Profiling miRNAs involved in Human Oligodendrocyte Precursor Cell Differentiation and Maturation

Embryonic stem (ES) cells require a network of transcription factors and signaling molecules to maintain their pluripotency. The transcription factor Oct4, one of pluripotency genes, is required for self-renewal and maintains the undifferentiated state of ES cells. Loss of expression of Oct4 initiates ES cell differentiation. Germ cell nuclear factor (GCNF), an orphan nuclear receptor, binds to a DR0 site within Oct4 gene promoter in human ES cells. Retinoic acid induces expression of GCNF, which binds the promoter of Oct4 gene, and accelerates the decrease of Oct4 gene expression and human ES cells differentiation. Expression of other pluripotency genes, such as Sox2, also decrease during human ES cell differentiation induced with retinoic acid. These findings increase our understanding of the mechanism of regulation of gene expression and in maintaining human ES cell pluripotence and during the differentiation process. Results 1: Tera-2 cells and human ES (H9) cells were induced to differentiate by Retinoic Acid (1μM). Expression of GCNF, Oct4, Sox2, Dax1, LRH1, SF1, Actin was determined by RT-PCR (A), and by western blot (B) in Tera-2 cells and human ES cells. GCNF expression increased by day 2 of differentiation in Tera-2 cells and human ES cells when treated with Retinoic Acid (1μM). GCNF expression is transient and decreased earlier in Tera-2 cells than in human ES cells. Expression of Oct4, Sox2 and SF-1 was gradually repressed during differentiation. Expression of the orphan receptor LRH1 increased when Tera-2 and human ES cells differentiated, and subsequently decreased. DAX1 expression was up-regulated when Tera-2 cells and human ES cells differentiate, and maintained the same level until day 6 in Tera-2 cells but gradually increased in human ES cells. Result 2: GCNF binding the DR0 site within the Oct4 promoter in Tera-2 cells and human ES cells is induced upon differentiation and is transient in nature. Detection of GCNF binding to the Oct4 promoter in vivo using Chromatin immunoprecipitation (ChIP) assays: Tera-2 cells and human ES cells were treated with Retinoic Acid (1 μM) and induced to differentiate. GCNF binding to the DR0 site of Oct4 promoter can be detected in vivo when Tera-2 cell and human ES cells differentiate, and then decreases at later stages of differentiation. Result 3: Analysis of GCNF binding to the DR0 element in the Oct4 promoter by electrophoretic mobility shift assays (EMSAs). No GCNF binding to the DR0 element was detected at day 0 in the Tera-2 cells and human ES cells. GCNF has been at the highest level of binding to the DR0 site at day 3 of RA-induced differentiation (arrow) and is shifted (arrow head) when GCNF-specific antibody is added to the EMSAs. GCNF DNA binding activity disappeared by day 5 of differentiation in Tera-2 cells. In human ES cells, GCNF displayed a higher level of binding to the DR0 site at day 3 of differentiation and decreased at day 5 of RA treatment. This result shows that GCNF directly binds to the conserved DR0 element in the Oct4 promoter in Tera-2 cells and human ES cell. (platform)

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Human embryonic stem (ES) cells are routinely cultured under atmospheric oxygen (~20%), a concentration that is known to impair embryo development in vitro and is likely to be suboptimal for maintaining human ES cells compared with physiological (~5%) oxygen conditions. Conflicting reports exist on the effect of oxygen during human ES cell culture and studies have been largely limited to characterisation of typical stem cell markers or analysis of global expression changes. This study aimed to identify physiological markers that could be used to evaluate the metabolic impact of oxygen on the MEL-2 human ES cell line after adaptation to either 5% or 20% oxygen in extended culture. ES cells cultured under atmospheric oxygen displayed decreased glucose consumption and lactate production when compared with those cultured under 5% oxygen, indicating an overall higher flux of glucose through glycolysis under physiological conditions. Higher glucose utilisation at 5% oxygen was accompanied by significantly increased expression of all glycolytic genes analysed. Analysis of amino acid turnover highlighted differences in the consumption of glutamine and threonine and in the production of proline. The expression of pluripotency and differentiation markers was, however, unaltered by oxygen and no observable difference in proliferation between cells cultured in 5% and 20% oxygen was seen. Apoptosis was elevated under 5% oxygen conditions. Collectively these data suggest that culture conditions, including oxygen concentration, can significantly alter human ES cell physiology with coordinated changes in gene expression, in the absence of detectable alterations in undifferentiated marker expression.

Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

BackgroundThe first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation.ResultsBased on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5), 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome.ConclusionThese results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.

Human embryonic stem (ES) cells provide a novel opportunity to study early developmental events in a human system. We have used human ES cell lines, including clonally derived lines, to evaluate haematopoiesis. Co-culture of the human ES cells with irradiated bone marrow stromal cell lines in the presence of fetal bovine serum (FBS), but without other exogenous cytokines, leads to differentiation of the human ES cells within a matter of days. A portion of these differentiated cells express CD34, the best-defined marker for early haematopoietic cells. Haematopoietic colony-forming cells (CFCs) are demonstrated by methylcellulose assay. Myeloid, erythroid, megakaryocyte and multipotential CFCs can all be derived under these conditions. Enrichment of CD34+ cells derived from the human ES cells markedly increases the yield of CFCs, as would be expected for cells derived from adult bone marrow or umbilical cord blood. Transcription factors are also expressed in a manner consistent with haematopoietic differentiation. This system now presents the potential to evaluate specific conditions needed to induce or support events in early human blood development. Human ES cells are also a novel source of cells for transplantation therapies. The immunogenicity of ES cell-derived cells is unknown. The unique properties of ES cells afford the opportunity to explore novel mechanisms to prevent immune-mediated rejection. Potential strategies to overcome rejection will be presented, including creation of haematopoietic chimerism as a means to successfully transplant cells and tissues derived from human ES cells.

Limited knowledge of the changes in estrogen receptor (ER) signaling during the transformation of the normal mammary gland to breast cancer hinders the development of effective prevention and treatment strategies. Differences in estrogen signaling between normal human primary breast epithelial cells and primary breast tumors obtained immediately following surgical excision were explored. Transcriptional profiling of normal ER+ mature luminal mammary epithelial cells and ER+ breast tumors revealed significant difference in the response to estrogen stimulation. Consistent with these differences in gene expression, the normal and tumor ER cistromes were distinct and sufficient to segregate normal breast tissues from breast tumors. The selective enrichment of the DNA binding motif GRHL2 in the breast cancer-specific ER cistrome suggests that it may play a role in the differential function of ER in breast cancer. Depletion of GRHL2 resulted in altered ER binding and differential transcriptional responses to estrogen stimulation. Furthermore, GRHL2 was demonstrated to be essential for estrogen-stimulated proliferation of ER+ breast cancer cells. DLC1 was also identified as an estrogen-induced tumor suppressor in the normal mammary gland with decreased expression in breast cancer. In clinical cohorts, loss of DLC1 and gain of GRHL2 expression are associated with ER+ breast cancer and are independently predictive for worse survival. This study suggests that normal ER signaling is lost and tumor-specific ER signaling is gained during breast tumorigenesis. Unraveling these changes in ER signaling during breast cancer progression should aid the development of more effective prevention strategies and targeted therapeutics.

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

Mammalian cell identity or cell fate is governed by transcription factors that recruit co-activators to regulate cell-specific gene expression programs. The histone acetyltransferase (HAT) paralogs EP300 and CREB binding protein (CBP) are master transcriptional co-activators that catalyze acetylation of key histones that mark active enhancers, thereby operating at a fundamental step of transcriptional control. Enhancers are often co-opted by cancer cells to control expression of oncogenic transcriptional programs. Identifying cancer-specific dependencies will greatly help prioritize clinical indications that are uniquely vulnerable to HAT inhibition, allowing for precise targeting of cancer cells while sparing normal tissue to maximize the therapeutic window and clinical efficacy. Estrogen receptor (ER) signaling is a validated clinical pathway in over 70% of breast cancer. ER-directed therapies are foundational for the treatment of ER+ breast cancer. However, these therapies are limited by residual ER signaling, which remains online through late-stage disease in a significant fraction of patients. EP300/CBP are critical components of the core ER signaling complex, which is recruited to target DNA by ER and other coactivators, to potentiate ER signaling. Furthermore, CBP and EP300 are dependency factors in the context of ESR1 mutations that arise in metastatic disease to facilitate disease progression. We report for the first time the activity of a novel, potent, selective and in vivo active EP300/CBP HAT inhibitor for the treatment of ER-positive (ER+) breast cancer. CPI-1 has pM biochemical and nM activity in ER+ breast cancer cell lines. Analysis of nascent and steady-state RNA levels facilitated by PRO-seq and RNA-seq technologies revealed a time and dose-dependent suppression of the ER transcriptome. Dynamic gene regulation was coupled with enhancer RNA (eRNA) transcription and enhancer acetylation, to identify enhancers that were hypersensitive to CBP/EP300 HAT inhibition. CPI-1 demonstrates significant tumor growth inhibition in a xenograft mouse model of ER+ breast cancer as a monotherapy and together with an ER targeting agent, the combination displays superior efficacy to either treatment alone. These results highlight the potential of targeting key oncogenic transcriptional nodes with complementary therapeutic mechanisms to achieve superior results and reveals the potential utility of inhibiting the HAT domain of EP300/CBP in molecularly refined cancer populations. Citation Format: Archana Bommi-Reddy, Jonathan Wilson, Annissa J. Huhn, Esteban Terzo, Florence Poy, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Andrew R. Conery, Richard Cummings, Julian Levell, Robert J. Sims. Efficacy of a novel EP300/CBP histone acetyltransferase inhibitor in hormone responsive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4722.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

The breast cancer cells response to chronic hypoxia involves the opposite regulation of NF-kB and estrogen receptor signaling

Endocrine therapies designed to block estrogen-dependent activation of the estrogen receptor (ER) signaling pathway have, and will continue to be, the cornerstone of therapeutic approaches to treat luminal breast cancer. These agents work by either inhibiting the synthesis of gonadal/extragonadal estrogens or by directly blocking the activity of ER and have had a very significant impact on mortality and morbidity in patients with breast cancer, when used in the preventative, adjuvant, or treatment settings. However, as with most targeted therapies, de novo and acquired resistance remain impediments to durable clinical responses. Whereas the mechanisms underlying resistance to endocrine therapies are complex and multifactorial, most can be attributed to changes in the biochemical properties of the receptor or to adaptive changes in cancer cells that alter the way ER-ligand complexes are recognized. Mutations in ER that permit it to activate transcription in a ligand-independent manner and/or which enable pathological neomorphic activities have received considerable attention of late. These mutations, which occur infrequently in primary tumors are present in ~30-40% of metastatic lesions. Whereas it remains to be determined if these mutations are causally responsible for endocrine therapy failure their presence is at least a surrogate for the acute ER dependence of a tumor. Notable also are emerging data suggesting that ~1-2% of lesions from patients with advanced disease harbor gene arrangements that result in the formation of pathological ESR1-fusions. There is abundant evidence from both preclinical and clinical studies that resistance to endocrine therapies may result from changes in the absolute expression or activity of receptor-associated coregulators (coactivators or corepressors) and/or to dysregulated activity of signaling pathways that converge on ER-coregulator complexes. These activities negate the inhibitory effects of aromatase inhibitors and competitive antagonists like tamoxifen. A key feature of the primary resistance mechanisms that have been described to date is the continued dependence of tumors on ER signaling. This observation, together with the positive clinical activity of first generation Selective Estrogen Receptor Downregulators (SERDs), has encouraged the development of the next generation of endocrine therapies. Currently, there are 13 SERDs in clinical development for breast cancer, several of which have already shown efficacy in early clinical studies. It is likely that several of these SERDs, which accomplish ER downregulation/degradation by different mechanisms, will be approved for clinical use and that it will be issues associated with tolerability, effects on bone and cognitive function, and the ability to manage off-target activities which will differentiate these drugs. In addition to these drugs which target ER directly there is considerable enthusiasm for emerging new classes of drugs that directly target ER-coregulators or exploit collateral vulnerabilities that emerge in cancer cells as they become therapy resistant. Most endocrine therapies have been developed with the goal of inhibiting the cancer cell intrinsic actions of ER. However, we believe that it is important to consider that drugs of this class also inhibit cancer cell extrinsic actions of ER (a) in stromal cells within tumors and (b) at sites distal to the tumor (i.e. bone), that may impact tumor biology. Understanding these complexities. will inform the best use of current medicines and will be instructive as to the development of the next generation of endocrine therapies. Citation Format: DP McDonnell. Leveraging our most contemporary understanding of estrogen signaling in breast cancer to inform the development of next generation endocrine therapies. [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr TF1-1.