Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

Human pre-implantation embryo development.

Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals.

Status of human germ cell differentiation from pluripotent stem cells.

Historically, the quality of life of infertile couples has been greatly diminished by the loss of opportunity to conceive. However, beginning with the advent of IVF in the late 1970s, novel clinical interventions have greatly changed the outlook for those with severe forms of infertility. Yet, in cases in which the quality and quantity of germ cells are most compromised, there are few options. In the present paper, the current status of germ cell development from stem cells is reviewed in light of potential utility for basic science and clinical applications.

Alpha-synuclein null mutation exacerbates the phenotype of a model of Menkes disease in female mice.

Genetic modifier screens provide a useful tool, in diverse organisms from Drosophila to C. elegans and mice, for recovering new genes of interest that may reduce or enhance a phenotype of interest. This study reports a modifier screen, based on N-ethyl-N-nitrosourea (ENU) mutagenesis and outcrossing, designed to increase understanding of the normal function of murine α-synuclein (Snca). Human SNCA was the first gene linked to familial Parkinson's disease. Since the discovery of the genetic link of SNCA to Parkinson's nearly three decades ago, numerous studies have investigated the normal function of SNCA protein with divergent roles associated with different cellular compartments. Understanding of the normal function of murine Snca is complicated by the fact that mice with homozygous null mutations live a normal lifespan and have only subtle synaptic deficits. Here, we report that the first genetic modifier (a sensitized mutation) that was identified in our screen was the X-linked gene, ATPase copper transporting alpha (Atp7a). In humans, mutations in Atp7a are linked to to Menkes disease, a disease with pleiotropic phenotypes that include a severe neurological component. Atp7a encodes a trans-Golgi copper transporter that supplies the copper co-factor to enzymes that pass through the ER-Golgi network. Male mice that carry a mutation in Atp7a die within 3 weeks of age regardless of Snca genotype. In contrast, here we show that Snca disruption modifies the phenotype of Atp7a in female mice. Female mice that carry the Atp7a mutation, on an Snca null background, die earlier (prior to 35 days) at a significantly higher rate than those that carry the Atp7a mutation on a wildtype Snca background ATPase copper transporting alpha. Thus, Snca null mutations sensitize female mice to mutations in Atp7a, suggesting that Snca protein may have a protective effect in females, perhaps in neurons, given the co-expression patterns. Although data has suggested diverse functions for human and mouse α-synuclein proteins in multiple cell compartments, this is the first demonstration via use of genetic screening to demonstrate that Snca protein may function in the ER-Golgi system in the mammalian brain in a sex-dependent manner.

Can cellular models revolutionize drug discovery in Parkinson's disease?

The study of mechanisms that underlie Parkinson's disease (PD), as well as translational drug development, has been hindered by the lack of appropriate models. Both cell culture systems and animal models have limitations, and to date none faithfully recapitulate all of the clinical and pathological phenotypes of the disease. In this review we examine the various cell culture model systems of PD, with a focus on different stem cell models that can be used for investigating disease mechanisms as well as drug discovery for PD. We conclude with a discussion of recent discoveries in the field of stem cell biology that have led to the ability to reprogram somatic cells to a pluripotent state via the use of a combination of genetic factors; these reprogrammed cells are termed "induced pluripotent stem cells" (iPSCs). This groundbreaking technique allows for the derivation of patient-specific cell lines from individuals with sporadic forms of PD and also those with known disease-causing mutations. Such cell lines have the potential to serve as a human cellular model of neurodegeneration and PD when differentiated into dopaminergic neurons. The hope is that these iPSC-derived dopaminergic neurons can be used to replicate the key molecular aspects of neural degeneration associated with PD. If so, this approach could lead to transformative new tools for the study of disease mechanisms. In addition, such cell lines can be potentially used for high-throughput drug screening. While not the focus of this review, ultimately it is envisioned that techniques for reprogramming of somatic cells may be optimized to a point sufficient to provide potential new avenues for stem cell-based restorative therapies.

Intact fetal ovarian cord formation promotes mouse oocyte survival and development.

BACKGROUND: Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. RESULTS: Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. CONCLUSIONS: Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the human fetal ovary, the identification of genetic components and molecular mechanisms of pre-follicle stage germ and somatic cell structures may be important for understanding human female infertility. In addition, this work provides a foundation for development of a robust fetal ovarian niche and transplantation based system to direct stem cell-derived oocyte differentiation as a potential therapeutic strategy for the treatment of infertility.

Characterization of six new human embryonic stem cell lines (HSF7, -8, -9, -10, -12, and -13) derived under minimal-animal component conditions.

Human embryonic stem cells (hESCs) provide a renewable source of a variety of cell types with the potential for use in both scientific research and clinical cell-based therapy. Several hESC lines have previously been isolated and characterized, however, the majority of these lines were generated in the presence of animal serum and animal-derived feeder cells. Therefore, the exposure of the hESC to animal products may have induced phenotypic and/or genomic changes in the hESC lines not characteristic of normal hESC. Moreover, those hESC lines exposed to animal components may not be used for therapeutic applications due to the risk of graft rejection and pathogenic transmission from animal sources. In this study, we characterized six new hESC lines derived from human blastocysts under minimal-animal component conditions and cultured with human fetal lung fibroblasts. The hESC lines retained the ability to self-renew, are karytopically normal, and express stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, and TRA-1-81, but not SSEA-1, markers of pluripotent hESC. In addition, we show that telomerase activity decreased in each of the hESC lines following differentiation into embryoid bodies, albeit to different degrees. Finally, we demonstrate that the hESC lines are capable of differentiating into the three embryonic germ layers in vitro and form complex teratomas in vivo. This suggests that the hESC lines described here are valuable models for both future in vitro and in vivo studies, which may aid in the progression toward clinical-grade cell therapy.

A method for single-cell sorting and expansion of genetically modified human embryonic stem cells.

Genetic modification of human embryonic stem (hES) cells is essential for studies of gene function and differentiation. The expression of transgenes may direct tissue-specific differentiation and aid in the identification of various differentiated cell types. Stable genomic integration of transgenes is optimal because hES cell differentiation can span several days to weeks and include numerous cell divisions, and establishing homogeneous modified cell lines will facilitate research studies. Herein we provide a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors. Using this method, we have established enhanced green fluorescent protein (eGFP)-expressing hES cell lines that are pluripotent, contain a diploid chromosomal content, and stably express eGFP following more than 2 months of routine culture and in vivo differentiation.

Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells.

The growth factors bone morphogenetic protein-4 (BMP4), BMP7, and BMP8b are required for specification of primordial germ cells (PGCs) in mice. Disruption of the genes that encode these factors leads to a severe reduction in number, or the complete absence, of PGCs. In addition, several studies have demonstrated that human BMP4 can promote PGC differentiation from mouse embryonic stem (ES) cells and in organ cultures. Here, we sought to determine whether recombinant human BMPs could induce differentiation of germ cells from human (h) ES cells. We found that addition of recombinant human BMP4 increased the expression of the germ cell-specific markers VASA and SYCP3 during differentiation of hES cells to embryoid bodies (EBs). In addition, BMP7 and BMP8b showed additive effects on germ cell induction when added together with BMP4. Finally, we observed that addition of BMPs to differentiating ES cells also increased the percentage of cells that stained positively for VASA. We note that the effects of recombinant BMPs were modest but reproducible and suggest that addition of BMPs to differentiation media increases differentiation of human germ cells from hES cells.

Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization.

The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage.

Importance of investigating epigenetic alterations for industry and regulators: An appraisal of current efforts by the Health and Environmental Sciences Institute.

Recent technological advances have led to rapid progress in the characterization of epigenetic modifications that control gene expression in a generally heritable way, and are likely involved in defining cellular phenotypes, developmental stages and disease status from one generation to the next. On November 18, 2013, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) held a symposium entitled "Advances in Assessing Adverse Epigenetic Effects of Drugs and Chemicals" in Washington, D.C. The goal of the symposium was to identify gaps in knowledge and highlight promising areas of progress that represent opportunities to utilize epigenomic profiling for risk assessment of drugs and chemicals. Epigenomic profiling has the potential to provide mechanistic information in toxicological safety assessments; this is especially relevant for the evaluation of carcinogenic or teratogenic potential and also for drugs that directly target epigenetic modifiers, like DNA methyltransferases or histone modifying enzymes. Furthermore, it can serve as an endpoint or marker for hazard characterization in chemical safety assessment. The assessment of epigenetic effects may also be approached with new model systems that could directly assess transgenerational effects or potentially sensitive stem cell populations. These would enhance the range of safety assessment tools for evaluating xenobiotics that perturb the epigenome. Here we provide a brief synopsis of the symposium, update findings since that time and then highlight potential directions for future collaborative efforts to incorporate epigenetic profiling into risk assessment.

Current and future genetic screening for male infertility.

Although much of male infertility is currently unexplained, it is likely that underlying defects in critical genes or entire gene pathways are responsible. Because powerful technologies exist to bypass severe male-factor infertility, improving the diagnosis of genetic infertility is important for the infertile couple, not only to explain the problem but also to inform them of conditions potentially transmissible to offspring.

Germ cell differentiation from pluripotent cells.

Infertility is a medical condition with an increasing impact in Western societies with causes linked to toxins, genetics, and aging (primarily delay of motherhood). Within the different pathologies that can lead to infertility, poor quality or reduced quantity of gametes plays an important role. Gamete donation and therefore demand on donated sperm and eggs in fertility clinics is increasing. It is hoped that a better understanding of the conditions related to poor gamete quality may allow scientists to design rational treatments. However, to date, relatively little is known about human germ cell development in large part due to the inaccessibility of human development to molecular genetic analysis. It is hoped that pluripotent human embryonic stem cells and induced pluripotent stem cells may provide an accessible in vitro model to study germline development; these cells are able to differentiate to cells of all three primary embryonic germ layers, as well as to germ cells in vitro. We review the state of the art in germline differentiation from pluripotent stem cells.

Ethical and legal issues arising in research on inducing human germ cells from pluripotent stem cells.

Derivation of eggs or sperm from pluripotent stem cells or direct reprogramming from somatic cells would have huge effects on assisted reproductive technology. Here we discuss important ethical, legal, and social issues that would be raised by the development of such female or male gametes for clinical use.

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.

INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.

Pumilio-2 function in the mouse nervous system.

Coordinated mRNA translation at the synapse is increasingly recognized as a critical mechanism for neuronal regulation. Pumilio, a translational regulator, is known to be involved in neuronal homeostasis and memory formation in Drosophila. Most recently, the mammalian Pumilio homolog Pumilio-2 (Pum2) has been found to play a role in the mammalian nervous system, in particular in regulating morphology, arborization and excitability of neuronal dendrites, in vitro. However, the role of Pum2 in vivo remains unclear. Here, we report our investigation of the functional and molecular consequences of Pum2 disruption in vivo using an array of neurophysiology, behavioral and gene expression profiling techniques. We used Pum2-deficient mice to monitor in vivo brain activity using EEG and to study behavior traits, including memory, locomotor activity and nesting capacities. Because of the suspected role of Pum2 in neuronal excitability, we also examined the susceptibility to seizure induction. Finally, we used a quantitative gene expression profiling assay to identify key molecular partners of Pum2. We found that Pum2-deficient mice have abnormal behavioral strategies in spatial and object memory test. Additionally, Pum2 deficiency is associated with increased locomotor activity and decreased body weight. We also observed environmentally-induced impairment in nesting behavior. Most importantly, Pum2-deficient mice showed spontaneous EEG abnormalities and had lower seizure thresholds using a convulsing dosage of pentylenetetrazole. Finally, some genes, including neuronal ion channels, were differentially expressed in the hippocampus of Pum2-deficient mice. These findings demonstrate that Pum2 serves key functions in the adult mammalian central nervous system encompassing neuronal excitability and behavioral response to environmental challenges.