X-inactivation status varies in human embryonic stem cell lines

Human embryonic stem cells (hESCs) derived from human blastocysts have an apparently unlimited proliferative capacity and can differentiate into ectoderm, mesoderm, and endoderm. As such, hESC lines have enormous potential for use in cell replacement therapies. It must first be demonstrated, however, that hESCs maintain a stable karyotype and phenotype and that gene expression is appropriately regulated. To date, different hESC lines exhibit similar patterns of expression of markers associated with pluripotent cells. However, the evaluation of epigenetic status of hESC lines has only recently been initiated. One example of epigenetic gene regulation is dosage compensation of the X chromosome in mammalian females. This is achieved through an epigenetic event referred to as X-chromosome inactivation (XCI), an event initiated upon cellular differentiation. We provide the first evidence that undifferentiated hESC lines exhibit different patterns of XCI.     

Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.     

Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment

Background : Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. Results: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC‐enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor‐atypical expression profiles of neuron‐, glia‐, stem cell‐, and tumor‐related genes. Conclusions: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti‐tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment. Developmental Dynamics 242:1078‐1093, 2013. © 2013 Wiley Periodicals, Inc.     

Human Embryonic Stem Cells: Mechanisms to Escape Replicative Senescence?

Human embryonic stem cells (hESCs) are unique in that they can proliferate indefinitely in culture in an undifferentiated state as well as differentiate into any somatic cells. Undifferentiated hESCs do not appear to undergo senescence and remain nontransformed over multiple passages. Culture hESCs maintain telomere length and exhibit high telomerase activity after prolonged in vitro culture. The ability of hESCs to bypass senescence is lost as hESCs differentiate into fully differentiated somatic cells. This loss of immortality upon differentiation may be due to a variety aging related factors such as reduction in telomere length, alteration of telomerase activity, changes in cell cycle regulation and decrease in DNA repair ability. Absence of such aging factors as well as the lack of genomic, mitochondrial and epigenetic changes, may contribute to the lack of senescence in hESCs. In this review, we will summarize recent advances in determining changes in these aspects in prolonged hESC cultures. We will in particular discuss the potential roles of several cellular pathways including the telomerase, p53, and Rb pathways in escaping senescence in hESCs. We will also discuss the genomic and epigenetic changes in long-term hESC culture and their potential roles in bypassing senescence, as well as alternative sources of pluripotent stem cells.     

Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.     

Unique gene expression signature by human embryonic stem cells cultured under serum-free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage

Understanding the interaction between human embryonic stem cells (hESCs) and their microenvironment is crucial for the propagation and the differentiation of hESCs for therapeutic applications. hESCs maintain their characteristics both in serum-containing and serum-replacement (SR) media. In this study, the effects of the serum-containing and SR culture media on the gene expression profiles of hESCs were examined. Although the expression of many known embryonic stem cell markers was similar in cells cultured in either media, surprisingly, 1,417 genes were found to be differentially expressed when hESCs cultured in serum-containing medium were compared with those cultured in SR medium. Several genes upregulated in cells cultured in SR medium suggested increased metabolism and proliferation rates in this medium, providing a possible explanation for the increased growth rate of nondifferentiated cells observed in SR culture conditions compared with that in serum medium. Several genes characteristic for cells with differentiated phenotype were expressed in cells cultured in serum-containing medium. Our data clearly indicate that the manipulation of hESC culture conditions causes phenotypic changes of the cells that were reflected also at the level of gene expression. Such changes may have fundamental importance for hESCs, and gene expression changes should be monitored as a part of cell culture optimization aiming at a clinical use of hESCs for cell transplantation.     

Analysis of the factors that limit the ability of feeder cells to maintain the undifferentiated state of human embryonic stem cells

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.     

Maintenance of human embryonic stem cells in mesenchymal stem cell-conditioned media augments hematopoietic specification

The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.     

Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells

The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.     

Human embryonic stem cells: lessons from stem cell niches in vivo

In vivo the stem cell niche is an essential component in controlling and maintaining the stem cells' ability to survive and respond to injury. Human embryonic stem cells (hESCs) appear to be an exception to this rule as they can be removed from their blastocytic microenvironment and maintained indefinitely in vitro. However, recent observations reveal the existence of an autonomously derived in vitro hESC niche. This provides a previously unappreciated mechanism to control hESC expansion and differentiation. Recognizing this, it may now be possible to take aspects of in vivo stem cell niches, namely extracellular matrices, paracrine signals and accessory cell types, and exploit them in order to gain fidelity in directed hESC differentiation. In doing so, routine customization of hESC lines and their application in regenerative therapies may be further enhanced using unique hESC niche-based approaches.     

Modeling human germ cell development with embryonic stem cells

There has previously been no robust cell-based model for examining the genetic and epigenetic mechanisms of human germ cell formation. Human embryonic stem cells (hESCs) could potentially fill this need, as all cell types analyzed to date (including mature germ cells) can be identified by marker analysis during hESC differentiation. Furthermore, hESCs could also be used to differentiate mature female germ cells (oocytes) in culture as an alternate reprogramming cell for somatic cell nuclear transfer. However, to differentiate and isolate a functional germ cell from hESCs, the mechanisms that regulate germ cell formation need to be understood. The purpose of this review is to summarize the current understanding of the earliest events in human germ cell formation and to describe some of the known genetic pathways that regulate germ cell specification and development in the mouse. Finally, the current literature on the formation of germ cells from ESCs will be described.     

The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.

Certain aggressive melanoma cell lines exhibit a dedifferentiated phenotype, expressing genes that are characteristic of various cell types including endothelial, neural, and stem cells. Moreover, we have shown that aggressive melanoma cells can participate in neovascularization in vivo and vasculogenic mimicry in vitro, demonstrating that these cells respond to microenvironmental cues and manifest developmental plasticity. To explore this plasticity further, we transplanted human metastatic melanoma cells into zebrafish blastula-stage embryos and monitored their behavior post-transplantation. The data show that human metastatic melanoma cells placed in the zebrafish embryo survive, exhibit motility, and divide. The melanoma cells do not form tumors nor integrate into host organs, but instead become scattered throughout the embryo in interstitial spaces, reflecting the dedifferentiated state of the cancer cells. In contrast to the fate of melanoma cells, human melanocytes transplanted into zebrafish embryos most frequently become distributed to their normal microenvironment of the skin, revealing that the zebrafish embryo contains possible homing cues that can be interpreted by normal human cells. Finally, we show that within the zebrafish embryo, metastatic melanoma cells retain their dedifferentiated phenotype. These results demonstrate the utility of the zebrafish embryonic model for the study of tumor cell plasticity and suggest that this experimental paradigm can be a powerful one in which to investigate tumor-microenvironment interactions.     

Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.     

Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so-called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial-like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial-like cells. Further differentiation results in the formation of terminally differentiated epithelial-like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC-derived and adult limbal epithelial-like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss. Disclosure of potential conflicts of interest is found at the end of this article.     

Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease

Human ESCs provide access to the earliest stages of human development and may serve as an unlimited source of functional cells for future cell therapies. The optimization of methods directing the differentiation of human embryonic stem cells (hESCs) into tissue-specific precursors becomes crucial. We report an efficient enrichment of mesenchymal stem cells (MSCs) from hESCs through specific inhibition of SMAD-2/3 signaling. Human ESC-derived MSCs (hESC-MSCs) emerged as a population of fibroblastoid cells expressing a MSC phenotype: CD73+ CD90+ CD105+ CD44+ CD166+ CD45- CD34- CD14- CD19- human leucocyte antigen-DR (HLA-DR)-. After 28 days of SMAD-2/3 inhibition, hESC cultures were enriched (>42%) in multipotent MSCs. CD73+CD90+ hESC-MSCs were fluorescence activated cell sorting (FACS)-isolated and long-term cultures were established and maintained for many passages displaying a faster growth than somatic tissue-derived MSCs while maintaining MSC morphology and phenotype. They displayed osteogenic, adipogenic, and chondrocytic differentiation potential and exhibited potent immunosuppressive and anti-inflammatory properties in vitro and in vivo, where hESC-MSCs were capable of protecting against an experimental model of inflammatory bowel disease. Interestingly, the efficient enrichment of hESCs into MSCs through inhibition of SMAD-2/3 signaling was not reproducible with distinct induced pluripotent stem cell lines. Our findings provide mechanistic insights into the differentiation of hESCs into immunosuppressive and anti-inflammatory multipotent MSCs with potential future clinical applications.     

Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR?1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel?-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR?1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel?) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.