeder cells or treatment of the cells with cytokines is necessary to maintain stem cells in an undifferentiated state and to keep their pluripotency. In particular, the cytokine leukemia inhibitory factor (LIF) has been used to cultivate murine embryonic stem (ES) cells in the absence of feeder cells. For unknown reasons, LIF does not evoke the same effect on rat or human stem cells. This article summarizes what is known about, and the problems associated with, the cultivation of stem cells and suggests experimental strategies that might help to overcome these difficulties.     

Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.