Embryonic Stem Cell Biology Research Articles

Human embryonic stem cells: 10 years on

Substantial advances in the biology of human embryonic stem (ES) cells, and the technology for working with them, have been made over the past 10 years. Regulatory frameworks for their study are well developed, although some countries remain particularly restrictive. Markers and criteria for characterising human ES cells are also generally agreed, and protocols for promoting their differentiation are being established, providing the groundwork for the development of applications over the next 10 years. The recent appearance of technology to convert somatic cells to ‘induced Pluripotent Stem Cells' closely resembling ES cells will certainly speed up these developments.

A novel embryonic stem cell line derived from the common marmoset monkey (Callithrix jacchus) exhibiting germ cell-like characteristics

Embryonic stem cells (ESC) hold great promise for the treatment of degenerative diseases. However, before clinical application of ESC in cell replacement therapy can be achieved, the safety and feasibility must be extensively tested in animal models. The common marmoset monkey (Callithrix jacchus) is a useful preclinical non-human primate model due to its physiological similarities to human. Yet, few marmoset ESC lines exist and differences in their developmental potential remain unclear. Blastocysts were collected and immunosurgery was performed. cjes001 cells were tested for euploidy by karyotyping. The presence of markers for pluripotency was confirmed by immunofluorescence staining and RT-PCR. Histology of teratoma, in vitro differentiation and embryoid body formation revealed the differentiation potential. cjes001 cells displayed a normal 46,XX karyotype. Alkaline phosphatase activity, expression of telomerase and the transcription factors OCT4, NANOG and SOX2 as well as the presence of stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigens (TRA)-1-60, and TRA-1-81 indicated pluripotency. Teratoma formation assay displayed derivatives of all three embryonic germ layers. Upon non-directed differentiation, the cells expressed the germ cell markers VASA, BOULE, germ cell nuclear factor and synaptonemal complex protein 3 and showed co-localization of VASA protein within individual cells with the germ line stem cell markers CD9, CD49f, SSEA-4 and protein gene product 9.5, respectively. The cjes001 cells represent a new pluripotent ESC line with evidence for enhanced spontaneous differentiation potential into germ cells. This cjes001 line will be very valuable for comparative studies on primate ESC biology.

Expression Profiling of Nuclear Receptors in Human and Mouse Embryonic Stem Cells

Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERRbeta, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation.

Expression of Functional Toll-Like Receptor 2 on Mouse Embryonic Stem Cells

Embryonic stem (ES) cells have the capacity to produce all cell types of the body. Understanding murine ES (mES) cell proliferation, survival, differentiation, and self renewal will enhance knowledge of developmental biology and essential use of ES cells. Recently, Toll Like Receptor (TLR) activation has been shown by others to play a role in influencing the differentiation of hematopoietic stem cells. Previous studies have also shown that TLR activation prevents mesenchymal stem cell differentiation into adipocytes, chondrocytes, and osteocytes and plays a role in bone repair. We hypothesized that certain TLR's would be expressed on mES cells and that the ligands for these expressed TLR's would induce functional activity in the mESC's. Therefore, we wanted to determine if TLRs are expressed on mES cells and if so, are they functional. Three different mES cell lines (R1, CGR8, and E14) were used to determine if TLRs are expressed at the mRNA level using primers for murine TLR1-9 mRNA. We found that TLR's 1, 2, 3, 6, and 9 were expressed at the mRNA level, but TLR's 4, 5, 7, and 8 were not. Based on the availability of antibodies to TLR's, and using flow cytometry, we found expression of TLR2 but not TLR 4 on the surface of all three mES cell lines. TLR ligands were used to treat mES cells in the presence of leukemia inhibitory factor (LIF) for an hour. Activation of TLR2 by its ligand Pam3Cys, a synthetic tri-acyl lipoprotein, on mES cells induced NF-κβ nuclear translocation when compared to ES cells not stimulated with TLR ligands. LPS, the ligand for TLR4 did not induce NF-κβ nuclear translocation on ES cells, consistent with lack of cell surface expression of TLR4 on mES cells. TLR expression and TLR ligand interaction were not associated with changes in the morphology of the mES cells or expression of Oct-4, SSEA-1, KLF-4, or Sox-2, markers for maintenance of the undifferentiated state of mES cells. This suggests that the cells remain in an undifferentiated state even after TLR activation by Pam3Cys in the presence of LIF. Thus our study has identified functionally active TLR2 on the surface of mES cells, information that may be of use to further defining a role for TLR's on ES cells, and for manipulation of other ES cell functions.

Cell surface biomarkers of embryonic stem cells

Embryonic stem cells (ESCs) can give rise to any adult cell type and thus offer enormous potential for regenerative medicine and drug discovery. Molecular biomarkers serve as valuable tools to classify and isolate ESCs and to monitor their differentiation state by antibody-based techniques. A number of biomarkers, such as certain cell surface antigens, are used to assign pluripotent ESCs; however, accumulating evidence suggests that ESCs are heterogeneous in morphology, phenotype and function, and are thereby classified into subpopulations characterized by multiple sets of molecular biomarkers. Biomarker discovery is also important for ESC biology to elucidate the molecular mechanisms that regulate pluripotency and differentiation. This review summarizes studies of ESC biomarker discovery. "Genome-wide" expression profiling of ESC mRNAs and proteins and direct analyses of the cell surface subproteome have demonstrated that ESCs express a diverse range of biomarkers, cell surface antigens, and signaling molecules found in different cell lineages, as well as a number of key molecules that assure "stemness". Clearly, future quantitative proteomics approaches will enhance our knowledge of the stage- and lineage-specific expression of the proteome and its temporal changes upon differentiation, and provide a more detailed view of nascent and clonally amplified ESCs.

Zfp143 Regulates Nanog Through Modulation of Oct4 Binding

Identification of regulators governing the maintenance of embryonic stem (ES) cells is crucial to the understanding of ES cell biology. We identified a zinc finger protein, Zfp143, as a novel regulator for self-renewal. Depletion of Zfp143 by RNA interference causes loss of self-renewal of ES cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays show the direct binding of Zfp143 to the Nanog proximal promoter. Knockdown of Zfp143 or mutation of the Zfp143 binding motif significantly downregulates Nanog proximal promoter activity. Importantly, enforced expression of Nanog is able to rescue the Zfp143 knockdown phenotype, indicating that Nanog is one of the key downstream effectors of Zfp143. More interestingly, we further show that Zfp143 regulates Nanog expression through modulation of Oct4 binding. Coimmunoprecipitation experiments revealed that Zfp143 and Oct4 physically interact with each other. This interaction is important because Oct4 binding to the Nanog promoter is promoted by Zfp143. Our study reveals a novel regulator functionally important for the self-renewal of ES cells and provides new insights into the expanded regulatory circuitry that maintains ES cell pluripotency. Disclosure of potential conflicts of interest is found at the end of this article.

Silencing of the Expression of Pluripotent Driven-Reporter Genes Stably Transfected into Human Pluripotent Cells

Marking of human embryonic stem (ES) and embryonal carcinoma (EC) cells with pluripotent promoter-driven reporter gene cassettes provides an important tool for studies related to maintenance of pluripotency, cell differentiation and cell selection. OCT4, TERF1 and telomerase reverse transcriptase component (TERT) are considered as pluripotent marker genes since they are expressed in both human ES and EC cells and significantly downregulated during the differentiation process. Our aim was to use core promoter regions from such pluripotent genes to drive expression of reporter genes that would be suitable for human ES cell selection amongst differentiated cells. Human ES and EC cells were stably transfected with a number of TERT, OCT4 and TERF1 promoter-driven EGFP or NTR gene cassettes. Gradual loss of reporter gene expression was observed from 24 h post-transfection during transient transfection studies, while almost complete loss of reporter expression was observed upon stable transfections. The loss of reporter gene expression was partly reversed by addition of a histone deacetylase inhibitor and a demethylating agent, suggesting that in vitro methylation of these exogenous constructs and the epigenetic architecture around the site of integration are likely to play a major role in their transcriptional activity. Inclusion of gene-regulatory elements in addition to the core promoters has been shown to minimize such effects and should be considered as an important strategy in such studies. Together our data suggest that human ES and EC cells are able to silence pluripotent promoter-driven reporter genes with high efficiency. Whether differentiated cells derived from human ES and EC cells retain this activity is unknown and need to be investigated before large-scale comparative reporter-based transfection studies can be used as a tool in human embryonic stem cell biology.

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

Variation in Homeodomain DNA Binding Revealed by High-Resolution Analysis of Sequence Preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

Biology and potential of human embryonic stem cells

Human embryonic stem cells (hESC) are obtained from the inner cell mass from the early embryo at blastocyste stage. Derived in cell lines for the first time in 1998, they can be maintained in culture in an undifferentiated state indefinitely under certain conditions. Two essential properties characterize hESC: pluripotency and self-renewal. Pluripotency is convey by the expression of specific transcription factors such as OCT4 and NANOG, and is under the control of growth factors such as IGF2 and FGFb. Markers used to characterize these cells include surface antigens, notably SSEA-3 and SSEA-4, and nuclear markers such as OCT4. HESC can differentiate into different cell types in vitro. They represent a unique and essential model for early human development research and for regenerative medicine. By their self-renewal capacity and their potential to differentiate into several cell types, hESC are an unlimited source of cells enabling to replace or restore lost or damaged cells in numerous diseases. Even if it is not conceivable today to use them in clinical practice for ethic and scientific reasons, it seems essential to explore the numerous potentialities of these cells. This knowledge might be relevant to handle adult stem cells in vitro and will be mandatory for a therapeutic use of hESC in the future.

Canine embryo-derived stem cells and models for human diseases

Embryonic stem (ES) cells are pluripotent and permanent cell lines which can differentiate into cell types of all the three germ layers. These features imply multiple opportunities for clinical applications in tissue engineering and regenerative medicine. Most of our knowledge on the biology and technology of ES cells is derived from studies with mouse ES cells. While appropriate for proof-of-principle studies, the mouse model has limitations in its application in translational, pre-clinical studies. This is particularly true for studies evaluating the safety and efficacy of stem cell therapies. For this purpose, large animal models more closely mimicking important aspects of human anatomy, physiology and pathology than mouse models are urgently needed. In this context, the dog is an excellent candidate: the plethora of different dog breeds offer a large phenotypic and genetic variability, which can be exploited increasingly well due to the advanced status of the dog genome project and the rapidly growing box of genomic tools. Recently, the first pluripotent canine embryo-derived stem cells have been described, further increasing the potential of the dog as a model system for regenerative medicine. Although these cells express alkaline phosphatase, NANOG and OCT4, and can be differentiated in vitro towards endoderm-, mesoderm- and ectoderm-lineages (typical features of human and mouse ES cells), their in vivo differentiation capability, i.e. formation of teratomas in immunodeficient mice or contribution to chimeric animals, remains to be demonstrated. Here, we discuss the features of reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.

Cell–cell interaction modulates neuroectodermal specification of embryonic stem cells

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.

CPKC isozyme specific substrates in murine embryonic stem cells.

PKCs are intracellular signalling enzymes that participate in embryonic stem cells (ES) biology and most likely modify targets associated with key processes including self‐renewal and differentiation. We first identified the PKC isozymes that are present in ES and used an exploratory approach to select potential substrates using cell based assays and phosphoproteomic. Murine ES express mainly PKCs α, βI, βII, δ, ε and ζ/λ. PMA or cPKC peptide modulators were used to identify cPKC substrates, which were resolved in 2D gels and developed with phospho‐specific dye. Upon treatment with cPKC‐selective activator peptide, ψβRACK, 41‐unique protein spots were identified, 9 increased phosphorylation. In contrast, treatment with the cPKC inhibitor, βC2‐4, resulted in a decreased phosphorylation of 51 spots and disappearance of 6. Furthermore, 27 spots disappeared upon treatment with βIPKC‐selective inhibitor, βIV5‐3, and 11 had a decrease in phosphorylation, whereas 10 spots disappeared with βIIPKC‐specific inhibitor, βIIV5‐3, and 11 ‐ decreased phosphorylation. Nine spots increased their intensity with the cPKC activator and inhibited with either beta PKC=specific inhibitor peptide and 8 spots were inhibited by more than one inhibitor peptide. Preliminary data using MS/MS revealed an increased phosphorylation of splicing factor 1 following treatment with either PMA or cPKC activator peptide while elongation factor 1 δ increased phosphorylation with PMA, but decreased with cPKC inhibitor treatment. Taken together, we began the identification of substrates of specific PKC isozymes in mES using isozyme‐selective inhibitors and activators, which deserve to be investigated in the future.

The Preimplantation Embryo: Handle with Care

The past decade has seen considerable advances in our understanding of intrinsic developmental mechanisms associated with gametogenesis and embryogenesis and accompanying applications in the fields of reproductive medicine, embryonic stem cell biology, and nuclear reprogramming. However, a new focus has recently emerged concerning the homeostatic regulation of embryonic cells, how this is set, and how it may influence the longitudinal progression and optimization of the developmental program and indeed the phenotype of the offspring. Attention has been drawn to the preimplantation stage of development as a sensitive "window" when in vitro and in vivo manipulations, such as culture conditions or maternal diet, may have critical consequences. In this article, we review how changes in environmental conditions, mediated via a range of epigenetic, cellular, and metabolic mechanisms in the preimplantation embryo, may alter the pattern of cell division, gene expression, morphology, and potential. We consider how fetal and postnatal phenotype may become susceptible to the plasticity of the preimplantation embryo and the risks for adult health and physiology.

Engineering tissue from human embryonic stem cells

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.

The standpoint of stem cell research

The 6th Annual Meeting of the International Society for Stem Cell Research focused on several issues, such as cancer stem cells, tissue regeneration, stem cell transplantation, biology of embryonic and adult stem cells of various species, regulation of stem cells epigenetic state and reprogramming of somatic cells for the production of induced pluripotent stem cells. This report summarizes the standpoint of stem cell research, based on the proceedings of the meeting.

Nuclear Proteomics and Directed Differentiation of Embryonic Stem Cells

During the past decade, regenerative medicine has been the subject of intense interest due, in large part, to our growing knowledge of embryonic stem (ES) cell biology. ES cells give rise to cell lineages from the three primordial germ layers--endoderm, mesoderm, and ectoderm. This process needs to be channeled if these cells are to be differentiated efficiently and used subsequently for therapeutic purposes. Indeed, an important area of investigation involves directed differentiation to influence the lineage commitment of these pluripotent cells in vitro. Various strategies involving timely growth factor supplementation, cell co-cultures, and gene transfection are used to drive lineage specific emergence. The underlying goal is to control directly the center of gene expression and cellular programming--the nucleus. Gene expression is enabled, managed, and sustained by the collective actions and interactions of proteins found in the nucleus--the nuclear proteome--in response to extracellular signaling. Nuclear proteomics can inventory these nuclear proteins in differentiating cells and decipher their dynamics during cellular phenotypic commitment. This review details what is currently known about nuclear effectors of stem cell differentiation and describes emerging techniques in the discovery of nuclear proteomics that will illuminate new transcription factors and modulators of gene expression.

Human embryonic stem cells: An experimental and therapeutic resource for neurological disease

There is a great and unmet need for meaningful therapies that will deliver restorative solutions to patients with neurological disorders such as multiple sclerosis (MS), Parkinson's disease and stroke. The emergence of human embryonic stem cells as an experimental and therapeutic resource represents a major opportunity for brain repair. Embryonic stem cells offer the potential to study human cells, model disease, accelerate drug discovery and of themselves act as a cell-based therapy. In contrast to other organs, a "one size fits all" approach is inappropriate for repair of the brain; rather therapies need to be "bespoke". The design and development of embryonic stem-cell based CNS reparative strategies pose many challenges, both conceptual and practical. Using multiple sclerosis as an example, this paper addresses the needs for the translation of embryonic stem cell biology to regenerative neurology.

Prospects of Embryonic Stem Cells in Treatment of Hematopoietic Disorders

Cellular therapies derived from embryonic stem (ES) cells have gained a renewed interest with the experimental demonstration that an embryonic stem cell lines can be established from human blastocyst-stage embryos and prompted to differentiate into almost all types of cells present in the body including hematopoietic cells. Hematopoiesis is a series of cellular processes whereby short-lived mature blood cells are continuously replenished from a pool of rare pluripotential hematopoietic stem cells, in a highly orchestrated process. Aberrances in this intricate process may lead to a malignancy of essential blood-forming organs, causing diseases such as leukemia, aplastic anemia, lymphoma, myelodysplasia and myeloproliferative disorders. Embryonic stem cells show great potential and it may be technologically feasible to transplant differentiated ES cells and to cure various kinds of blood disorders. Understanding the biology of ES cell derived hematopoiesis may lead to the development of co-transplantation protocols that will result in a decreased morbidity and mortality by providing safer and simpler transplantation procedures for patients with malignant and non-malignant conditions. The potential utility of ES cells for gene therapy, tissue engineering and the treatment of a wide variety of currently untreatable diseases is simply too essential to ignore, however, our knowledge and ability to deliver these forms of therapy in a safe and efficient manner requires additional advances in the understanding of the basic biology of ES cells. In this article, we will discuss the factors and methodologies responsible for the differentiation of ES cells into hematopoietic progenitors and their potential to treat different blood related diseases.

TGF-β Signaling in Development

The transforming growth factor-beta (TGF-beta) superfamily comprises nearly 30 growth and differentiation factors that include TGF-betas, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-beta superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-beta inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-beta members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-beta and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-beta and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.