Dahlke, Flemming T; Leo, Elettra; Mark, Felix C; Pörtner, Hans-Otto; Bickmeyer, Ulf; Frickenhaus, Stephan; Storch, Daniela
Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO2 -driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO2 conditions (400 μatm vs. 1100 μatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO2 ) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid-base-relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3-6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO2 caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO2 and mitochondrial capacities. Elevated PCO2 stimulated MO2 at cold and intermediate temperatures, but exacerbated warming-induced constraints on MO2 , indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO2 . Increased MO2 in response to elevated PCO2 was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO2 treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO2 conditions and suggest that acclimation to elevated PCO2 requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains
Henningson, Carl T; Stanislaus, Marisha A; Gewirtz, Alan M
Stem cells are characterized by the ability to remain undifferentiated and to self-renew. Embryonic stem cells derived from blastocysts are pluripotent (able to differentiate into many cell types). Adult stem cells, which were traditionally thought to be monopotent multipotent, or tissue restricted, have recently also been shown to have pluripotent properties. Adult bone marrow stem cells have been shown to be capable of differentiating into skeletal muscle, brain microglia and astroglia, and hepatocytes. Stem cell lines derived from both embryonic stem and embryonic germ cells (from the embryonic gonadal ridge) are pluripotent and capable of self-renewal for long periods. Therefore embryonic stem and germ cells have been widely investigated for their potential to cure diseases by repairing or replacing damaged cells and tissues. Studies in animal models have shown that transplantation of fetal, embryonic stem, or embryonic germ cells may be able to treat some chronic diseases. In this review, we highlight recent developments in the use of stem cells as therapeutic agents for three such diseases: Diabetes, Parkinson disease, and congestive heart failure. We also discuss the potential use of stem cells as gene therapy delivery cells and the scientific and ethical issues that arise with the use of human stem cells.
Resnik, David B
This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells.
Resnik, David B.
This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198
Landry, Donald W; Zucker, Howard A
The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.
The appearance of diploidy, the presence of two genomes or chromosome sets, is a fundamental hallmark of eukaryotic evolution and bisexual reproduction, because diploidy offers the basis for the bisexual life cycle, allowing for oscillation between diploid and haploid phases. Meiosis produces haploid gametes. At fertilization, male and female gametes fuse to restore diploidy in a zygote, which develops into a new life. At sex maturation, diploid cells enter into meiosis, culminating in the production of haploid gametes. Therefore, diploidy ensures pluripotency, cell proliferation, and functions, whereas haploidy is restricted only to the post-meiotic gamete phase of germline development and represents the end point of cell growth. Diploidy is advantageous for evolution. Haploidy is ideal for genetic analyses, because any recessive mutations of essential genes will show a clear phenotype in the absence of a second gene copy. Recently, my laboratory succeeded in the generation of medaka haploid embryonic stem (ES) cells capable of whole animal production. Therefore, haploidy in a vertebrate is able to support stable cell culture and pluripotency. This finding anticipates the possibility to generate haploid ES cells in other vertebrate species such as zebrafish. These medaka haploid ES cells elegantly combine haploidy and pluripotency, offering a unique yeast-like system for in vitro genetic analyses of molecular, cellular, and developmental events in various cell lineages. This chapter is aimed to describe the strategy of haploid ES cell derivation and their characteristics, and illustrate the perspectives of haploid ES cells for infertility treatment, genetic screens, and analyses.
Olsvik, Pål A; Berntssen, Marc H G; Hylland, Ketil; Eriksen, Dag Ø; Holen, Elisabeth
The aim of this study was to investigate whether (226)Ra, a radionuclide present in produced water from oil platforms in the North Sea and other offshore drilling areas, could affect vulnerable early life stages of Atlantic cod (Gadus morhua). Blastula-stage embryonic cells (EC) from fertilized eggs of Atlantic cod were isolated and exposed to environmental relevant concentrations of (226)Ra and transcription of selected genes quantified. The results showed a weak, but significant up-regulation of GPx3 and HSP70 transcripts after 48 h of exposure to 2.11 Bq/L. In EC exposed to three (226)Ra concentrations (2.11, 23 and 117 Bq/L) for 12 h, metallothionein, HSP90AA, thioredoxin and caspase 8 were significantly up-regulated in cells exposed to 117 Bq/L, whereas thioredoxin was also significantly up-regulated in EC exposed to 23 Bq/L. When EC were exposed to the same (226)Ra concentrations for 48 h, only heme oxygenase was significantly up-regulated in the 23 Bq/L exposure group. The results suggest that environmentally relevant activities of (226)Ra may induce oxidative stress and apoptosis in fish ECs. Exposure of Atlantic cod EC to Cd, selected as a model toxicant, supported the ability of EC around blastula stage to respond to toxicants by altered transcription. Due to dilution, environmentally relevant concentrations of radionuclides present in produced water would be expected to pose a minor threat to early life stages of fish. Copyright © 2012 Elsevier Ltd. All rights reserved.
The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state.
Hadjimichael, Christiana; Chanoumidou, Konstantina; Papadopoulou, Natalia; Arampatzi, Panagiota; Papamatheakis, Joseph; Kretsovali, Androniki
Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies. PMID:26516408
Aznar, J; Gómez, I
Are embryonic stem cells being used for therapeutic purposes? The aim of this short report is to review to what extent are embryonic stem cells currently used for therapeutic purposes. To the best of our knowledge, only four clinical trials have been authorised so far to use human embryonic stem cells for therapeutic purposes; two of these are included in the ClinicalTrials.gov data base, and the other two, the study sponsored by Geron and the last one initiated by Advanced Cell Technology in the United Kingdom, are not. But, in addition, Geron withdrew the clinical trial which had been originally proposed by the company itself. This brief review focuses the debate on the use of embryonic stem cells in human cell therapy. To the best of our knowledge, only three trials are ongoing with therapeutic purposes thus far, with not all of them having begun to include patients, and of course without any of them yet having obtained evaluable clinical results. Copyright © 2012 Elsevier España, S.L. All rights reserved.
Varanou, A; Page, C P; Minger, S L
Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically.
Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark
Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC. PMID:22110659
Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J; Wolvetang, Ernst; Prescott, Mark
Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.
Amit, Michal; Itskovitz-Eldor, Joseph
Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.
Andersson, Anna-Karin M
This article contributes to the current debate on human embryonic stem cell researchers' possible complicity in the destruction of human embryos and the relevance of such complicity for the issue of commodification of human embryos. I will discuss if, and to what extent, researchers who destroy human embryos, and researchers who merely use human embryos destroyed by others, have moral use rights, and/or moral property rights, in these embryos. I argue that the moral status of the human embryo, however justified, places few restrictions on the latter researchers' use of it, and property rights in it, once it is destroyed. I argue that the former researchers have no property rights in the destroyed embryo but use rights in it to the extent allowed by the legitimate owners of the destroyed embryo. I discuss the implications of this account for previous and current US federal law regulating human embryonic stem cell research.
Sage, William M
Embryonic stem cells are actively debated in political and public policy arenas. However, the connections between stem cell innovation and overall health care policy are seldom elucidated. As with many controversial aspects of medical care, the stem cell debate bridges to a variety of social conversations beyond abortion. Some issues, such as translational medicine, commercialization, patient and public safety, health care spending, physician practice, and access to insurance and health care services, are core health policy concerns. Other issues, such as economic development, technologic progress, fiscal politics, and tort reform, are only indirectly related to the health care system but are frequently seen through a health care lens. These connections will help determine whether the stem cell debate reaches a resolution, and what that resolution might be.
Hynes, Richard O
The United States is a federal union with separate state jurisdictions. In part owing to the sometimes heated debate about public support for human embryonic stem-cell (ESC) research, there has been restricted federal support and little central regulation of this research to date. Instead, guidelines developed by scientific organizations have set principles for oversight and good practice for this research. These guidelines are functioning well, have influenced developing state regulations and, one hopes, will affect any future federal regulation.
Pera, Martin F; Trounson, Alan O
It is widely anticipated that human embryonic stem (ES) cells will serve as an experimental model for studying early development in our species, and, conversely, that studies of development in model systems, the mouse in particular, will inform our efforts to manipulate human stem cells in vitro. A comparison of primate and mouse ES cells suggests that a common underlying blueprint for the pluripotent state has undergone significant species-specific modification. As we discuss here, technical advances in the propagation and manipulation of human ES cells have improved our understanding of their growth and differentiation, providing the potential to investigate early human development and to develop new clinical therapies.
Many health care issues generate minimal passion, promoting benign commentary and support from the various stakeholders involved. Stem cell research does not fall into this category, and on the contrary, embryonic stem cell (ESC) research has continued to foster controversy and emotion. Since 1998, which marked the first successful laboratory isolation of ESCs, this research continues to ignite moral, ethical, and legal debate over its efficacy. The focus of this policy analysis is to introduce the issues, examine and address the various perspectives that surround ESC research, and present policy options and/or solutions that may be used to successfully create a policy consensus regarding this much debated topic.
Embryonic stem cells can give rise to all somatic cells, making them an attractive cell source for tissue engineering applications. The propensity of cells to form tissue-like structures in a culture dish has been well documented. We and others made use of this intrinsic property to generate bioartificial heart muscle. First proof-of-concept studies involved immature heart cells mainly from fetal chicken, neonatal rats and mice. They eventually provided evidence that force-generating heart muscle can be engineered in vitro. Recently, the focus shifted to the application of stem cells to eventually enable the generation of human heart muscle and reach following long-term goals: (1) development of a simplified in vitro model of heart muscle development; (2) generation of a human test-bed for drug screening and development; (3) allocation of surrogate heart tissue to myocardial repair applications. This overview will provide the background for cell-based myocardial repair, introduce the main myocardial tissue engineering concepts, discuss the use of embryonic and non-embryonic stem cells, and lays out the potential direct and indirect therapeutic use of human tissue engineered myocardium.
Deb, Kaushik Dilip; Jayaprakash, Anitha Devi; Sharma, Vijay; Totey, Satish
ABSTRACT Embryonic stem cells are considered the mother of all kinds of tissues and cells and it is envisioned as the holy grail of regenerative medicine. However, their use in cell replacement therapies (CRT) has so far been limited and their potentials are yet to be fully realized. The use of human embryonic stem cells (hESC) involves many safety issues pertaining to culture conditions and epigenetic changes. The role and importance of an epigenomic signature in derivation and maintenance of hESC are discussed. We provide a list of important epigenetic markers, which should be studied for evaluation of safety in hESC-based cell replacement therapies. These genes also need to be screened to determine an epigenetic signature for pluripotency in the hESCs. Finally a comprehensive list of all known stemness signature genes and the marker genes for different germ line lineages are presented. This review aims at summing up most of the intriguing molecules that can play a role in the maintenance of pluripotency and can help in determining hESC differentiation to various lineages. Extensive understanding of these markers will eventually help the researchers to transform the hESC research from bench to the bedside. The use of hESCs in CRTs is still in its infancy; much effort is warranted to turn them into the much dreamed about magic wand of regenerative medicine.
Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles
Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.
Research using embryonic stem cells raises a variety of ethical questions, which will be explored in this article. At the core of the ethical controversy is the question of the status of the embryo and its availability for research. A range of countries have approved the use of "supernumerous" embryos from in-vitro fertilization. But ethical problems also arise in reproduction medicine, the informed consent of affected couples, and the targeted production of embryos and egg cell donation for research. The author discuss some of these neglected issues and develops suggestions for comprehensive ethical reflection.
This study investigates the emotional and behavioral effects of interpersonal online communication, focusing on the controversy surrounding the loosening of restrictions on human embryonic stem cell research. The issue, central to national and statewide elections in 2008, generated heated debate among candidates and voters and evoked strong emotional sentiments among partisans. Using the theory of affective intelligence, this study proposes a predictive model connecting levels of enthusiasm and anxiety with behavioral and information-seeking outcomes. Cognitive appraisal theory is also employed to provide a role for political emotion in accounting for interactive media effects. To investigate the ways that online deliberation may influence discussions surrounding stem cell research, a between-subjects experimental study was conducted that systematically varied the tone of feedback received (reinforcing or challenging) and type of interaction (synchronous or asynchronous) experienced by users. Results indicate that emotional responses play a significant role in predicting behavioral intentions arising from the user-to-user interactive experience.
We are currently facing an unprecedented level of public interest in research on embryonic stem cells, an area of biomedical research that until recently was small, highly specialized and of limited interest to anyone but experts in the field. Real and imagined possibilities for the treatment of degenerative and other diseases are of special interest to our rapidly ageing population; real and imagined associations of stem cells to cloning, embryos and reproduction stir deeply held beliefs and prejudices. The conjunction of these factors could explain the recent sudden interest in embryonic stem cells but we ought to remember that this research has a long and convoluted history, and that the findings described today in the scientific and popular press are firmly grounded in research that has been going on for several decades. Here I briefly recapitulate this fascinating history.
Zhu, Wei-Zhong; Hauch, Kip D; Xu, Chunhui; Laflamme, Michael A
The muscle lost after a myocardial infarction is replaced with noncontractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. Human embryonic stem cell-derived cardiomyocytes exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain in the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-derived cardiomyocytes and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges.
Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena
Mammalian embryogenesis requires intricate interactions between embryonic and extra-embryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combine mouse embryonic stem cells (ESCs) and extra-embryonic trophoblast stem cells (TSCs) in a 3D-scaffold to generate structures whose morphogenesis is remarkably similar to natural embryos. By using genetically-modified stem cells and specific inhibitors, we show embryogenesis of ESC- and TSC-derived embryos, ETS-embryos, depends on crosstalk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extra-embryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell-types resemble natural embryos.
Mallick, Bibekanand; Chakrabarti, Jayprokas; Ghosh, Zhumur
MicroRNAs represents a new layer of gene regulation in stem cells by controlling the molecular mechanisms involved in modulating stem cell fate and behavior. Such a role of microRNA is seen in embryonic stem cell as well, maintaining a delicate balance between survival, proliferation, and self-renewal signals. Further, dysregulation of stem cell self-renewal is a likely requirement for the initiation and formation of cancer stem cells that probably pose resistance to current cancer treatments. In fact, the precise mechanism that regulates embryonic as well as cancer stem cell self-renewal and pluripotency remains largely unknown. Understanding the miRNA related stem cell biology and pathways offers great promise for improving stem cell mediated regenerative therapy as well as cancer therapies. Here we summarize some of the emerging evidences demonstrating the role of these molecular switches in embryonic and cancer stem cells.
Fu, Xuemei; Cui, Ke; Yi, Qiuxiang; Yu, Lili; Xu, Yang
Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.
Siemen, Henrike; Nix, Michael; Endl, Elmar; Koch, Philipp; Itskovitz-Eldor, Joseph; Brüstle, Oliver
Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.
Deb, Kaushik Dilip; Sarda, Kanchan
Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic. PMID:18230169
Sakano, Daisuke; Shiraki, Nobuaki; Kume, Shoen
Pluripotent stem cells are considered as a cell source for replacement therapies for pancreatic beta cells and other organs.We identified tetrabenazine (TBZ), vesicular monoamine transporter 2 (VMAT2) inhibitor as a promoter of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Ngn3-positive endocrine progenitor cells. A cell-permeable cAMP analog, dBu-cAMP promotes beta cell maturation in late stage of differentiation. The induced beta cells can secrete insulin in a glucose-dependent manner.Our protocol consists of a three -step differentiation process. ES cell recapitulate embryonic developmental processes in vitro. Therefore, the ES cell differentiation system is a useful model for the understanding of molecular mechanism of beta-cell differentiation and are useful for application for future regenerative medicine.
Nakatsuji, Norio; Suemori, Hirofumi
Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is
Cardiac muscle engineering is evolving rapidly and may ultimately be exploited to (1) model cardiac development, physiology, and pathology; (2) identify and validate drug targets; (3) assess drug safety and efficacy; and (4) provide therapeutic substitute myocardium. The ultimate success in any of these envisioned applications depends on the utility of human cells and their assembly into myocardial equivalents with structural and functional properties of mature heart muscle. Embryonic stem cells appear as a promising cell source in this respect, because they can be cultured reliably and differentiated robustly into cardiomyocytes. Despite their unambiguous cardiogenicity, data on advanced maturation and seamless myocardial integration of embryonic stem cell-derived cardiomyocytes in vivo are sparse. Additional concerns relate to the limited control over cardiomyogenic specification and cardiomyocyte maturation in vitro as well as the risk of teratocarcinoma formation and immune rejection of stem cell implants in vivo. Through the invent of embryonic-like stem cells - such as parthenogenetic stem cells, male germline stem cells, and induced pluripotent stem cells - some but certainly not all of these issues may be addressed, albeit at the expense of additional concerns. This review will discuss the applicability of embryonic and embryonic-like stem cells in myocardial tissue engineering and address issues that require particular attention before the potential of stem cell-based heart muscle engineering may be fully exploited. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
Czerwinska, Areta M; Nowacka, Joanna; Aszer, Magdalena; Gawrzak, Sylwia; Archacka, Karolina; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Koblowska, Marta; Ciemerych, Maria A; Grabowska, Iwona
The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.
Lavial, Fabrice; Pain, Bertrand
Embryonic stem cells (ESCs) were isolated in the early 1980s from mouse and in the late 1990s from primate and human. These cells present the unique property of self-renewal and the ability to generate differentiated progeny in all embryonic lineages both in vitro and in vivo. The mESCs (mouse embryonic stem cells) can contribute to both somatic and germinal lineages once re-injected into a recipient embryo at the blastocyst stage. In avian species, chicken embryonic stem cells (cESCs) have been isolated from the in vitro culture of early chicken blastodermal cells (cBCs) taken from stage X embryo (EG&K) These cESCs can be maintained under specific culture conditions and have been characterized on the basis of their morphology, biochemical features, in vitro differentiation potentialities and in vivo morphogenetic properties. The relationship between these cESCs and some of the chicken germ cells identified and grown under specific culture conditions are still under debate, in particular with the identification of the Cvh gene as a key factor for germ cell determination. Moreover, by cloning the avian homologue of the Oct4 mammalian gene, we have demonstrated that this gene, as well as the chicken Nanog gene, was involved in the characterization and maintenance of the chicken pluripotency. These first steps toward the understanding of pluripotency control in a non-mammalian species opens the way for the development and characterization of putative new cell types such as chicken EpiSC and raises the question of the existence of reprogramming in avian species. These different points are discussed.
Seiler, Andrea E M; Buesen, Roland; Visan, Anke; Spielmann, Horst
The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.
While some people claim that the present disagreement over embryonic stem (ES) cell research cannot be resolved, others argue that developing transparency and trust are key elements that could resolve the existing disagreements over such research. This paper reveals that transparency is not necessarily a requirement for advancing ES cell research, since in Israel, for instance, there is (almost) no transparency, and research nevertheless flourishes. Moreover, trust is not independent of cultural values and religious beliefs. Because of these beliefs, the environment in Israel for ES cell research has been pragmatic and liberal. The Israeli case illustrates the key role that culture and religion can play in biomedical research; it also suggests that as far as cultural values or religious beliefs of people in Western countries strongly oppose research on embryonic tissue, it would be very difficult, if not impossible, to overcome the disagreements.
Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H
The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome
Saniei, Mansooreh; De Vries, Raymond
Various successes in regenerative medicine by therapeutic cloning have given rise to expectations that treatments will soon be developed for incurable diseases. But using embryonic stem cells for this purpose raises many ethical dilemmas including those about the beginning of human life. Arguments concerning stem cell research and therapeutic cloning in different countries are influenced by both the religious and bioethical traditions which dominate in these cultures. This article examines how these traditions have influenced stem cell research in Iran through an account of scientific advances and the development of regulations on embryonic stem cell research in Iran.
Mignone, John L; Kreutziger, Kareen L; Paige, Sharon L; Murry, Charles E
Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.
Kraushaar, Daniel C; Zhao, Keji
Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes. Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.
Stanford, William L; Epp, Trevor; Reid, Tammy; Rossant, Janet
Gene trapping in embryonic stem cells (ESCs) generates random, sequence-tagged insertional mutations, which can often report the gene expression pattern of the mutated gene. This mutagenesis strategy has often been coupled to expression or function-based assays in gene discovery screens. The availability of the mouse genome sequence has shifted gene trapping from a gene discovery platform to a high-throughput mutagenesis platform. At present, a concerted worldwide effort is underway to develop a library of loss-of-function mutations in all mouse genes. The International Gene Trap Consortium (IGTC) is leading the way by making a first pass of the genome by random mutagenesis before a high-throughput gene targeting program takes over. In this chapter, we provide a methods guidebook to exploring and using the IGTC resource, explain the different kinds of vectors and insertions that reside in the different libraries, and provide advice and methods for investigators to design novel expression-based "cottage industry" screens.
Narsinh, Kazim H; Cao, Feng; Wu, Joseph C
Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogenic cells. Investigating in vivo hESC behaviour and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), mat be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogenous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.
Strulovici, Yael; Leopold, Philip L; O'Connor, Timothy P; Pergolizzi, Robert G; Crystal, Ronald G
Human embryonic stem cells (hESCs) theoretically represent an unlimited supply of normal differentiated cells to engineer diseased tissues to regain normal function. However, before hESCs can be useful as human therapeutics, technologies must be developed to provide them with the specific signals required to differentiate in a controlled fashion, to regulate and/or shut down the growth of hESCs and their progeny once they have been transferred to the recipient, and to circumvent the recognition of non-autologous hESC-derived cells as foreign. In the context that gene therapy technologies represent strategies to deliver biological signals to address all of these challenges, this review sets out a framework for combined gene transfer/hESC therapies. We discuss how hESCs are derived, characterized, and differentiated into specific cell lineages, and we summarize the characteristics of the 500 hESC lines reported to date. The successes and failures of gene transfer to hESCs are reviewed for both non-viral and viral vectors, as are the challenges to successful use of gene transfer in developing hESC therapy. We also consider gene transfer as a means of facilitating growth and isolation of genetically modified hESCs and as a mechanism for mitigating adverse effects associated with administration of hESCs or their derivatives. Finally, we evaluate the challenges that are likely to be encountered in translating the promise of hESCs to the clinic.
The purpose of this essay is to stimulate academic discussion about the ethical justification of using human primordial stem cells for tissue transplantation, cell replacement, and gene therapy. There are intriguing alternatives to using embryos obtained from elective abortions and in vitro fertilisation to reconstitute damaged or dysfunctional human organs. These include the expansion and transplantation of latent adult progenitor cells. Key Words: Primordial stem cell research • embryonic stem cells • pluripotent stem cells • embryo research PMID:10860206
Smith, G D; Swain, J E; Bormann, C L
Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.
Campagnolo, Luisa; Fenoglio, Ivana; Massimiani, Micol; Magrini, Andrea; Pietroiusti, Antonio
Due to the increasing use of engineered nanoparticles in many consumer products, rapid and economic tests for evaluating possible adverse effects on human health are urgently needed. In the present chapter the use of mouse embryonic stem cells as a valuable tool to in vitro screen nanoparticle toxicity on embryonic tissues is described. This in vitro method is a modification of the embryonic stem cell test, which has been widely used to screen soluble chemical compounds for their embryotoxic potential. The test offers an alternative to animal experimentation, reducing experimental costs and ethical issues.
Robertson, John A
Embryonic stem cells, which have the potential to save many lives, must be recovered from aborted fetuses or live embyros. Although tissue from aborted fetuses can be used without moral complicity in the underlying abortion, obtaining stem cells from embryos necessarily kills them, thus raising difficult questions about the use of embryonic human material to save others. This article draws on previous controversies over embryo research and distinctions between intrinsic and symbolic moral status to analyze these issues. It argues that stem cell research with spare embryos produced during infertility treatment, or even embryos created specifically for research or therapeutic purposes, is ethically acceptable and should receive federal funding.
The Roman Catholic Church in Australia has lobbied politicians to prohibit embryonic stem cell research, on the grounds that such research violates the sanctity and inherent dignity of human life. I suggest, however, that reasoned reflection does not uniquely support such conclusions about the morality of stem cell research. A recent parliamentary standing committee report recommended that embryonic stem cell research be allowed to proceed in certain circumstances, and there appears to be widespread support in the Australian community for this position. I argue that the moral value of democracy requires parliamentarians to acknowledge the informed views of the wider community here, and to resist lobbying by church leaders on this issue.
Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405
Rise, Matthew L; Nash, Gordon W; Hall, Jennifer R; Booman, Marije; Hori, Tiago S; Trippel, Edward A; Gamperl, A Kurt
Early life stage mortality is an important issue for Atlantic cod aquaculture, yet the impact of the cod maternal (egg) transcriptome on egg quality and mortality during embryonic development is poorly understood. In the present work, we studied embryonic mortality and maternal transcript expression using eggs from 15 females. Total mortality at 7days post-fertilization (7 dpf, segmentation stage) was used as an indice of egg quality. A 20,000 probe (20K) microarray experiment compared the 7hours post-fertilization (7 hpf, ~2-cell stage) egg transcriptome of the two lowest quality females (>90% mortality at 7 dpf) to that of the highest quality female (~16% mortality at 7 dpf). Forty-three microarray probes were consistently differentially expressed in both low versus high quality egg comparisons (25 higher expressed in low quality eggs, and 18 higher expressed in high quality eggs). The microarray experiment also identified many immune-relevant genes [e.g. interferon (IFN) pathway genes ifngr1 and ifrd1)] that were highly expressed in eggs of all 3 females regardless of quality. Twelve of the 43 candidate egg quality-associated genes, and ifngr1, ifrd1 and irf7, were included in a qPCR study with 7 hpf eggs from all 15 females. Then, the genes that were confirmed by qPCR to be greater than 2-fold differentially expressed between 7 hpf eggs from the lowest and highest quality females (dcbld1, ddc, and acy3 more highly expressed in the 2 lowest quality females; kpna7 and hacd1 more highly expressed in the highest quality female), and the 3 IFN pathway genes, were included in a second qPCR study with unfertilized eggs. While some maternal transcripts included in these qPCR studies were associated with extremes in egg quality, there was little correlation between egg quality and gene expression when all females were considered. Both dcbld1 and ddc showed greater than 100-fold differences in transcript expression between females and were potentially influenced by
Czerwinska, Areta M.; Nowacka, Joanna; Aszer, Magdalena; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Grabowska, Iwona
ABSTRACT The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E. PMID:27610933
Chapman, Audrey R
Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.
Limaye, Advait; Hall, Bradford; Kulkarni, Ashok B
The establishment of mouse embryonic stem (ES) cell liness has allowed for the generation of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. PMID:19731225
Hornstein, Eran; Benvenisty, Nissim
The recent isolation of human embryonic stem (ES) cells is evoking great hopes for their future utilization in cell-replacement therapies and human development research. The hallmarks of ES cells, pluripotency and self-renewal capacity, suggest an infinite source for tissues of virtually all desired types. Specifically, human ES cells may potentially be the basis for effective treatments of a wide range of human neurodegenerative disorders. To enable the translation of this novel biomedical field into the clinic, mechanisms that control the differentiation of human embryonic stem cells into fully functional neuronal cells should be analyzed and controlled.
In 1998, researchers discovered that embryonic stem cells could be derived from early human embryos. This discovery has raised a series of ethical and public-policy questions that are now being confronted by multiple international organizations, nations, cultures, and religious traditions. This essay surveys policies for human embryonic stem cell research in four regions of the world, reports on the recent debate at the United Nations about one type of such research, and reviews the positions that various religious traditions have adopted regarding this novel type of research. In several instances the religious traditions seem to have influenced the public-policy debates.
Moore, Kim E; Mills, Jeanette F; Thornton, Melissa M
The complex moral and ethical debate surrounding the definition of the origins of human life, together with conflicting current and proposed legislation on state and federal levels, is hindering the course of research into the therapeutic uses of human embryonic stem cells. However, newly identified sources of adult stem cells, free from many of the ethical and legal concerns attached to embryonic stem cell research, may offer great promise for the advancement of medicine. These alternative sources may alleviate the need to resolve the stem cell debate before further therapeutic benefits of stem cell research can be realized. While legislation and ethics evolve to address the legal and moral issues of embryonic stem cell research, innovative researchers will continue to search for and find real and present solutions for cell-based therapies using adult stem cells.
Přikrylová, Terézia; Pacherník, Jiří; Kozubek, Stanislav; Bártová, Eva
The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson’s disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells. PMID:23951389
Talk of policy has dominated talk of science for those interested in embryonic stem cell science. But research is continuing, and the advances are making clear why embryonic stem cells are such an important scientific and medical resource. PMID:16966416
Lott, Jason P; Savulescu, Julian
An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Human embryonic stem cell technology could solve the organ shortage problem by restoring diseased or damaged tissue across a range of common conditions. However, such technology faces several largely ignored immunological challenges in delivering cell lines to large populations. We address some of these challenges and argue in favor of encouraging contribution or intentional creation of embryos from which widely immunocompatible stem cell lines could be derived. Further, we argue that current immunological constraints in tissue transplantation demand the creation of a global stem cell bank, which may hold particular promise for minority populations and other sub-groups currently marginalized from organ procurement and allocation systems. Finally, we conclude by offering a number of practical and ethically oriented recommendations for constructing a human embryonic stem cell bank that we hope will help solve the ongoing organ shortage problem.
AD_________________ Award Number: W81XWH-04-1-0366 TITLE: Developing human embryonic stem cells ...Mar 04 – 28 Feb 07 4. TITLE AND SUBTITLE Developing human embryonic stem cells for grafting in Parkinson’s disease 5a...TERMS Parkinson’s disease, transplantation, embryonic stem cell , neuronal differentiation, dopamine, xenograft, functional recovery 16. SECURITY
Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...
Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...
At the heart of the extensive ethical and regulatory debates that have surrounded human embryonic stem cells is the human pre-implantation embryo. Advances in the understanding of cellular reprogramming, both by cell nuclear replacement and by potential new protocols, should lead to methods that circumvent the use of a practicably viable embryo.
Lee, Jae-Young; Park, Sunghyun; Kim, Kwang-Soo; Ko, Jeong-Jae; Lee, Soohong; Kim, Keun Pil; Park, Kyung-Soon
Sprouty (Spry) genes encode inhibitors of the receptor tyrosine kinase signaling cascade, which plays important roles in stem cells. However, the role of Spry4 in the stemness of embryonic stem cells has not been fully elucidated. Here, we used mouse embryonic stem cells (mESCs) as a model system to investigate the role of Spry4 in the stem cells. Suppression of Spry4 expression results in the decreases of cell proliferation, EB formation and stemness marker expression, suggesting that Spry4 activity is associated with stemness of mESCs. Teratoma assay showed that the cartilage maturation was facilitated in Spry4 knocked down mESCs. Our results suggest that Spry4 is an important regulator of the stemness and differentiation of mESCs. PMID:27660833
Hosseinkhani, Mohsen; Shirazi, Reza; Rajaei, Farzad; Mahmoudi, Masoud; Mohammadi, Navid; Abbasi, Mahnaz
Context Stem cells have the potential to generate a renewable source of cells for regenerative medicine due to their ability to self-renew and differentiate to various functional cell types of the adult organism. The extracellular microenvironment plays a pivotal role in controlling stem cell fate responses. Therefore, identification of appropriate environmental stimuli that supports cellular proliferation and lineage-specific differentiation is critical for the clinical application of the stem cell therapies. Evidence Acquisition Traditional methods for stem cells culture offer limited manipulation and control of the extracellular microenvironment. Micro engineering approaches are emerging as powerful tools to control stem cell-microenvironment interactions and for performing high-throughput stem cell experiments. Results In this review, we provided an overview of the application of technologies such as surface micropatterning, microfluidics, and engineered biomaterials for directing stem cell behavior and determining the molecular cues that regulate cell fate decisions. Conclusions Stem cells have enormous potential for therapeutic and pharmaceutical applications, because they can give rise to various cell types. Despite their therapeutic potential, many challenges, including the lack of control of the stem cell microenvironment remain. Thus, a greater understanding of stem cell biology that can be used to expand and differentiate embryonic and adult stem cells in a directed manner offers great potential for tissue repair and regenerative medicine. PMID:23682319
Azarin, Samira M.; Palecek, Sean P.
The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. PMID:20161686
Jensen, David A
Can one consistently deny the permissibility of abortion while endorsing the killing of human embryos for the sake of stem cell research? The question is not trivial; for even if one accepts that abortion is prima facie wrong in all cases, there are significant differences with many of the embryos used for stem cell research from those involved in abortion--most prominently, many have been abandoned in vitro, and appear to have no reasonably likely meaningful future. On these grounds one might think to maintain a strong position against abortion but endorse killing human embryos for the sake of stem cell research and its promising benefits. I will argue, however, that these differences are not decisive. Thus, one who accepts a strong view against abortion is committed to the moral impermissibility of killing human embryos for the sake of stem cell research. I do not argue for the moral standing of either abortion or the killing of embryos for stem cell research; I only argue for the relation between the two. Thus the conclusion is relevant to those with a strong view in favor of the permissibility of killing embryos for the sake of research as much as for those who may strongly oppose abortion; neither can consider their position in isolation from the other.
The recent discussions about alternative sources of human embryonic stem cells (White Paper of the US President's Council on Bioethics, 2005), while stirring new interest in the developmental potential of the various abnormal embryos or constructs proposed as such sources, also raise questions about the potential of the derived embryonic stem cells. The data on the developmental potential of embryonic stem cells that seem relevant for ethical considerations and aspects of patentability are discussed. Particular attention is paid to the meaning of "totipotency, omnipotency and pluripotency" as illustrated by a comparison of the developmental potential of three-dimensional clusters of blastomeres (morula), embryonic stem cells, somatic or (adult) stem cells or other somatic (non-stem) cells. This paper focuses on embryoid bodies and on direct cloning by tetraploid complementation. Usage and patenting of these cells cannot be considered to be ethically sound as long as totipotency and tetraploid complementability of embryonic stem cells are not excluded for the specific cell line in question. Testing this poses an ethical problem in itself and needs to be discussed in the future.
The recent discussions about alternative sources of human embryonic stem cells (White Paper of the US President's Council on Bioethics, 2005), while stirring new interest in the developmental potential of the various abnormal embryos or constructs proposed as such sources, also raise questions about the potential of the derived embryonic stem cells. The data on the developmental potential of embryonic stem cells that seem relevant for ethical considerations and aspects of patentability are discussed. Particular attention is paid to the meaning of “totipotency, omnipotency and pluripotency” as illustrated by a comparison of the developmental potential of three‐dimensional clusters of blastomeres (morula), embryonic stem cells, somatic or (adult) stem cells or other somatic (non‐stem) cells. This paper focuses on embryoid bodies and on direct cloning by tetraploid complementation. Usage and patenting of these cells cannot be considered to be ethically sound as long as totipotency and tetraploid complementability of embryonic stem cells are not excluded for the specific cell line in question. Testing this poses an ethical problem in itself and needs to be discussed in the future. PMID:17074826
Vilchez, David; Boyer, Leah; Morantte, Ianessa; Lutz, Margaret; Merkwirth, Carsten; Joyce, Derek; Spencer, Brian; Page, Lesley; Masliah, Eliezer; Berggren, W. Travis; Gage, Fred H.; Dillin, Andrew
Embryonic stem cells are able to replicate continuously in the absence of senescence and, therefore, are immortal in culture1,2. While genome stability is central for survival of stem cells; proteome stability may play an equally important role in stem cell identity and function. Additionally, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. We hypothesized that stem cells have an increased proteostasis ability compared to their differentiated counterparts and asked whether proteasome activity differed among human embryonic stem cells (hESCs). Notably, hESC populations exhibit a high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11/RPN-63–5 and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. Proteasome inhibition affects pluripotency of hESCs inducing differentiation towards specific cell lineages. FOXO4, an insulin/IGF-1 responsive transcription factor associated with long lifespan in invertebrates6,7, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Our results establish a novel regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates with hESC function and identity. PMID:22972301
Leeb, C; Jurga, M; McGuckin, C; Forraz, N; Thallinger, C; Moriggl, R; Kenner, L
Although stem cell research is a rather new field in modern medicine, media soon popularized it. The reason for this hype lies in the potential of stem cells to drastically increase quality of life through repairing aging and diseased organs. Nevertheless, the essence of stem cell research is to understand how tissues are maintained during adult life. In this article, we summarize the various types of stem cells and their differentiation potential in vivo and in vitro. We review current clinical applications of stem cells and highlight problems encountered when going from animal studies to clinical practice. Furthermore, we describe the current state of induced pluripotent stem cell technology and applications for disease modelling and cell replacement therapy. © 2011 Blackwell Publishing Ltd.
A wide variety of cell and tissue types that are sought in regenerative medicine can be generated from embryonic stem cells (ESCs), and currently two derivatives of human embryonic stem cells (hESCs) have entered human clinical trials. However, the ethical controversy surrounding this technology, which uses preimplantation human embryos to generate cell lines, is limiting research and the development of new therapies. Several new technologies such as induced pluripotent cells or parthenogenetically derived pluripotent cells hold great promise, but more research is needed before their derivatives can be proven to be safe and functional for use in human patients. The blastomere biopsy-based technique allows the derivation of human ESClines without sacrificing a human embryo and was shown to be robust and produce safe and functional derivatives of therapeutic value.
Kerman, Bilal E.; Kim, Hyung Joon; Padmanabhan, Krishnan; Mei, Arianna; Georges, Shereen; Joens, Matthew S.; Fitzpatrick, James A. J.; Jappelli, Roberto; Chandross, Karen J.; August, Paul; Gage, Fred H.
Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation. PMID:26015546
Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert
Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.
Cavazzana-Calvo, Marina; André-Schmutz, Isabelle; Lagresle, Chantal; Fischer, Alain
In the last 30 years, allogeneic bone marrow transplantation has become the treatment of choice for many hematologic malignancies or inherited disorders and a number of changes have been registered in terms of long-term survival rate of transplanted patients as well as of available sources of hematopoietic stem cell (HSC). In parallel to the publication of better results in HSC transplantation, several recent discoveries have opened a scientific and ethical debate on the therapeutical potential of stem cells isolated from adult or embryonic tissues. One of the major discoveries in this field is the capacity of bone marrow-derived stem cells to treat a genetic liver disease in a mouse model, thus justifying the concept of transdifferentiation of adult stem cell and raising hopes on its possible therapeutical applications. We have tried here to summarise the advances in this field and to discuss the limits of these biological data.
Merrill, Bradley J
Embryonic stem cells (ESCs) can generate all of the cell types found in the adult organism. Remarkably, they retain this ability even after many cell divisions in vitro, as long as the culture conditions prevent differentiation of the cells. Wnt signaling and β-catenin have been shown to cause strong effects on ESCs both in terms of stimulating the expansion of stem cells and stimulating differentiation toward lineage committed cell types. The varied effects of Wnt signaling in ESCs, alongside the sometimes unconventional mechanisms underlying the effects, have generated a fair amount of controversy and intrigue regarding the role of Wnt signaling in pluripotent stem cells. Insights into the mechanisms of Wnt function in stem cells can be gained by examination of the causes for seemingly opposing effects of Wnt signaling on self-renewal versus differentiation.
Ghosh, Dhimankrishna; Yan, Xiaowei; Tian, Qiang
Embryonic stem cells (ESCs) are endowed with the ability to generate multiple cell lineages and carries great therapeutic potentials in regenerative medicines. Future application of ESCs in human health and diseases will embark on the delineation of molecular mechanisms that define the biology of ESCs. Here we discuss how the finite ESC components mediate the intriguing task of brain development and exhibits biomedical potentials to cure diverse neurological disorders. PMID:19530135
O'Rourke, P Pearl; Abelman, Melinda; Heffernan, Kate Gallin
Centralized banking of human embryonic stem (hES) cells is an endeavor that can benefit individual research efforts and enhance international collaboration but is complicated by the fact that the science is rapidly evolving in an environment of heterogeneous laws, guidelines, and ethical standards. Written from the vantage point of regulatory professionals, this article provides an overview of the benefits of and challenges facing hESC banking enterprises in general with a focus on a global centralized banking effort.
Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H
The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.
Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.
The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications. PMID:25961451
Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira
Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.
Manzar, Nabeel; Manzar, Bushra; Hussain, Nuzhat; Hussain, M Fawwad Ahmed; Raza, Sajjad
To determine the knowledge, attitude, and ethical concerns of medical students and graduates with regard to Embryonic Stem Cell (ESC) research. This questionnaire based descriptive study was conducted at the Civil Hospital Karachi (CHK), Pakistan from February to July 2008. A well structured questionnaire was administered to medical students and graduate doctors, which included their demographic profile as well as questions in line with the study objective. Informed consent was taken and full confidentiality was assured to the participants. Data were entered in a Statistical Package for Social Sciences (SPSS version.12) and analyzed. A total of 204 male and 216 female medical students and doctors were administered questionnaires out of which 105 males (51.4%) and 108 females (50%) were aware of the embryonic stem cell research and its ethical implications. Forty percent males and 47% of females were of the opinion that life begins at conception. Forty-six percent males and 39% females were in favor of stem cell research while only 31% males and 28% females supported the ESC research. Less than 1/3 of students supported using frozen embryos for research purposes while more than 2/3 indicated that they were unlikely to support abortion for stem cell research purposes. The majority of the students were in favor of stem cell research with some reservations regarding ESC research. A sizeable number of students withheld their views, reflecting their poor understanding of medical ethics. The result of the study indicates a need for incorporating bioethics into the medical curriculum.
Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells. PMID:28373885
Acosta, Nefi D; Golub, Sidney H
Stem cell policy in the United States is an amalgam of federal and state policies. The scientific development of human pluripotent embryonic stem cells (ESCs) triggered a contentious national stem cell policy debate during the administration of President George W. Bush. The Bush "compromise" that allowed federal funding to study only a very limited number of ESC derived cell lines did not satisfy either the researchers or the patient advocates who saw great medical potential being stifled. Neither more restrictive legislation nor expansion of federal funding proved politically possible and the federal impasse opened the door for a variety of state-based experiments. In 2004, California became the largest and most influential state venture into stem cell research by passing "Prop 71," a voter initiative that created a new stem cell agency and funded it with $3 billion. Several states followed suit with similar programs to protect the right of investigators to do stem cell research and in some cases to invest state funding in such projects. Other states devised legislation to restrict stem cell research and in five states, criminal penalties were included. Thus, the US stem cell policy is a patchwork of multiple, often conflicting, state and federal policies. © 2016 American Society of Law, Medicine & Ethics.
Oottamasathien, Siam; Wang, YongQing; Williams, Karin; Franco, Omar E.; Wills, Marcia L.; Thomas, John C.; Saba, Katrina; Sharif-Afshar, Ali-Reza; Makari, John H.; Bhowmick, Neil A; DeMarco, Romano T.; Hipkens, Susan; Magnuson, Mark; Brock, John W.; Hayward, Simon W.; Pope, John C.; Matusik, Robert J.
Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies. PMID:17289017
Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T.; Pan, Chenyi; McDevitt, Todd C.; Fan, Yuhong
Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes. PMID:22589736
Wainwright, Steven P; Williams, Clare; Michael, Mike; Farsides, Bobbie; Cribb, Alan
Most accounts of the ethics of stem cell research are de- contextualised reviews of the ethical and legal literature. In this chapter we present a socially embedded account of some of the ethical implications of stem cell research, from the perspectives of scientists directly involved in this area. Based on an ethnography of two leading embryonic stem cell laboratories in the UK, our data form part of the findings from a larger project mapping the scientific, medical, social and ethical dimensions of innovative stem cell treatment, focusing on the areas of liver cell and pancreatic islet cell transplantation. We explore three key issues: what individual scientists themselves view as ethical sources of human embryos and stem cells; their perceptions of human embryos and stem cells; and how scientists perceive regulatory frameworks in stem cell research. We argue that these dimensions of laboratory practice are all examples of 'ethical boundary-work', which is becoming an integral part of the routine practice and performance of biomedical science. Our work adds to the relatively few sociological studies that explore ethics in clinical settings and to an even smaller body of work that explores scientists' views on the ethical issues relating to their research.
Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy discussions relating to their establishment or maintenance. It is argued (1) that 'ethical arguments' are often more important in the establishment phase and 'efficiency arguments' more important in the maintenance phase, and (2) that arguments relating to the interests of embryo and gamete donors are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being ignored in the policy making process.
Singla, Dinender K
Cell transplantation is a subject of fast-growing research with a potential of a therapeutic approach for the treatment of heart diseases. Clinical applications require preparation of large number of donor cells. Stem cell studies published to date demonstrate that scientists have not reached the general consensus to use an optimal cell type for better cardiac repair and regeneration. We used embryonic stem (ES) cells and their released factors for cardiac repair and regeneration. The major concern of cardiac regeneration with stem cells includes engraftment, differentiation, and teratoma formation after ES cell transplantation. Our current knowledge of ES cell transplantation in the heart is very limited. This review discusses the use of various growth factors to enhance ES cells engraftment and differentiation, as well as the issue of teratoma formation.
Li, X.; Zhao, L.; Oh, Steve K. W.; Chong, W. K.; Ong, J. K.; Chen, Allen K.; Choo, Andre B. H.
A measurement and imaging system has been developed for in-line continuous measurement of live, unmodified, human embryonic stem cells (hESC). The measurement will not affect cell growth, structure, sterility and suitability for clinical use. The stem cell imaging system (SCIS) can be used to support the optimization of automated stem cell growth for invitro study and for high-volume bio-manufacture. This paper present the experimental and analysis for the optimization of system parameters. A non-linear lighting is developed to obtain a clear images. The individual cluster can be traced from day one to day two. The whole system is calibrated with measurement microscope and haemacytometer. The measurement accuracy is better than 90%.
While the argument of complicity is only rarely discussed in bioethics, it is of obvious relevance to the issue of imported embryonic stem cells in countries in which the derivation of stem cells from early human embryos is legally prohibited and/or morally rejected. Complicity means that making use of the results or products of an illegal or morally problematic activity is itself morally problematic, although generally to a lesser degree than the original activity. The question arises as to which conditions make the argument of complicity plausible, thus supporting attacks against legislation that aims to promote research based on 'fruits of a forbidden tree'. This paper distinguishes a number of different variants of complicity, proposes that they deserve different kinds of moral valuation and applies the results to the ongoing debate about the German stem cell research law.
Wang, Xiaoying; Li, Shenglin; Cao, Tong; Fu, Xin; Yu, Guangyan
To investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from human embryonic stem cells (H9) via embryonic body (EB) and treated with Sodium fluoride (NaF) and Formaldehyde (FA). Proliferation, specific gene and protein expression and karyotype of cells were analyzed by MTT assay, RT-PCR, immunocytochemistry and karyotype analysis, respectively. Cytotoxicity was detected by MTT assay and flow cytometry, and genotoxicity was studied by micronucleus test (MNT), sister chromatid exchange (SCE) and comet assay. EBf-H9s were spindle-shaped with a diploid karyotype. They expressed the fibroblast markers prolyl 4-hydroxylase β and vimentin but did not express Oct-4 and Sox-2, and decreased expression of Nanog. The proliferation of EBf-H9 and murine L929 cells was inhibited by sodium fluoride (NaF) and formaldehyde (FA), and the cell cycle was arrested in different phases with the treatments. In genotoxicity assays with NaF and FA, positive responses were detected in human EBf-H9s comparable to those in the murine L929 cell line. EBf-H9 may be a suitable new cell source for toxicity research on biomaterials and other agents. Copyright © 2012 Elsevier Ltd. All rights reserved.
Wei, Quan-Xiang; van der Hoeven, Franciscus; Hollstein, Monica; Odell, Adam F
This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.
Amoh, Yasuyuki; Katsuoka, Kensei; Hoffman, Robert M
Multipotent adult stem cells have many potential therapeutic applications. Our recent findings suggest that hair follicles are a promising source of easily accessible multipotent stem cells. Stem cells in the hair follicle area express the neural stem cell marker nestin, suggesting that hair-follicle stem cells and neural stem cells have common features. Nestin-expressing hair follicle stem cells can form neurons and other cell types, and thus adult hair follicle stem cells could have important therapeutic applications, particularly for neurologic diseases. Transplanted hair follicle stem cells promote the functional recovery of injured peripheral nerve and spinal cord. Recent findings suggest that direct transplantation of hair-follicle stem cells without culture can promote nerve repair, which makes them potentially clinically practical. Human hair follicle stem cells as well as mouse hair follicle stem cells promote nerve repair and can be applied to test the hypothesis that human hair follicle stem cells can provide a readily available source of neurologically therapeutic stem cells. The use of hair follicle stem cells for nerve regeneration overcomes critical problems of embryonic stem cells or induced pluripotent stem cells in that the hair follicle stem cells are multipotent, readily accessible, non-oncogenic, and are not associated with ethical issues.
Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat
Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.
Hertsenberg, Andrew J.; Funderburgh, James L.
Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. PMID:26026882
Zhao, Xiaoyang; Lv, Zhuo; Liu, Lei; Wang, Liu; Tong, Man; Zhou, Qi
Knockout Brown Norway (BN) rat could be a useful disease model for human disorders, however, a failure to derive embryonic stem (ES) cells disturbs the further development of the model. In this study, we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley (SD) rats. The BN rat ES cells expressed the key transcription factors, and were able to form embryonic bodies (EBs) when being differentiated in vitro. After injecting the BN rat ES cells into the SD rat blastocysts, high-contribution chimeric rats were generated and could survive to their adulthood. Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.
Kovacs, Gergo; Szabo, Viktoria; Pirity, Melinda K.
Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp +/+) and rybp null mutant (rybp −/−) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found that rybp null mutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack of rybp coincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes. PMID:26788067
Dai, Wangde; Kloner, Robert A
Embryonic stem cells are a promising source for myocardial regeneration due to their pluripotency and plasticity. In theory, embryonic stem cells are capable of self-renewal in an unlimited fashion, and can differentiate into any cell type required for cell-based therapy, including cardiac myocytes. In recent years, embryonic stem cells have been transplanted for cardiac regeneration in animal models, and the results are encouraging. However, there are still many hurdles to be overcome for the clinical application of embryonic stem cells.
Jiang, Yuanda; Hang, Haiying
There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG. PMID:22216215
Panzica-Kelly, Julieta M; Brannen, Kimberly C; Ma, Yan; Zhang, Cindy X; Flint, Oliver P; Lehman-McKeeman, Lois D; Augustine-Rauch, Karen A
The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values < 22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.
Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Gariglio, Patricio
The wide range of invasive and noninvasive lesion phenotypes associated with high-risk human papillomavirus (HR-HPV) infection in cervical cancer (CC) indicates that not only the virus but also specific cervical epithelial cells in the transformation zone (TZ), such as stem cells (SCs), play an important part in the development of cervical neoplasia. In this review, we focused in an expression signature that is specific to embryonic SCs and to poorly differentiated cervical malignant tumors and we hypothesize that this expression signature may play an important role to promote cell growth, survival, colony formation, lack of adhesion, as well as cell invasion and migration in CC.
Sumer, Huseyin; Verma, Paul J
Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.
Kawahara, Yumi; Manabe, Tomotaka; Matsumoto, Masaya; Kajiume, Teruyuki; Matsumoto, Masayasu; Yuge, Louis
Background Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. Methodology/Principal Findings We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. Conclusions/Significance Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells. PMID:19626124
Ishiuchi, Takashi; Torres-Padilla, Maria-Elena
Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.
Collas, Philippe; Noer, Agate; Timoskainen, Sanna
In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state.
Murray, Patricia; Edgar, David
The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis. PMID:15306413
Guenther, Matthew G
Embryonic stem cells (ESCs) have the potential to generate virtually any cell type or tissue type in the body. This remarkable plasticity has yielded great interest in using these cells to understand early development and in treating human disease. In an effort to understand the basis of ESC pluripotency, genetic and genomic studies have revealed transcriptional regulatory circuitry that maintains the pluripotent cell state and poises the genome for downstream activation. Critical components of this circuitry include ESC transcription factors, chromatin regulators, histone modifications, signaling molecules and regulatory RNAs. This article will focus on our current understanding of these components and how they influence ESC and induced pluripotent stem cell states. Emerging themes include regulation of the pluripotent genome by a core set of transcription factors, transcriptional poising of developmental genes by chromatin regulatory complexes and the establishment of multiple layers of repression at key genomic loci.
Baizabal, José-Manuel; Covarrubias, Luis
Specific neuronal differentiation of Embryonic Stem Cells (ESCs) depends on their capacity to interpret environmental cues. At present, it is not clear at which stage of differentiation ESCs become competent to produce multiple neuronal lineages in response to the niche of the embryonic brain. To unfold the developmental potential of ESC-derived precursors, we transplanted these cells into the embryonic midbrain explants, where neurogenesis occurs as in normal midbrain development. Using this experimental design, we show that the transition from ESCs to Embryoid Body (EB) precursors is necessary to differentiate into Lmx1a(+)/Ptx3(+)/TH(+) dopaminergic neurons around the ventral midline of the midbrain. In addition, EB cells placed at other dorsal-ventral levels of the midbrain give rise to Nkx6.1(+) red nucleus (RN) neurons, Nkx2.2(+) ventral interneurons and Pax7(+) dorsal neurons at the correct positions. Notably, differentiation of ESCs into Neural Precursor Cells (NPCs) prior to transplantation markedly reduces specification at the Lmx1a, Nkx6.1 and Pax7 expression domains, without affecting neuronal differentiation. Finally, exposure to Fgf8 and Shh in vitro promotes commitment of some ESC-derived NPCs to differentiate into putative Lmx1a(+) dopaminergic neurons in the midbrain. Our data demonstrate intrinsic developmental potential differences among ESC-derived precursor populations.
Macfarlan, Todd S; Gifford, Wesley D; Driscoll, Shawn; Lettieri, Karen; Rowe, Helen M; Bonanomi, Dario; Firth, Amy; Singer, Oded; Trono, Didier; Pfaff, Samuel L
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.
Ding, Hong; Xu, Xin; Qin, Xian; Yang, Chengjian; Feng, Qiuting
Embryonic stem cells (ESCs) are capable to differentiate into cardiomyocytes, with the potential to treat cardiovascular diseases. However, directed differentiation is still a challenge faced by scientists. As a natural substance in grapes, resveratrol (RV) is important for cardiovascular protection. The studies of RV and its effects on ESC differentiation have potential clinical applications. Using mouse embryonic stem cells (mESCs), we investigated the effects of different concentrations of RV (5, 10, 20, 50, and 100 μmol/L) exposure on mESCs viability, expression levels of cardiac marker genes in embryoid bodies (EBs) derived from mESCs, expression levels of maturity indicative cardiac markers in cardiomyocytes derived from mESCs, and the beating properties of EBs. About 10 μmol/L of RV showed no toxicity on cell viability and was the optimal concentration to promote mESC differentiation, induce mESC differentiation to cardiomyocytes, and gain the beating properties of EBs. RV can successfully direct the differentiation of mESCs into cardiomyocytes, shedding light on its future applications to treat cardiovascular diseases. © 2016 John Wiley & Sons Ltd.
Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J.
The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. PMID:26890252
Shekari, Faezeh; Nezari, Hossein; Larijani, Mehran Rezaei; Han, Chia-Li; Baharvand, Hossein; Chen, Yu-Ju; Salekdeh, Ghasem Hosseini
As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells. Copyright © 2017. Published by Elsevier B.V.
The experience accumulated in cardiac cell therapy suggests that regeneration of extensively necrotic myocardial areas is unlikely to be achieved by the sole paracrine effects of the grafted cells but rather requires the conversion of these cells into cardiomyocytes featuring the capacity to substitute for those which have been irreversibly lost. In this setting, the use of human pluripotent embryonic stem cells has a strong rationale. The experimental results obtained in animal models of myocardial infarction are encouraging. However, the switch to clinical applications still requires to address some critical issues, among which optimizing cardiac specification of the embryonic stem cells, purifying the resulting progenitor cells so as to graft a purified population devoid from any contamination by residual pluripotent cells which carry the risk of tumorigenesis and controlling the expected allogeneic rejection by clinically acceptable methods. If the solution to these problems is a pre-requisite, the therapeutic success of this approach will also depend on the capacity to efficiently transfer the cells to the target tissue, to keep them alive once engrafted and to allow them to spatially organize in such a way that they can contribute to the contractile function of the heart.
The experience accumulated in cardiac cell therapy suggests that regeneration of extensively necrotic myocardial areas is unlikely to be achieved by the sole paracrine effects of the grafted cells but rather requires the conversion of these cells into cardiomyocytes featuring the capacity to substitute for those which have been irreversibly lost. In this setting, the use of human pluripotent embryonic stem cells has a strong rationale. The experimental results obtained in animal models of myocardial infarction are encouraging. However, the switch to clinical applications still requires to address some critical issues, among which the optimization of the cardiac specification of the embryonic stem cells, the purification of the resulting progenitor cells so as to graft a purified population devoid from any contamination by residual pluripotent cells which carry the risk of tumorigenesis, and the control of the expected allogeneic rejection by clinically acceptable methods. If the solution to these problems is a prerequisite, the therapeutic success of this approach will also depend on the capacity to efficiently transfer the cells to the target tissue, to keep them alive once engrafted, and to allow them to spatially organize in such a way that they can contribute to the contractile function of the heart.
Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser
Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.
Willems, Erik; Bushway, Paul J.
Debilitating cardiomyocyte loss underlies the progression to heart failure. Although there have been significant advances in treatment, current therapies are intended to improve or preserve heart function rather than regenerate lost myocardium. A major hurdle in implementing a cell-based regenerative therapy is the inefficient differentiation of cardiomyocytes from either endogenous or exogenous stem cell sources. Moreover, cardiomyocytes that develop in human embryonic stem cell (hESC) or human-induced pluripotent stem cell (hIPSC) cultures are comparatively immature, even after prolonged culture, and differences in their calcium handling, ion channel, and force generation properties relative to adult cardiomyocytes raise concerns of improper integration and function after transplantation. Thus, the discovery of natural and novel small molecule synthetic regulators of differentiation and maturation would accelerate the development of stem-cell-based myocardial therapies. Here, we document recent advances in defining natural signaling pathways that direct the multistep cardiomyogenic differentiation program and the development of small molecules that might be used to enhance differentiation as well as the potential characteristics of lead candidates for pharmaceutical stimulation of endogenous myocardial replacement. PMID:19319460
Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng
The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.
Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio
The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. PMID:28068409
Zhou, Shufeng; Abdouh, Mohamed; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio
The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells.
Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combin...
The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...
The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...
Fins, Joseph J; Schachter, Madeleine
This essay considers the implications of President George W. Bush's proposal for human embryonic stem cell research. Through the perspective of patent law, privacy, and informed consent, we elucidate the ongoing controversy about the moral standing of human embryonic stem cells and their derivatives and consider how the inconsistencies in the president's proposal will affect clinical practice and research.
The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES ...
The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES ...
Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combin...
Jain, Neha; Lee, Eun Jung
The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes.
Dormeyer, Wilma; van Hoof, Dennis; Braam, Stefan R; Heck, Albert J R; Mummery, Christine L; Krijgsveld, Jeroen
Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosomal abnormalities in culture are essentially indistinguishable from hECC. Direct comparison of karyotypically normal hESCs with hECCs could lead to understanding differences between their mechanisms of growth control and contribute to implementing safe therapeutic use of stem cells without the development of germ cell cancer. While several comparisons of hECCs and hESCs have been reported, their cell surface proteomes are largely unknown, partly because plasma membrane proteomics is still a major challenge. Here, we present a strategy for the identification of plasma membrane proteins that has been optimized for application to the relatively small numbers of stem cells normally available, and that does not require tedious cell fractionation. The method led to the identification of 237 and 219 specific plasma membrane proteins in the hESC line HUES-7 and the hECC line NT2/D1, respectively. In addition to known stemness-associated cell surface markers like ALP, CD9, and CTNNB, a large number of receptors, transporters, signal transducers, and cell-cell adhesion proteins were identified. Our study revealed that several Hedgehog and Wnt pathway members are differentially expressed in hESCs and hECCs including NPC1, FZD2, FZD6, FZD7, LRP6, and SEMA4D, which play a pivotal role in stem cell self-renewal and cancer growth. Various proteins encoded on chromosome 12p, duplicated in testicular cancer, were uniquely identified in hECCs. These included GAPDH, LDHB, YARS2, CLSTN3, CSDA, LRP6, NDUFA9, and NOL1, which are known to be upregulated in testicular cancer. Distinct HLA molecules were revealed on the surface of hESCs and hECCs, despite their low abundance. Results were
Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota
Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.
Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang
In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.
Blomberg, L A; Telugu, B P V L
Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that an ESC can maintain an undifferentiated state indefinitely, self-renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the terminally differentiated cells of a conceptus. These attributes make the ESC an ideal source for genome editing for both agricultural and biomedical applications. Although, ESC lines have been successfully established from rodents and primates, authentic ungulate stem cell lines on the contrary are still not available. Outstanding issues including but not limited to differences in pluripotency characteristics among the existing ESC lines, pre-implantation embryo development, pluripotency pathways, and culture conditions plague our efforts to establish authentic ESC lines from farm animals. In this review, we highlight some of these issues and discuss how the recent derivation of induced pluripotent stem cells (iPSCs) might augur the establishment of robust authentic ESC lines from farm animals. © 2012 This article is a U.S. Government work and is in the public domain in the USA.
Garcia-Alegria, Eva; Iluit, Maria; Stefanska, Monika; Silva, Claudio; Heeg, Sebastian; Kimber, Susan J.; Kouskoff, Valerie; Lacaud, Georges; Vijayaraghavan, Aravind; Batta, Kiran
Pluripotent stem cells represent a promising source of differentiated tissue-specific stem and multipotent progenitor cells for regenerative medicine and drug testing. The realisation of this potential relies on the establishment of robust and reproducible protocols of differentiation. Several reports have highlighted the importance of biomaterials in assisting directed differentiation. Graphene oxide (GO) is a novel material that has attracted increasing interest in the field of biomedicine. In this study, we demonstrate that GO coated substrates significantly enhance the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. GO does not affect cell proliferation or survival of differentiated cells but rather enhances the transition of haemangioblasts to haemogenic endothelial cells, a key step during haematopoietic specification. Importantly, GO also improves, in addition to murine, human ES cell differentiation to blood cells. Taken together, our study reveals a positive role for GO in haematopoietic differentiation and suggests that further functionalization of GO could represent a valid strategy for the generation of large numbers of functional blood cells. Producing these cells would accelerate haematopoietic drug toxicity testing and treatment of patients with blood disorders or malignancies. PMID:27197878
Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi
There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal's ureter to the cloacal-developed bladder, a technique we called the "stepwise peristaltic ureter" (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.
Lorincz, Matthew T
Nine neurodegenerative disorders are caused by CAG/polyglutamine (polyQ) repeat expansions. The underlying molecular mechanisms responsible for disease specific neurodegeneration remain elusive. In vivo and in vitro models utilizing rodent tissues, immortalized human cell lines, and human post mortem samples have provided insight into disease mechanisms. Concern that cellular and molecular processes observed in these models may not faithfully reproduce human diseases or be useful to identify compounds of therapeutic utility has driven development of new disease models. In addition to their therapeutic potential, stem cells represent a renewable source of tissue that can be directed into neurons and glia and can be used to study neurodegenerative cascades from their inception. Neuronally differentiated human stem cells containing CAG repeat expansions have the potential to accurately replicate human CAG repeat diseases and may be a faithful predictor of which compounds will be of human benefit. As a first step in development of this type of model, we developed murine embryonic stem cell models to study the mechanisms of polyQ tract induced neuronal degeneration.
Risner-Janiczek, Jessica R.; Ungless, Mark A.; Li, Meng
In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation. PMID:21887381
Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.
Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453
Garcia-Alegria, Eva; Iluit, Maria; Stefanska, Monika; Silva, Claudio; Heeg, Sebastian; Kimber, Susan J; Kouskoff, Valerie; Lacaud, Georges; Vijayaraghavan, Aravind; Batta, Kiran
Pluripotent stem cells represent a promising source of differentiated tissue-specific stem and multipotent progenitor cells for regenerative medicine and drug testing. The realisation of this potential relies on the establishment of robust and reproducible protocols of differentiation. Several reports have highlighted the importance of biomaterials in assisting directed differentiation. Graphene oxide (GO) is a novel material that has attracted increasing interest in the field of biomedicine. In this study, we demonstrate that GO coated substrates significantly enhance the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. GO does not affect cell proliferation or survival of differentiated cells but rather enhances the transition of haemangioblasts to haemogenic endothelial cells, a key step during haematopoietic specification. Importantly, GO also improves, in addition to murine, human ES cell differentiation to blood cells. Taken together, our study reveals a positive role for GO in haematopoietic differentiation and suggests that further functionalization of GO could represent a valid strategy for the generation of large numbers of functional blood cells. Producing these cells would accelerate haematopoietic drug toxicity testing and treatment of patients with blood disorders or malignancies.
Germany since 1990 has one of the strictest human embryo protection laws, yet according to the Stem Cell Act of 2002 allows, under strict conditions, the import and use of human embryonic stem cells (hESC) for high priority research goals. The author tries to show how this is taken to be coherent by the parliamentary majority (though not necessarily by the general public) in Germany. In doing so, he firstly looks into the chronicle of the debate in Germany showing its different stages since 1999, then dwells upon the relation between the law and the role of ethics in this issue, and thirdly presents the two fundamentally different positions of the German debate, that is, that the human embryo created for IVF purposes is a human being and stands from its very beginnings under the constitutional principles of respect for, and protection of, human life versus the position that before being implanted the human embryo may become a human being and therefore belongs to the human species only potentially, so that its right to life protection may be assessable over against other high priority goals, such as research aiming at possible help for patients with life-endangering diseases. In spite of the Stem Cell Act of 2002, the debate of the German general public goes on, especially due to the recent EU 6th Research Framework Program which plans to also fund hESC research.
Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi
There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney. PMID:26392557
... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in...
Sherley, J L
Misinformation erodes the legitimacy of any public debate. Since the start of human embryonic stem cell research deliberations in the USA, misinformation concerning the nature of human embryos, their availability for research, and the potential for using them to develop new medical therapies have been widespread and persistent. Basic facts, well understood by physicians and biologists, have been so misstated and misrepresented in the news media and political speeches that the general public has been put in a state of constant uncertainty. The solution to the present troubling condition is better education in the form of diligent, honest, and complete scientific disclosure by responsible scientists and physicians; and more care given to accurate reporting by news media. Several key aspects of newly emerging embryonic and non-embryonic stem cell technologies are defined and discussed as they relate to the debate over the use of human embryos for medical research. An important topic for consideration is how to disclose with clarity the scientific basis for human embryonic life. Thereafter, failings in proposed technologies for developing new therapies with human embryonic stem cells, that have been grossly under-reported, are examined. Finally, properties of adult stem cells are presented in contradistinction to embryonic stem cells, both in terms of adult stem cells as a scientifically better alternative to embryonic stem cells and in terms of the technological challenges that must be overcome to realize the potential of adult stem cells for new medical therapies.
De Trizio, Ella; Brennan, Christopher S
Few sciences have held out such therapeutic promise and correspondingly stirred so much controversy in countries throughout the world as the developing science surrounding human embryonic stem cells. Since the first reported development of several lines of human embryonic stem cells in 1988, many governments around the world have attempted to address the thorny ethical issues raised by human embryonic stem cell research by the passage of laws. In some cases these laws have directly regulated governmental funding of the science; in other cases they have created a legal environment that has either encouraged or discouraged both governmental and private funding of the science. This article first differentiates human embryonic stem cells from other types of stem cells and frames the ethical controversy surrounding human embryonic stem cell research, then surveys laws governing human embryonic stem cell research in various scientifically advanced countries located throughout the Pacific Rim, Europe and North America and explains the impact these laws have had on governmental and private funding of human embryonic stem cell research.
Pagliara, Stefano; Franze, Kristian; McClain, Crystal R; Wylde, George W; Fisher, Cynthia L; Franklin, Robin J M; Kabla, Alexandre J; Keyser, Ulrich F; Chalut, Kevin J
Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.
Tichy, Elisia D. Stambrook, Peter J.
Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.
Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E
Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.
Embryonic stem cells provide a potential resource for research and drug screening. To make such a resource feasible, it is necessary to generate cells of sufficient quality and quantity. The challenge is to expand cell numbers while maintaining the fidelity of phenotype and to control and direct differentiation to produce the cell type of interest in a format that is suitable for drug screening. At present, large-scale culturing of human ES cell lines is problematic and provides substantial challenges. This article provides an overview of current bioprocessing techniques that could be used to generate cells for drug discovery applications. This will generate further technical expertise that can be applied in the production of cells for potential therapeutic applications.
Takebe, Naoko; Ivy, S Percy
Selectively targeting cancer stem cells (CSC) or tumor-initiating cells (TIC; from this point onward referred to as CSCs) with novel agents is a rapidly emerging field of oncology. Our knowledge of CSCs and their niche microenvironments remains a nascent field. CSC's critical dependence upon self-renewal makes these regulatory signaling pathways ripe for the development of experimental therapeutic agents. Investigational agents targeting the Notch, Hedgehog, and Wnt pathways are currently in late preclinical development stages, with some early phase 1-2 testing in human subjects. This series of articles will provide an overview and summary of the current state of knowledge of CSCs, their interactive microenvironment, and how they may serve as important targets for antitumor therapies. We also examine the scope and stage of development of early experimental agents that specifically target these highly conserved embryonic signaling pathways.
Cell transplantation approaches offer the potential to promote regenerative growth of diseased hearts. It is well established that donor cardiomyocytes stably engraft into recipient hearts when injected directly into the myocardial wall. Moreover, the transplanted donor cardiomyocytes participate in a functional syncytium with the host myocardium. Thus, transplantation of donor cardiomyocytes resulted in at least partial restoration of lost muscle mass. It is also well established that embryonic stem (ES) cells differentiate into cells of ecto-, endo-, and mesodermal lineages when cultured under appropriate conditions in vitro. Robust cardiomyogenic differentiation was frequently observed in spontaneously differentiating ES cultures. Cellular, molecular and physiologic analyses indicated that ES-derived cells were bona fide cardiomyocytes, with in vitro characteristics typical for cells obtained from early stages of cardiac development. Thus, ES-derived cardiomyocytes constitute a viable source of donor cells for cell transplantation therapies. PMID:16370325
Saladi, Srinivas Vinod; de la Serna, Ivana L
Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure.
Kehat, Izhak; Khimovich, Leonid; Caspi, Oren; Gepstein, Amira; Shofti, Rona; Arbel, Gil; Huber, Irit; Satin, Jonathan; Itskovitz-Eldor, Joseph; Gepstein, Lior
Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.
Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.
Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.
Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.
A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.
Eckersley-Maslin, Mélanie A.; Thybert, David; Bergmann, Jan H.; Marioni, John C.; Flicek, Paul; Spector, David L.
Summary Random autosomal monoallelic gene expression refers to the transcription of a gene from one of two homologous alleles. We assessed the dynamics of monoallelic expression during development through an allele-specific RNA sequencing screen in clonal populations of hybrid mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We identified 67 and 376 inheritable autosomal random monoallelically expressed genes in ESCs and NPCs respectively, a 5.6-fold increase upon differentiation. While DNA methylation and nuclear positioning did not distinguish the active and inactive alleles, specific histone modifications were differentially enriched between the two alleles. Interestingly, expression levels of 8% of the monoallelically expressed genes remained similar between monoallelic and biallelic clones. These results support a model in which random monoallelic expression occurs stochastically during differentiation, and for some genes is compensated for by the cell to maintain the required transcriptional output of these genes. PMID:24576421
Amit, Michal; Itskovitz-Eldor, Joseph
Abstract Embryonic stem (ES) cells are unique cells derived from the inner cell mass of the mammalian blastocyst. These cells are immortal and pluripotent, retain their developmental potential after prolonged culture, and can be continuously cultured in an undifferentiated state. Many in vitro differentiation systems have been developed for mouse ES cells, including reproducible methods for mouse ES cell differentiation into haematopoietic and neural precursors, cardiomyocytes, insulin-secreting cells, endothelial cells and various other cell types. The derivation of new human ES cell lines provides the opportunity to develop unique models for developmental research and for cell therapies. In this review we consider the derivation and spontaneous differentiation of human ES cells. PMID:12033726
Sun, Yubing; Villa-Diaz, Luis G; Lam, Raymond H W; Chen, Weiqiang; Krebsbach, Paul H; Fu, Jianping
Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.
Sun, Yubing; Villa-Diaz, Luis G.; Lam, Raymond H. W.; Chen, Weiqiang; Krebsbach, Paul H.; Fu, Jianping
Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function. PMID:22615930
Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.
We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.
Abdelalim, Essam M
Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.
Wang, Jian; Alexander, Peter; Wu, Leeju; Hammer, Robert; Cleaver, Ondine; McKnight, Steven L.
Measurements of the abundance of common metabolites in cultured embryonic stem (ES) cells revealed an unusual state with respect to one-carbon metabolism. These findings led to the discovery of copious expression of the gene encoding threonine dehydrogenase (TDH) in ES cells. TDH-mediated catabolism of threonine takes place in mitochondria to generate glycine and acetyl–coenzyme A (CoA), with glycine facilitating one-carbon metabolism via the glycine cleavage system and acetyl-CoA feeding the tricarboxylic acid cycle. Culture media individually deprived of each of the 20 amino acids were applied to ES cells, leading to the discovery that ES cells are critically dependent on one amino acid—threonine. These observations show that ES cells exist in a high-flux backbone metabolic state comparable to that of rapidly growing bacterial cells. PMID:19589965
Nichols, Jennifer; Smith, Austin
Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.
Many who believe that human embryos have moral status are convinced that their use in human embryonic stem cell (hESC) research can be morally justified as long as they are discarded embryos left over from fertility treatments. This is one reason why this view about discarded embryos has played such a prominent role in the debate over publicly funding hESC research in the United States and other countries. Many believe that this view offers the best chance of a compromise between the different sides in this debate. This paper focuses on what seems to be the most plausible argument for this view about discarded embryos. It shows that this argument is unsound regardless of how one understands the claim that embryos have moral status. It also discusses the implications of this conclusion for attempts to use this argument as a basis for public policy.
This paper examines the debate on human embryonic stem cell (hES) research. In Japan, as in many Western countries, the moral status of the human embryo has been the main focus. There is a dichotomy between the advocates of research on hES cells and the advocates of the dignity of the embryos. Advocates of research on hES cells claim that the act of abandoning the embryo and the act of using it for research have the same moral status. Critics of research using embryos reject this position, claiming that the embryo has a status incommensurable with other values. This paper points out that the standpoint of the woman providing the embryos has been overlooked in this discussion.
Wang, Han; Luo, Xie; Leighton, Jake
Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study. PMID:26462244
Longworth-Mills, Emma; Koehler, Karl R; Hashino, Eri
This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage. After 16-20 days, vesicles containing inner ear sensory hair cells and supporting cells arise from the cultured aggregates. Aggregates may be analyzed using immunohistochemistry and electrophysiology techniques. This system serves as a simple and relatively inexpensive in vitro model of inner ear development.
Yu, Guang-yan; Cao, Tong; Ouyang, Hong-wei; Peng, Shuang-qing; Deng, Xu-liang; Li, Sheng-lin; Liu, He; Zou, Xiao-hui; Fu, Xin; Peng, Hui; Wang, Xiao-ying; Zhan, Yuan
The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.
Li, Wen-Zhong; Ai, Zhi-Ying; Wang, Zhi-Wei; Chen, Lin-Lin; Guo, Ze-Kun; Zhang, Yong
Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.
Macia, Angela; Muñoz-Lopez, Martin; Cortes, Jose Luis; Hastings, Robert K.; Morell, Santiago; Lucena-Aguilar, Gema; Marchal, Juan Antonio; Badge, Richard M.; Garcia-Perez, Jose Luis
Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated. PMID:21041477
Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra
Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.
Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra
Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705
Macia, Angela; Muñoz-Lopez, Martin; Cortes, Jose Luis; Hastings, Robert K; Morell, Santiago; Lucena-Aguilar, Gema; Marchal, Juan Antonio; Badge, Richard M; Garcia-Perez, Jose Luis
Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.
Ma, Risheng; Latif, Rauf; Davies, Terry F
The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be committed to thyroid cell speciation under
Mehta, Rajvi H
The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.
Unser, Andrea Mannarino
The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.
Mouse embryonic stem cells derive from the inner cell mass of the blastocyst and give rise to the three primitive embryonic layers, which later will form all the different tissue types of an adult. Embryonic stem cells are thus defined as totipotent cells. In vitro, these cells can give rise to all the somatic cells. Different laboratories have now shown that cultured embryonic stem cells can also differentiate into germline cells. By using the transcription factor Oct-4 as a tool for the visualization of germ cells, it has been shown the derivation of oocytes from mouse embryonic stem cells. These works should contribute to various areas, including therapeutic cloning which associates nuclear transfer and selective production of a specific cell type.
González, Sheyla; Ibáñez, Elena
Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536
Shinde, Vaibhav; Brungs, Sonja; Henry, Margit; Wegener, Lucia; Nemade, Harshal; Rotshteyn, Tamara; Acharya, Aviseka; Baumstark-Khan, Christa; Hellweg, Christine E; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios
Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs) under simulated microgravity within a fast-rotating clinostat (clinorotation) and capture of microarray-based gene signatures. The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g) after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs). Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The most significant simulated microgravity-affected genes, signal
Matveeva, Natalia M; Kizilova, Elena A; Serov, Oleg L
The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.
Radan, Lida; Hughes, Chris S.; Teichroeb, Jonathan H.; Zamora, Flora M. Vieira; Jewer, Michael; Postovit, Lynne-Marie
Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δβ hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms. PMID:24749509
Antonica, Francesco; Kasprzyk, Dominika Figini; Opitz, Robert; Iacovino, Michelina; Liao, Xiao-Hui; Dumitrescu, Alexandra Mihaela; Refetoff, Samuel; Peremans, Kathelijne; Manto, Mario; Kyba, Michael; Costagliola, Sabine
The primary function of thyroid gland is to metabolize iodide by synthesizing thyroid hormones that are critical regulators of growth, development and metabolism in virtually all tissues. To date, research on thyroid morphogenesis was missing an efficient stem-cell model system which allows to recapitulate in vitro the molecular and morphogenic events regulating thyroid follicular cells differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2.1 and PAX8 is sufficient to direct mouse embryonic stem-cells (mESC) differentiation into thyroid follicular cells which organized into three-dimensional follicular structures when treated with thyrotropin. Those in vitro derived follicles showed significant iodide organification activity. Importantly, when grafted in vivo into athyreoid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mESC can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue. PMID:23051751
Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.
Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499
Turksen, Kursad; Troy, Tammy-Claire
The isolation of pluripotent human embryonic stem (hES) cells having the capacity to differentiate in vitro to numerous cell types generated much excitement and promise in the field of regenerative medicine. However, along with great enthusiasm came hot controversy for stem cell research and researchers alike because available hES cell lines were isolated from "excess" embryos from in vitro fertilization clinics. Despite ethical and political debates, the methods and protocols to study diverse lineages are developing. Furthermore, strategies using specific growth factor combinations, cell-cell and cell-extracellular matrix induction systems are being explored for directed differentiation along a desired lineage. However, there is a great need to characterize the mechanisms that control self-renewal and differentiation and a necessity to improve methodologies and develop new purification protocols for the potential future clinical application of hES cells. After the scientific and political obstacles are overcome, it is anticipated that the hES cell field will make a tremendous difference in conditions, such as burn traumas and diabetic foot ulcers, as well a number of degenerative diseases such as Parkinson's disease, type 1 diabetes, rheumatoid arthritis, and myocardial infarction. In this introductory chapter, we will summarize and review recent progress in the field of hES cell differentiation protocols and discuss some of the current issues surrounding hES cell research.
Cohen, Netta Mendelson; Dighe, Vikas; Landan, Gilad; Reynisdóttir, Sigrún; Palsson, Arnar; Mitalipov, Shoukhrat; Tanay, Amos
DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.
Miszta-Lane, Helena; Mirbolooki, Mohammadreza; James Shapiro, A M; Lakey, Jonathan R T
Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.
The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...
The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...
Nairn, Alison V.; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Xie, Jin; Harris, Kyle; Dalton, Stephen; Kulik, Michael; Pierce, J. Michael; Toida, Toshihiko; Moremen, Kelley W.; Linhardt, Robert J.
Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of
Kallur, Therése; Blomberg, Pontus; Stenfelt, Sonya; Tryggvason, Kristian; Hovatta, Outi
For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).
Park, Siyeon; Im, Gun-Il
Tissue engineering for skeletal tissues including bone and cartilage have been focused on the use of adult stem cells. Although there are several pioneering researches on skeletal tissue regeneration from embryonic stem cells (ESCs), ethical issues and the possibility of immune rejection clouded further attention to the application of ESCs for nonlethal orthopedic conditions. However, the recent discovery of induced pluripotent stem cells (iPSCs) led to reconsider the use of these pluripotential cells for skeletal regeneration. The purpose of this review was to summarize the current knowledge of osteogenic and chondrogenic induction from ESCs and iPSCs and to provide a perspective on the application of iPSCs for skeletal regeneration.
Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E
Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.
Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much
Castro-Diaz, Nathaly; Ecco, Gabriela; Coluccio, Andrea; Kapopoulou, Adamandia; Yazdanpanah, Benyamin; Friedli, Marc; Duc, Julien; Jang, Suk Min; Turelli, Priscilla; Trono, Didier
Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with, at any given time, a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage, likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem (hES) cells, KAP1 (KRAB [Krüppel-associated box domain]-associated protein 1), the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI–piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether, these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected. PMID:24939876
Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta
Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.
Kuai, Xiao Ling; Gagliardi, Christine; Flaat, Mette; Bunnell, Bruce A.
The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14–16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), β-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and β-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, β-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen β-tublinIII (73±4.7%) and some expressed intermediate neuron antigen MAP2 (18±7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%±1.2%), GFAP (4.93±1.9%), S100 (7±3.5%) and O4 (0.2±70.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be
Patient: Male, 21 Final Diagnosis: Crohn’s disease Symptoms: Intolerance to specific foods • abdominal pain and diarrhea Medication: Human embryonic stem cell therapy Clinical Procedure: Human embryonic stem cell transplantation Specialty: Gastroenterology Objective: Unusual or unexpected effect of treatment Background: Crohn’s disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565 000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn’s disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn’s disease. Case Report: A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Conclusions: Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn’s disease. PMID:26923312
Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari
We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.
Latif, Rauf; Davies, Terry F.
Objective: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Methods: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Results: Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. Conclusion: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be
Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.
We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.
Cheng, Aixin; Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J; Hardingham, Timothy E; Kimber, Susan J
In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions.
Jensen, Janne; Hyllner, Johan; Björquist, Petter
Development of new drugs is costly and takes huge resources into consideration. The big pharmaceutical companies are currently facing increasing developmental costs and a lower success-rate of bringing new compounds to the market. Therefore, it is now of outmost importance that the drug-hunting companies minimize late attritions due to sub-optimal pharmacokinetic properties or unexpected toxicity when entering the clinical programs. To achieve this, a strong need to test new candidate drugs in assays of high human relevance in vitro as early as possible has been identified. The traditionally used cell systems are however remarkably limited in this sense, and new improved technologies are of greatest importance. The human embryonic stem cells (hESC) is one of the most powerful cell types known. They have not only the possibility to divide indefinitely; these cells can also differentiate into all mature cell types of the human body. This makes them potentially very valuable for pharmaceutical development, spanning from use as tools in early target studies, DMPK or safety assessment, as screening models to find new chemical entities modulating adult stem cell fate, or as the direct use in cell therapies. This review illustrates the use of hESC in the drug discovery process, today, as well as in a future perspective. This will specifically be exemplified with the most important cell type for pharmaceutical development-the hepatocyte. We discuss how hESC-derived hepatocyte-like cells could improve this process, and how these cells should be cultured if optimized functionality and usefulness should be achieved. J. Cell. Physiol. 219: 513-519, 2009. (c) 2009 Wiley-Liss, Inc.
Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J.; Hardingham, Timothy E.
In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions. PMID:25273540
Atkinson, Stuart P; Lako, Majlinda; Armstrong, Lyle
The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products that can be cost-prohibitive for large-scale production, and effective lineage-specific differentiation protocols also still remain relatively undefined and are inefficient at producing large amounts of cells for therapeutic use. Furthermore, the mechanisms and signalling pathways that mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large-scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents that can boost hESC pluripotency/self-renewal and survival and has greatly increased the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22515554
Renz, Peter F; Beyer, Tobias A
Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).
Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells. Conclusions The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further. PMID:22041030
Koch, Cody A; Jordan, Corinne E; Platt, Jeffrey L
The fetus has pluripotent stem cells that when transferred to mature individuals can generate tumors. However, for reasons yet unknown, tumors form rarely in the fetus and/or the mother during normal gestation. We questioned whether the complement system might protect against tumor formation by pluripotent stem cells. Murine embryonic stem cells were notably more susceptible than cardiomyocytes differentiated from those cells to lysis by complement in heterologous and homologous sera. Treatment of embryonic stem cells with heterologous serum averted tumor formation after residual cells were transplanted into mice. Confirming the importance of homologous complement in preventing formation of tumors, untreated embryonic stem cells formed tumors more quickly in C3-deficient than in wild-type mice. Susceptibility of embryonic stem cells to complement required an intact alternative pathway and was owed at least in part to a relative deficiency of sialic acid on cell surfaces compared with differentiated cells. Susceptibility to complement and resistance to tumors was inversely related to the number of cells transferred. These findings show that formation of tumors from embryonic stem cells is controlled in part by the alternative pathway of complement and suggest that susceptibility to complement might represent a general property of pluripotent stem cells that can be exploited to prevent tumor formation.
Kar, Srabani; Mitra, Shinjini; Banerjee, Ena Ray
Stem cells are cells capable of proliferation, self-renewal, and differentiation into specific phenotypes. They are an essential part of tissue engineering, which is used in regenerative medicine in case of degenerative diseases. In this chapter, we describe the methods of isolating and culturing various types of stem cells, like human embryonic stem cells (hESCs), human umbilical cord derived mesenchymal stem cells (hUC-MSCs), murine bone marrow derived mesenchymal stem cells (mBM-MSCs), murine adipose tissue derived mesenchymal stem cells (mAD-MSCs), and murine bone marrow derived dendritic cells (mBMDCs). All these cell types can be used in tissue engineering techniques.
Gauthaman, Kalamegam; Venugopal, Jayarama Reddy; Yee, Fong Chui; Peh, Gary Swee Lim; Ramakrishna, Seeram; Bongso, Ariff
Inadequate cell numbers in culture is one of the hurdles currently delaying the application of human embryonic stem cells (hESCs) for transplantation therapy. Nanofibrous scaffolds have been effectively used to expand and differentiate non-colony forming multipotent mesenchymal stem cells (MSC) for the repair of tissues or organs. In the present study, we evaluated the influence of nanofibrous scaffolds for hESC proliferation, increase in colony formation, self-renewal properties, undifferentiation and retention of ‘stemness’. Polycaprolactone/collagen (PCL/collagen) and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning technology. The hESCs were seeded on the nanofibrous scaffolds in the presence or absence of mitomycin-C treated mouse embryonic fibroblasts (MEFs). The hESCs grown on both scaffolds in the presence of the MEFs produced an increase in cell growth of 47.58% (P≤ 0.006) and 40.18% (P≤ 0.005), respectively, over conventional controls of hESCs on MEFs alone. The hESC colonies were also larger in diameter on the scaffolds compared to controls (PCL/collagen, 156.25 ± 7 μM and PCL/gelatin, 135.42 ± 5 μM). Immunohistochemistry of the hESCs grown on the nanofibrous scaffolds with MEFs, demonstrated positive staining for the various stemness-related markers (octamer 4 [OCT-4], tumour rejection antigen-1–60, GCTM-2 and TG-30), and semi-quantitative RT-PCR for the pluripotent stemness genomic markers (NANOG, SOX-2, OCT-4) showed that they were also highly expressed. Continued successful propagation of hESC colonies from nanofibrous scaffolds back to conventional culture on MEFs was also possible. Nanofibrous scaffolds support hESC expansion in an undifferentiated state with retention of stemness characteristics thus having tremendous potential in scaling up cell numbers for transplantation therapy. PMID:19228268
Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V; Bickmore, Wendy A; Brickman, Joshua M
Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene’s developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision. DOI: http://dx.doi.org/10.7554/eLife.14926.001 PMID:27723457
Munroe, Robert J.; Bergstrom, Rebecca A.; Zheng, Qing Yin; Libby, Brian; Smith, Richard; John, Simon W.M.; Schimenti, Kerry J.; Browning, Victoria L.; Schimenti, John C.
The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain1 and interlocus2 variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chi-maeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives. PMID:10700192
Lee, Jung Bok; Kim, Jin Mee; Kim, Sung Jong; Park, Jong Hyuk; Hong, Seok Ho; Roh, Sung Il; Kim, Moon Kyoo; Yoon, Hyun Soo
Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.
Paulose, Jiffin K; Rucker, Edmund B; Cassone, Vincent M
Recent attention on the early development of circadian rhythms has yielded several avenues of potential study regarding molecular and physiological rhythms in embryonic stem cells (ESCs) and their derivatives. While general guidelines of experimental design are-as always-applicable, there are certain idiosyncrasies with respect to experiments involving circadian rhythms that will be addressed. ESCs provide a number of challenges to the circadian biologist: growth rates are normally much higher than in established cell culture systems, the cells' innate drive towards differentiation and the lack of known synchronizing input pathways are a few examples. Some of these challenges can be addressed post hoc, such as normalization to total RNA or protein for transcript abundance studies. Most others, as outlined here, require special handling of the samples before and during experimentation in order to preserve any potential circadian oscillation that is present. Failure to do so may result in a disruption of endogenous oscillation(s) or, potentially worse, generation of an artificial oscillation that has no biological basis. This chapter begins with cultured ESCs, derived from primary blastocysts or in the form of cell lines, and outlines two methods of measuring circadian rhythms: the 2DG method of measuring glucose uptake (Sokoloff et al. J Neurochem 28:897-916, 1977) and real-time measurement of molecular rhythms using transgenic bioluminescence (Yoo et al. Proc Natl Acad Sci U S A 101:5339-5346, 2004).
Kalkan, Tüzer; Olova, Nelly; Roode, Mila; Mulas, Carla; Lee, Heather J; Nett, Isabelle; Marks, Hendrik; Walker, Rachael; Stunnenberg, Hendrik G; Lilley, Kathryn S; Nichols, Jennifer; Reik, Wolf; Bertone, Paul; Smith, Austin
Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
Perla, Venu; Perrin, Nancy A; Greenlee, Anne R
The objective of this in vitro study was to use a mouse embryonic stem (mES) cell model to better understand pesticide injury that may adversely affect early pregnancy and to evaluate an antioxidant intervention. Undifferentiated D3 mES cells were incubated 24h with control, reference dose (RfD), no observed effect level (NOEL), or lowest observed effect level (LOEL) of paraquat, a commonly used, toxic agricultural herbicide. Pesticide effects were evaluated at 0 and 24h using assays for cell proliferation, total reactive oxygen species (ROS), viability, and alkaline phosphatase activity. Compared to 0 h, cell proliferation increased significantly in the 24h control treatment and was stalled in all paraquat dilutions tested. ROS production and percent necrotic and apoptotic cells were significantly increased at all paraquat concentrations examined. Alkaline phosphatase activity suggested that cells remained undifferentiated during the study period. Experiments with ascorbic acid suggested that pesticide effects on cell viability and ROS production were minimized by the recommended daily allowance (RDA) of vitamin C. Data suggest pesticide-induced injury can occur very early in development and at concentrations predicted without health consequences. Mouse ES cells may provide a useful in vitro model for rapidly screening developmental toxicants and protective interventions.
Lu, Min; Lin, Reigh-Yi
Although TSH is the main regulator of thyroid growth and function, TSH binding activity in fat has long been reported. Since the TSH receptor (TSHR) has been detected in both preadipocytes and adipocytes, we hypothesized that it may play a role in adipose differentiation. Here, we use an in vitro model of adipogenesis from mouse embryonic stem (ES) cells to define TSH function. Directed differentiation of ES cells into the adipose lineage can be achieved over a 3-week period. Although adipocyte differentiation is initiated early in the development of cultured ES cells, TSHR up-regulation is precisely correlated with terminal differentiation of those adipocytes. The adipocytes express TSHR on the cell surface and respond to TSH with increased intracellular cAMP production, suggesting the activation of the protein kinase A signaling pathway. To determine whether TSH impacts adipogenesis, we examined how adipocytes responded to TSH at various points during their differentiation from cultured ES cells. We found that TSH greatly increases adipogenesis when added in the presence of adipogenic factors. More importantly, our data suggest that TSH also stimulates adipogenesis in cultured ES cells even in the absence of adipogenic factors. This finding provides the first evidence of TSH being a pro-adipogenic factor that converts ES cells into adipocytes. It further highlights the potential of ES cells as a model system for use in the study of TSH’s role in the regulation of physiologically relevant adipose tissue. PMID:18180327
Penkert, Rhiannon R; Kalejta, Robert F
Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.
Li, Xiajun; Leder, Philip
Background The mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood. Results To address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-Jkappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene. Conclusion Taken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells. PMID:17725840
Tsai, D F-C
Human embryonic stem cell research can bring about major biomedical breakthroughs and thus contribute enormously to human welfare, yet it raises serious moral problems because it involves using human embryos for experiment. The "moral status of the human embryo" remains the core of such debates. Three different positions regarding the moral status of the human embryo can be categorised: the "all" position, the "none" position, and the "gradualist" position. The author proposes that the "gradualist" position is more plausible than the other two positions. Confucius's moral principle of jen, which proposes a unique theory of "love of gradation", and the principle of yi, which advocates "due treatment for persons", are then explored. The author then argues that our moral obligations to do good to other living organisms, persons, and our families are different. Putting together the "gradualist" position on the human embryo, and Confucius's theories of "love of gradation" and "due treatment for persons", the author concludes that the early embryo has less ethical significance than the later fetus and adult human. The moral obligation we have toward persons is clearer and stronger than that which we have toward human embryos. Embryo research is justifiable if it brings enormous welfare to human persons that cannot be otherwise achieved. The "love of gradation" requires us, however, to extend love and respect towards other entities according to their different status. We should therefore be very cautious in using human embryos for research, acknowledging the gradualist nature of their moral status.
Chen Hao; Hattori, Fumiyuki; Murata, Mitsushige; Li Weizhen; Yuasa, Shinsuke; Onizuka, Takeshi; Shimoji, Kenichiro; Ohno, Yohei; Sasaki, Erika; Kimura, Kensuke; Hakuno, Daihiko
Common marmoset monkeys have recently attracted much attention as a primate research model, and are preferred to rhesus and cynomolgus monkeys due to their small bodies, easy handling and efficient breeding. We recently reported the establishment of common marmoset embryonic stem cell (CMESC) lines that could differentiate into three germ layers. Here, we report that our CMESC can also differentiate into cardiomyocytes and investigated their characteristics. After induction, FOG-2 was expressed, followed by GATA4 and Tbx20, then Nkx2.5 and Tbx5. Spontaneous beating could be detected at days 12-15. Immunofluorescent staining and ultrastructural analyses revealed that they possessed characteristics typical of functional cardiomyocytes. They showed sinus node-like action potentials, and the beating rate was augmented by isoproterenol stimulation. The BrdU incorporation assay revealed that CMESC-derived cardiomyocytes retained a high proliferative potential for up to 24 weeks. We believe that CMESC-derived cardiomyocytes will advance preclinical studies in cardiovascular regenerative medicine.
Xing, Yun; Xiong, Wei; Zhu, Lin; Osawa, Eiji; Hussin, Saber; Dai, Liming
Because of their unique photoluminescence and magnetic properties, nanodiamonds (NDs) are promising for biomedical imaging and therapeutical applications. However, these biomedical applications will hardly be realized unless the potential hazards of NDs to humans and other biological systems are ascertained. Previous studies performed in our group and others have demonstrated the excellent biocompatibility of NDs in a variety of cell lines without noticeable cytotoxicity. In the present paper, we report the first genotoxicity study on NDs. Our results showed that incubation of embryonic stem cells with NDs led to slightly increased expression of DNA repair proteins, such as p53 and MOGG-1. Oxidized nanodiamonds (O-NDs) were demonstrated to cause more DNA damage than the pristine/raw NDs (R-NDs), showing the surface chemistry specific genotoxicity. However, the DNA damages caused by either the O-NDs or the R-NDs are much less severe than those caused by multiwalled carbon nanotubes (MWNTs) observed in our previous study. These findings should have important implications for future applications of NDs in biological applications.
Munroe, R J; Bergstrom, R A; Zheng, Q Y; Libby, B; Smith, R; John, S W; Schimenti, K J; Browning, V L; Schimenti, J C
The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.
Hirano, Seiki; Tominaga, Yohei; Ichinoe, Akimasa; Ushijima, Yasuhiro; Tsuchimoto, Daisuke; Honda-Ohnishi, Yoko; Ohtsubo, Toshio; Sakumi, Kunihiko; Nakabeppu, Yusaku
To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.
Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.
The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500–2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation. PMID:22802633
Rouzbeh, Shaghayegh; Kobari, Ladan; Cambot, Marie; Mazurier, Christelle; Hebert, Nicolas; Faussat, Anne-Marie; Durand, Charles; Douay, Luc; Lapillonne, Hélène
While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs.
Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad
Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund
Underwood, Jean M; Becker, Klaus A; Stein, Gary S; Nickerson, Jeffrey A
The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
D'Amour, Kevin A; Agulnick, Alan D; Eliazer, Susan; Kelly, Olivia G; Kroon, Evert; Baetge, Emmanuel E
The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.
Fagan, Melinda Bonnie
Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. Copyright © 2015 Elsevier Ltd. All rights reserved.
Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. PMID:25547290
Tachibana, Masahito; Sparman, Michelle; Sritanaudomchai, Hathaitip; Ma, Hong; Clepper, Lisa; Woodward, Joy; Li, Ying; Ramsey, Cathy; Kolotushkina, Olena; Mitalipov, Shoukhrat
Mitochondria are found in all eukaryotic cells and contain their own genome (mitochondrial DNA or mtDNA). Unlike the nuclear genome, which is derived from both the egg and sperm at fertilization, the mtDNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin. Mutations in mtDNA contribute to a diverse range of currently incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we demonstrate here that the mitochondrial genome can be efficiently replaced in mature non-human primate oocytes (Macaca mulatta) by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg. The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization, embryo development and produced healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infants born so far originated from the spindle donors whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring. Spindle replacement is shown here as an efficient protocol replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.
Tian, L-P; Zhang, S; Xu, L; Li, W; Wang, Y; Chen, S-D; Ding, J-Q
Embryonic stem cells (ESC) transplantation is a potential therapeutic approach for Parkinson's disease (PD). However, one of the main challenges to this therapy is the post-transplantation survival of dopaminergic (DA) neurons. In this study, mouse ESC were differentiated into DA neurons by a modified serum free protocol. These ESC-derived neurons were then transplanted into striatum of 6-OHDA lesioned rat. The viability of grafted DA neurons was decreased, accompanied by activated microglia and high levels of proinflammatory factors, such as TNF-α and iNOS, in the graft niche. This suggested that the local neuroinflammation might be involved in the reduced cells viability. Selenite, the source of essential micronutrient selenium, could inhibit NF-κB p65 nuclear translocation and subsequently reduce iNOS, COX-2 and TNF-α expression in LPS-treated BV2 cells in a dose dependant manner. Before the transplantation of ESC-derived DA neurons, 6-OHDA lesioned rats were intraperitoneally injected with selenite. The expression levels of TNF-α and iNOS were decreased by 30% and 50%, respectively, in selenite treated group. The survival of implanted DA neurons and the rotational behavior of transplanted rats were also remarkably improved by selenite treatment. To sum up, selenite might benefit ESCs transplantation therapy in PD through anti-inflammation effects.
Sallam, Karim; Wu, Joseph C
Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.
Schlesinger, Sharon; Goff, Stephen P
Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.
Fujimoto, Akihisa; Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Wolf, Don P
Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.
Harikumar, Arigela; Meshorer, Eran
Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.
Guo, Jianli; Jauch, Anna; Heidi, Holtgreve-Grez; Schoell, Brigitte; Erz, Dorothee; Schrank, Martina; Janssen, Johannes W G
The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources.
Whyte, Warren A; Bilodeau, Steve; Orlando, David A; Hoke, Heather A; Frampton, Garrett M; Foster, Charles T; Cowley, Shaun M; Young, Richard A
Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states.
Whyte, Warren A.; Bilodeau, Steve; Orlando, David A.; Hoke, Heather A.; Frampton, Garrett M.; Foster, Charles T.; Cowley, Shaun M.; Young, Richard A.
Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development1,2. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation3,4. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1–NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. PMID:22297846
Ying, Qi-Long; Wray, Jason; Nichols, Jennifer; Batlle-Morera, Laura; Doble, Bradley; Woodgett, James; Cohen, Philip; Smith, Austin
In the three decades since pluripotent mouse embryonic stem (ES) cells were first described1,2 they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts1–7. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state. PMID:18497825
Schwindinger, W F; Reese, K J; Lawler, A M; Gearhart, J D; Levine, M A
Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (G alpha(s)) are present in subjects with Albright hereditary osteodystrophy, a syndrome of characteristic developmental defects and, in some patients, resistance to multiple hormones that stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the first step in generating a model of Albright hereditary osteodystrophy, the gene encoding G alpha(s) (Gnas) was disrupted in mouse embryonic stem (ES) cells by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G alpha(s) messenger RNA and G alpha(s) protein in targeted ES cells were approximately 50% of levels in untargeted ES cells. In response to 10 microM forskolin and to various concentrations of isoproterenol (0.1-3.0 microM), cAMP accumulation was reduced in the G alpha(s) knockout ES cell lines, relative to wild-type ES cells and to five of six ES cell lines with randomly integrated targeting vector. These results support the role of G alpha(s) haploinsufficiency in reducing the ability of hormones to generate cAMP in subjects with pseudohypoparathyroidism type Ia. The targeted disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G alpha(s) and cAMP coupled signal transduction in differentiation and development.
Kim, Eun Kyoung; Son, Mi-Young; Kang, Youngkuk; Lee, Chang-Hee; Kim, Hae Rim; Won, Youngsuk; Yoon, Wonkee; Kim, Hyoung-Chin
Embryonic stem cells (ESCs) are an emerging source for cell-based therapies aimed at repairing damaged organ tissues; however, the efficiency of directed differentiation is low and refinement of differentiation protocols is hampered by incomplete understanding of the mechanisms involved in this process. To find new compounds which can improve the efficiency of directed differentiation of ESCs to cardiomyocytes, we screened several thousand chemical compounds and identified a promising group. All of the compounds found have a common structure of 1H-pyrrole,2,2'-(phenylmethylene)bis. Here we report the potential mechanism of action for 31002 which showed the strongest activity among the compounds selected. In the presence of 31002, 15 times more cardiomyocytes differentiated from ESCs, i.e., 3.5% to 52% of total differentiated cells. Moreover, the cardiomyocytes showed functional characteristics including rhythmic beating and marker gene expression. 31002 inhibited the down-regulation of genes related to the three germ layers in the late stage of ESCs differentiation, implying that 31002 supports a continuous fate commitment of undifferentiated ESCs to the cardiac lineage by prolonging the three germ layer stages. Therefore, compounds in this group, including 31002, might be useful as directed cardiomyogenic differentiation-inducers to produce cells for use in cell therapy aimed at restoring damaged heart tissue. PMID:21998609
Vazin, Tandis; Freed, William J.
The greatest therapeutic promise of human embryonic stem cells (hESC) is to generate specialized cells to replace damaged tissue in patients suffering from various degenerative diseases. However, the signaling mechanisms involved in lineage restriction of ESC to adopt various cellular phenotypes are still under investigation. Furthermore, for progression of hESC-based therapies towards clinical applications, appropriate culture conditions must be developed to generate genetically stable homogenous populations of cells, to hinder possible adverse effects following transplantation. Other critical challenges that must be addressed for successful cell implantation include problems related to survival and functional efficacy of the grafted cells. This review initially describes the derivation of hESC and focuses on recent advances in generation, characterization, and maintenance of these cells. We also give an overview of original and emerging differentiation strategies used to convert hESC to different cell types. Finally, we will discuss transplantation studies of hESC-derived cells with respect to safety and functional recovery. PMID:20714081
Tompkins, Joshua D; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D
The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500-2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation.
Reid, L H; Shesely, E G; Kim, H S; Smithies, O
We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations. Images PMID:1850104
Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel
We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.
Facucho-Oliveira, Joao M; Alderson, Jon; Spikings, Emma C; Egginton, Stuart; St John, Justin C
Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the mitochondrial genome (mtDNA). MtDNA replication is regulated by the nuclear-encoded mitochondrial transcription factor A (TFAM) and the mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg, Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA replication, identified by 5-bromo-2'-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of replication and upregulation of Polg, Polg2 and Tfam, whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.
Siegel, Andrew W
There is a lack of consensus on whether the derivation and use of human embryonic stem cells (hESCs) from embryos remaining after infertility treatment morally require the informed consent of third-party gamete donors who contributed to the creation of the embryos. The principal guidelines for oversight and funding of hESC research in the United States make minimal or no demands for consent from gamete donors. In this article, I consider the arguments supporting and opposing gamete donor consent for hESC research and embryo research more broadly. I argue that it is not morally permissible to use leftover embryos in research without the informed consent of gamete donors, and that we should place restrictions on the use of existing hESC lines that may have been derived without informed consent. While the standard argument for this position relies on an appeal to gamete donors' interest in controlling what happens with their genetic material, I identify shortcomings with the standard approach and seek instead to locate the deeper moral foundations for gamete donor consent in rights to bodily integrity.
Bahmani, L; Taha, M F; Javeri, A
Embryonic stem (ES) cells secrete some soluble factors which may affect the differentiation potential of adult stem cells toward different lineages. In the present study, we evaluated neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) following coculture with ES cells. For this purpose, ADSCs were induced in a medium supplemented with a synthetic serum replacement and various concentrations of retinoic acid (RA). Then, third-passaged ADSCs were indirectly cocultured with ES cells, and the expression levels of pluripotency markers, OCT4 and Sox2, mesenchymal stem cell markers, CD73 and CD105, and proliferating cell nuclear antigen (PCNA), were assessed in the cocultured ADSCs. Moreover, the control and cocultured ADSCs were differentiated with or without RA treatment. We showed here that 2-week differentiated ADSCs expressed several neuron-specific genes, and RA treatment improved neural differentiation of the ADSCs. The expression levels of OCT4, Sox2 and PCNA were upregulated in the cocultured ADSCs. Moreover, coculture with the ES cells significantly improved neural differentiation of the ADSCs. Treatment of the cocultured ADSCs with RA diminished the expression of neural maturation markers. Coculture with the ES cells efficiently improves neural differentiation of the ADSCs. Non-contact coculture with the ES cells may be used as an efficient strategy to improve differentiation potential of adult stem cells for developmental studies and regenerative medicine.
Heng, Boon Chin; Liu, Hua; Cao, Tong
Human embryonic stem cells have tremendous potential in the newly emerging field of regenerative medicine. Recently, it was demonstrated that the rescue of lethal cardiac defects in Id knockout mutant mouse embryos was not due to the transplanted cells giving rise to functional new tissues within the defective embryonic heart. Instead, there is indirect evidence that the observed therapeutic effect was due to various secreted factors emanating from the transplanted cells. This therefore, introduces the exciting prospect of utilizing human embryonic stem cells as biological 'catalysts' to promote tissue repair and regeneration in transplantation therapy. However, the immunological barrier against allogenic transplantation, as well as the teratogenic potential of human embryonic stem cells poses major technical challenges. A possible strategy to overcome the immunological barrier may be to impose a temporary regimen of immunosuppressive drugs followed by their gradual withdrawal, once adequate tissue regeneration has been achieved. Other more novel alternatives include the use of microencapsulation to block interaction with the transplant recipient's immune system, and co-transplantation with bone marrow-derived mesenchymal stem cells, which have been demonstrated to possess immuno-suppressive properties. The teratogenic potential of human embryonic stem cells could possibly be alleviated by directing the differentiation of these cells to specific lineages prior to transplantation, or through mitotic inactivation (gamma irradiation or mitomycin C exposure). Co-transplantation with autologous adult stem cells may represent a novel strategy to further enhance the 'catalytic' effects of human embryonic stem cells. The various factors secreted by human embryonic stem cells could then have a concentrated localized effect on relatively large numbers of co-transplanted autologous adult stem cells, which may in turn lead to enhanced repair and regeneration of the damaged
From July 2002 to May 2008, 36 applications for the import and use of human embryonic stem cells (hES) were reviewed by the German Central Ethics Committee for Stem Cell Research (ZES). A flood of applications anticipated by opponents to human embryonic stem cell research has not occurred since the enactment of the German Stem Cell Act in 2002. On the contrary, German hES cell research is below international average in terms of project numbers. The current restrictions for using hES cells in Germany might be causative for the opinion that this type of research is not considered to be very promising. This could hold true especially for research aiming at clinical applications. Consequently, potential research goals of premium importance, especially those of potential clinical relevance, could be seriously jeopardized.
Mishra, Paras Kumar; Kuypers, Nicholas John; Singh, Shree Ram; Leiberh, Noel Diaz; Chavali, Vishalakshi; Tyagi, Suresh C
Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differentiate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating myocardial infarction (death of myocardial tissue) and type 1 diabetes (death of pancreatic beta cells). Understanding the components regulating ESC differentiation is the key to unlock the regenerative potential of ESC-based therapies. Both the stiffness of extracellular matrix (ECM) and surrounding niche/microenvironment play pivotal roles in ESC differentiation. Matrix metalloproteinase-9 (MMP9) induces fibrosis that causes stiffness of the ECM and impairs differentiation of cardiac stem cells into cardiomyocytes. Here, we describe the method of ESC culture and differentiation, and the expression of MMP9 and its inhibitor, tissue inhibitor of metalloproteinase-4 (TIMP4) in differentiating ESC.
Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella
Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.
Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J.; Fico, Annalisa; Minchiotti, Gabriella
Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544
Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of
In late 2005 the Legislation Review: Prohibition of Human Cloning Act 2002 (Cth) and the Research Involving Human Embryos Act 2002 (Cth) recommended the establishment of an Australian stem cell bank. This article aims to address a lack of discussion of issues surrounding stem cell banking by suggesting possible answers to the questions of whether Australia should establish a stem cell bank and what its underlying philosophy and functions should be. Answers are developed through an analysis of regulatory, scientific and intellectual property issues relating to embryonic stem cell research in the United Kingdom, United States and Australia. This includes a detailed analysis of the United Kingdom Stem Cell Bank. It is argued that a "guardian" model stem cell bank should be established in Australia. This bank would aim to promote the maximum public benefit from human embryonic stem cell research by providing careful regulatory oversight and addressing ethical issues, while also facilitating research by addressing practical scientific concerns and intellectual property issues.
Roche, Stephane; Delorme, Bruno; Oostendorp, Robert A J; Barbet, Romain; Caton, David; Noel, Daniele; Boumediene, Karim; Papadaki, Helen A; Cousin, Beatrice; Crozet, Carole; Milhavet, Ollivier; Casteilla, Louis; Hatzfeld, Jacques; Jorgensen, Christian; Charbord, Pierre; Lehmann, Sylvain
Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.
Roubal, Ivan; Park, Sun Joo; Kim, Yong
Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.
Neganova, Irina; Lako, Majlinda
It is well known that G1 to S phase transition is tightly regulated by the expression and phosphorylation of a number of well-characterized cyclins, cyclin-dependent kinases and members of the retinoblastoma gene family. In this review we discuss the role of these components in regulation of G1 to S phase transition in somatic cells and human embryonic stem cells. Most importantly, we discuss some new tenable links between maintenance of pluripotency and cell cycle regulation in embryonic stem cells by describing the role that master transcription factors play in this process. Finally, the differences in cell cycle regulation between murine and human embryonic stem cells are highlighted, raising interesting questions regarding their biology and stages of embryonic development from which they have been derived. PMID:18638068
Li, Hui; Gao, Shuai; Huang, Hua; Liu, Wenqiang; Huang, Huanwei; Liu, Xiaoyu; Gao, Yawei; Le, Rongrong; Kou, Xiaochen; Zhao, Yanhong; Kou, Zhaohui; Li, Jia; Wang, Hong; Zhang, Yu; Wang, Hailin; Cai, Tao; Sun, Qingyuan; Gao, Shaorong; Han, Zhiming
Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.
Lewandowski, Jarosław; Kurpisz, Maciej
Developing procedures for the derivation of human pluripotent stem cells (PSCs) gave rise to novel pathways into regenerative medicine research. For many years, stem cells have attracted attention as a potentially unlimited cell source for cellular therapy in neurodegenerative disorders, cardiovascular diseases, and spinal cord injuries, for example. In these studies, adult stem cells were insufficient; therefore, many attempts were made to obtain PSCs by other means. This review discusses key issues concerning the techniques of pluripotent cell acquisition. Technical and ethical issues hindered the medical use of somatic cell nuclear transfer and embryonic stem cells. Therefore, induced PSCs (iPSCs) emerged as a powerful technique with great potential for clinical applications, patient-specific disease modelling and pharmaceutical studies. The replacement of viral vectors or the administration of analogous proteins or chemical compounds during cell reprogramming are modifications designed to reduce tumorigenesis risk and to augment the procedure efficiency. Intensified analysis of new PSC lines revealed other barriers to overcome, such as epigenetic memory, disparity between human and mouse pluripotency, and variable response to differentiation of some iPSC lines. Thus, multidimensional verification must be conducted to fulfil strict clinical-grade requirements. Nevertheless, the first clinical trials in patients with spinal cord injury and macular dystrophy were recently carried out with differentiated iPSCs, encouraging alternative strategies for potential autologous cellular therapies.
Miranda, Alberto; Pericuesta, Eva
Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis. PMID:21483752
Wang, Zhichong; Ge, Jian; Huang, Bing; Gao, Qianying; Liu, Bingqian; Wang, Linghua; Yu, Ling; Fan, Zhigang; Lu, Xiaoming; Liu, Jingbo
Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.
Lee, Keun-Bae; Choi, Jin; Cho, Seong-Beom; Chung, Jae-Yoon; Moon, Eun-Sun; Kim, Nack-Sung; Han, Ho-Jae
The effects of embryonic stem cells (ESCs) on diabetic wound healing were investigated using an excisional skin wound model in 110 diabetes-induced rats. We transplanted a clonal population of ESCs (5 × 10(6)) by topical injection into full thickness skin wounds. Four study groups were used; nondiabetic rats as a control, non-insulin controlled diabetic rats not treated with ESCs, insulin controlled diabetic rats not treated with ESCs, and insulin controlled diabetic rats treated with ESCs. Five rats in each experimental group were sacrificed on days 1, 5, 10, 15, and 20 after wounding. Wounds images were acquired daily and wound sizes were calculated. We measured the mRNA levels of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), and fibronectin levels in extracellular matrix, and assessed wound healing by assessing histological parameters of epidermal regeneration, granulation tissue thickness, and angiogenesis. In the ESC-treated group, wound sizes were significantly smaller than in the insulin controlled diabetic group not treated with ESCs on days 5 and 10 (p < 0.05), and EGF and VEGF levels were markedly higher on days 5 and 10, fibronectin levels on day 5 after injection. All histological scores in the ESC-treated group were significantly higher than those of the insulin controlled diabetic group on day 5 (p < 0.05). Our results shows that topical ESCs enhance diabetic wound healing during the early stage, and suggest that ESCs transplantation offers a novel therapeutic modality for the treatment of diabetic wounds.
Liu, Haisong; Zhang, Donghui; Yang, Weifeng; Deng, Hongkui
Background Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Methodology and Principal Findings Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation. Conclusions and Significance The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification. PMID:19107197
Wei, Wei; Qing, Tingting; Ye, Xin; Liu, Haisong; Zhang, Donghui; Yang, Weifeng; Deng, Hongkui
Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation. The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.
Zhang, Junli; Rao, Rammohan V.; Spilman, Patricia; Mangada, Julie; Xie, Lin; Vitelli, Cathy; Gorostiza, Olivia F.; Madden, David T.; Zeng, Xianmin; Jin, Kunlin; Hart, Matthew J.; Bredesen, Dale E.; Galvan, Veronica
Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping ß-actin promoter. The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759–769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies. PMID:21874159
Zhang, Junli; Rao, Rammohan V; Spilman, Patricia; Mangada, Julie; Xie, Lin; Vitelli, Cathy; Gorostiza, Olivia F; Madden, David T; Zeng, Xianmin; Jin, Kunlin; Hart, Matthew J; Bredesen, Dale E; Galvan, Veronica
Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping β-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759-769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.
Kasuda, Shogo; Tatsumi, Kohei; Sakurai, Yoshihiko; Shima, Midori; Hatake, Katsuhiko
Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future.
Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey
Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272
Miranda, Alberto; Pericuesta, Eva; Ramírez, Miguel Ángel; Gutierrez-Adan, Alfonso
Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.
Poursani, Ensieh M; Mehravar, Majid; Soltani, Bahram Mohammad; Mowla, Seyed Javad
POU domain proteins are an important family of transcription factors that regulates cell type-specific gene expression. One of the most crucial members of this family that maintains pluripotency and self-renewal of embryonic stem cells is POU5F1/OCT4. The OCT4 gene can generate several variants under different situations/cell types includes OCT4A that is the major factor sustains pluripotency in embryonic stem and embryonic carcinoma cells, and also OCT4B and OCT4B1, which are transcribed from a different potential promoter located in intron1 and are expressed in various tissues and cell types. In present study, during expression check of OCT4B1 in embryonic carcinoma cells (NT2), we discovered a novel OCT4 transcript for the first time and designated it as OCT4B4. This variant is expressed in various human pluripotent cells and its expression is down-regulated upon induction of differentiation. Moreover, knocking down of OCT4B4 by shRNA resulted in increased accumulation of transfected cells in G0/G1 phase compared to the mock-transfected control cells. Copyright © 2017. Published by Elsevier B.V.
Chen, Lukui; Qiu, Rong; Li, Lushen
This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.
Koledova, Zuzana; Krämer, Alwin; Kafkova, Leona Raskova; Divoky, Vladimir
Embryonic stem cells seem to have the intriguing capacity to divide indefinitely while retaining their pluripotency. This self-renewal is accomplished by specialized mechanisms of cell-cycle control. In the last few years, several studies have provided evidence for a direct link between cell-cycle regulation and cell-fate decisions in stem cells. In this review, we discuss the peculiarities of embryonic stem cell-cycle control mechanisms, implicate their involvement in cell-fate decisions, and distinguish centrosomes as important players in the self-renewal versus differentiation roulette.
Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung
Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.
Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung
Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)-dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. Copyright © 2015 Elsevier Inc. All rights reserved.
Priddle, Helen; Jones, D Rhodri E; Burridge, Paul W; Patient, Roger
The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.
Domev, Hagit; Amit, Michal; Laevsky, Ilana; Dar, Ayelet; Itskovitz-Eldor, Joseph
Human mesenchymal stem cells (hMSCs) can be derived from various adult and fetal tissues. However, the quality of tissues for the isolation of adult and fetal hMSCs is donor dependent with a nonreproducible yield. In addition, tissue engineering and cell therapy require large-scale production of a pure population of lineage-restricted stem cells that can be easily induced to differentiate into a specific cell type. Therefore, human embryonic stem cells (hESCs) can provide an alternative, plentiful source for generation of reproducible hMSCs. We have developed efficient differentiation protocols for derivation of hMSCs from hESCs, including coculture with murine OP9 stromal cells and feeder layer-free system. Our protocols have resulted in the generation of up to 49% of hMSCs, which expressed CD105, CD90, CD29, and CD44. The hMSCs exhibited high adipogenic, chondrocytic, and osteogenic differentiation in vitro. The latter correlated with osteocalcin secretion and vascular endothelial growth factor (VEGF) production by the differentiating hMSCs. hMSC-derived osteoblasts further differentiated and formed ectopic bone in vivo, and induced the formation of blood vessels in Matrigel implants. Our protocol enables generation of a purified population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, and particularly into vasculogenesis-inducing osteoblasts, which can contribute to the development of bone repair protocols.
Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.
Zhang, Lijun; Hua, Qiuhong; Tang, Kaiyi; Shi, Changjie; Xie, Xin; Zhang, Ru
G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth, death, movement, transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study, we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol, we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially, the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted, whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4, knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together, our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine.
Power, Carl; Rasko, John E J
In the past few years, relatively straightforward laboratory techniques have been developed to reprogram normal body cells to enter an embryonic stem cell-like state. Not only do these induced pluripotent stem cells hold great medical promise--perhaps greater than that of embryonic stem cells--but they also have escaped the ethical controversy in which the latter is mired. This article examines how cell reprogramming is likely to transform regenerative and reproductive medicine and highlights some of the medical, moral, and political hurdles that it faces. It also argues that induced pluripotent stem cells are more ethically problematic than most people believe and that cell reprogramming will not solve the stem cell controversy but complicate it further.
Christou, Y A; Moore, H D; Shaw, P J; Monk, P N
Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.
Background Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. Results We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. Conclusions We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature. PMID:26694224
Menzorov, Aleksei G; Matveeva, Natalia M; Markakis, Marios N; Fishman, Venyamin S; Christensen, Knud; Khabarova, Anna A; Pristyazhnyuk, Inna E; Kizilova, Elena A; Cirera, Susanna; Anistoroaei, Razvan; Serov, Oleg L
Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.
Zhang Enming; Li Xiaolong; Zhang Shufang; Chen Liangqiang; Zheng Xiaoxiang . E-mail: email@example.com
Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation.
Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James
Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.
Goldstein, Ronald S
Xenografting of normal and transformed mammalian tissues and cells to chick embryos has been performed for almost 100 years. Embryonic stem cells, derived more than 25 years ago from murine, and more than 10 years ago from human blastocysts, have transformed many fields of biological research. There is a growing body of studies combining these two widely-used experimental systems. This review surveys those reports in which murine or human embryonic stem cells, or differentiated derivatives of these pluripotent stem cells, were transplanted to embryonated chick eggs. Many of these studies have utilized the unique characteristics of both experimental models to obtain answers to developmental questions that are difficult or impossible to approach with xenografting to adult rodents or tissue culture-only techniques.
Feng, Jun-Feng; Liu, Jing; Zhang, Xiu-Zhen; Zhang, Lei; Jiang, Ji-Yao; Nolta, Jan; Zhao, Min
Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, it is critical to investigate how human NSCs (hNSCs) respond to EF before any possible clinical attempt. Aiming to characterize the EF-stimulated and guided migration of hNSCs, we derived hNSCs from a well-established human embryonic stem cell line H9. Small applied DC EFs, as low as 16 mV/mm, induced significant directional migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode increased with the increase of field strength. (Rho-kinase) inhibitor Y27632 is used to enhance viability of stem cells and has previously been reported to inhibit EF-guided directional migration in induced pluripotent stem cells and neurons. However, its presence did not significantly affect the directionality of hNSC migration in an EF. Cytokine receptor [C-X-C chemokine receptor type 4 (CXCR4)] is important for chemotaxis of NSCs in the brain. The blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional migration. Applied EFs could potentially be further exploited to guide hNSCs to injured sites in the central nervous system to improve the outcome of various diseases.
Magown, Philippe; Rafuse, Victor F; Brownstone, Robert M
Motoneurons derived from embryonic stem cells can be transplanted in the tibial nerve, where they extend axons to functionally innervate target muscle. Here, we studied spontaneous muscle contractions in these grafts 3 mo following transplantation. One-half of the transplanted grafts generated rhythmic muscle contractions of variable patterns, either spontaneously or in response to brief electrical stimulation. Activity generated by transplanted embryonic stem cell-derived neurons was driven by glutamate and was modulated by muscarinic and GABAergic/glycinergic transmission. Furthermore, rhythmicity was promoted by the same transmitter combination that evokes rhythmic locomotor activity in spinal cord circuits. These results demonstrate that there is a degree of self-assembly of microcircuits in these peripheral grafts involving embryonic stem cell-derived motoneurons and interneurons. Such spontaneous activity is reminiscent of embryonic circuit development in which spontaneous activity is essential for proper connectivity and function and may be necessary for the grafts to form functional connections with muscle.NEW & NOTEWORTHY This manuscript demonstrates that, following peripheral transplantation of neurons derived from embryonic stem cells, the grafts are spontaneously active. The activity is produced and modulated by a number of transmitter systems, indicating that there is a degree of self-assembly of circuits in the grafts.
Roberts, Iwan; Baila, Stefano; Rice, R Brent; Janssens, Michiel Etienne; Nguyen, Kim; Moens, Nathalie; Ruban, Ludmila; Hernandez, Diana; Coffey, Pete; Mason, Chris
The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested.
Gao, Xiugong; Sprando, Robert L.; Yourick, Jeffrey J.
Thalidomide is a potent developmental toxicant that induces a range of birth defects, notably severe limb malformations. To unravel the molecular mechanisms underpinning the teratogenic effects of thalidomide, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on the differentiation of mouse embryonic stem cells (mESCs), and published the major findings in a research article entitled “Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells” . The data presented herein contains complementary information related to the aforementioned research article. PMID:26217789
This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.
Ji, Yuan; Ma, Yongbin; Chen, Xiang; Ji, Xianyan; Gao, Jianyi; Zhang, Lei; Ye, Kai; Qiao, Fuhao; Dai, Yao; Wang, Hui; Wen, Xiangmei; Lin, Jiang; Hu, Jiabo
Human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSCs) are able to inhibit proliferation of leukemia cells. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSC‑MVs) might play an important part in antitumor activity. Microvesicles were isolated by ultracentrifugation and identified under a scanning electron microscopy and transmission electron microscope separately. After 48-h cocultured with hESC‑MSCs and hESC‑MSC‑MVs, the number of K562 and HL60 was counted and tumor cell viability was measured by CCK8 assay. The expression of proteins Bcl-2 and Bax were estimated by western blotting. Transmission electron microscope and western blot analysis were adopted to evaluate the autophagy level. Results showed that both hESC‑MSCs and hESC‑MSC‑MVs inhibited proliferation of leukemia cells in a concentration-dependent manner. hESC‑MSC‑MVs reduced the ratio of Bcl/Bax, enhanced the protein level of Beclin-1 and LC3-II conversion, thus upregulating autophagy and apoptosis. In conclusion, microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibited tumor growth and stimulated autophagy and excessive autophagy might induce apoptosis.
Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R
Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.
Barad, Lili; Schick, Revital; Zeevi-Levin, Naama; Itskovitz-Eldor, Joseph; Binah, Ofer
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the capacity to differentiate into any specialized cell type, including cardiomyocytes. Therefore, hESC-derived and hiPSC-derived cardiomyocytes (hESC-CMs and hiPSC-CMs, respectively) offer great potential for cardiac regenerative medicine. Unlike some organs, the heart has a limited ability to regenerate, and dysfunction resulting from significant cardiomyocyte loss under pathophysiological conditions, such as myocardial infarction (MI), can lead to heart failure. Unfortunately, for patients with end-stage heart failure, heart transplantation remains the main alternative, and it is insufficient, mainly because of the limited availability of donor organs. Although left ventricular assist devices are progressively entering clinical practice as a bridge to transplantation and even as an optional therapy, cell replacement therapy presents a plausible alternative to donor organ transplantation. During the past decade, multiple candidate cells were proposed for cardiac regeneration, and their mechanisms of action in the myocardium have been explored. The purpose of this article is to critically review the comprehensive research involving the use of hESCs and hiPSCs in MI models and to discuss current controversies, unresolved issues, challenges, and future directions.
Jin, Jiali; Liu, Jian; Chen, Cong; Liu, Zhenping; Jiang, Cong; Chu, Hongshang; Pan, Weijuan; Wang, Xinbo; Zhang, Lingqiang; Li, Bin; Jiang, Cizhong; Ge, Xin; Xie, Xin; Wang, Ping
Nanog is a master pluripotency factor of embryonic stem cells (ESCs). Stable expression of Nanog is essential to maintain the stemness of ESCs. However, Nanog is a short-lived protein and quickly degraded by the ubiquitin-dependent proteasome system. Here we report that the deubiquitinase USP21 interacts with, deubiquitinates and stabilizes Nanog, and therefore maintains the protein level of Nanog in mouse ESCs (mESCs). Loss of USP21 results in Nanog degradation, mESCs differentiation and reduces somatic cell reprogramming efficiency. USP21 is a transcriptional target of the LIF/STAT3 pathway and is downregulated upon differentiation. Moreover, differentiation cues promote ERK-mediated phosphorylation and dissociation of USP21 from Nanog, thus leading to Nanog degradation. In addition, USP21 is recruited to gene promoters by Nanog to deubiquitinate histone H2A at K119 and thus facilitates Nanog-mediated gene expression. Together, our findings provide a regulatory mechanism by which extrinsic signals regulate mESC fate via deubiquitinating Nanog. PMID:27886188
Chin, Mark H.; Pellegrini, Matteo; Plath, Kathrin; Lowry, William E.
Recent work from our group and others has argued that human induced pluripotent stem cells (hiPSCs) generated by the introduction of four viruses bearing reprogramming factors differ from human embryonic stem cells (hESCs) at the level of gene expression. Many of the differences seen were common across independent labs and, at least to some extent, are thought to be a result of residual expression of donor cell-specific genes (Chin et al., 2009; Ghosh et al., 2010; Marchetto et al., 2009). Two new reports re-analyze similar expression datasets as those used in Chin et al., (Chin et al., 2009) and come to different conclusions (Newman et al., 2010, Guenther et al., 2010). Here, we compare various approaches to perform gene expression meta-analysis that all support our original conclusions and present new data to demonstrate that polycistronic delivery of the reprogramming factors and extended culture brings hiPSCs transcriptionally much closer to hESCs than older methods. PMID:20682452
... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Cell Line to be Approved for Use in NIH Funded Research. OMB No. 0925-0601-- Expiration Date 4/30/2013... Information Collection: The form is used by applicants to request that human embryonic stem cell lines...
Among the many promised and potential applications of embryonic stem cells, in vitro toxicology is one area in which ES cells have already proven their utility. In 2003, the Embryonic Stem Cell Test (EST) protocol was validated in Europe as an in vitro alternative to live animal...
Among the many promised and potential applications of embryonic stem cells, in vitro toxicology is one area in which ES cells have already proven their utility. In 2003, the Embryonic Stem Cell Test (EST) protocol was validated in Europe as an in vitro alternative to live animal...
Le Rolle, Anne-France; Chiu, Thang K; Zeng, Zhaoshi; Shia, Jinru; Weiser, Martin R; Paty, Philip B; Chiu, Vi K
Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer.
Takazawa, Tomonori; Croft, Gist F.; Amoroso, Mackenzie W.; Studer, Lorenz; Wichterle, Hynek; MacDermott, Amy B.
Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases. PMID:22802953
Tosca, Lucie; Feraud, Olivier; Magniez, Aurélie; Bas, Cécile; Griscelli, Frank; Bennaceur-Griscelli, Annelise; Tachdjian, Gérard
Human embryonic stem cells exhibit genomic instability that can be related to culture duration or to the passaging methods used for cell dissociation. In order to study the impact of cell dissociation techniques on human embryonic stem cells genomic instability, we cultured H1 and H9 human embryonic stem cells lines using mechanical/manual or enzymatic/collagenase-IV dissociation methods. Genomic instability was evaluated at early (
Recent efforts to resolve the political impasse over human embryonic stem cells (ESC) have generated proposals for obtaining ESC while avoiding the destruction of human embryos. This new chapter in the scientific and ethical debate provides an important opportunity to introduce additional ethical considerations to enhance public discourse.
Zhang, Muzi; Sun, Pingxin; Wang, Yusheng; Chen, Junnan; Lv, Linjie; Wei, Wanguo; Jin, Caixia; Li, Wenlin
Somatic stem cells play crucial roles in organogenesis and tissue homeostasis and regeneration and may ultimately prove useful for cell therapy for a variety of degenerative diseases and injuries; however, isolation and expansion of most types of somatic stem cells from tissues are technically challenging. Human pluripotent stem cells are a renewable source for any adult cell types, including somatic stem cells. Generation of somatic stem cells from human pluripotent stem cells is a promising strategy to get these therapeutically valuable cells. Previously, we developed a chemically defined condition for mouse hepatoblast self-renewal through a reiterative screening strategy. In the present study, we efficiently generated hepatoblasts from human embryonic stem cells by a stepwise induction strategy. Importantly, these human embryonic stem cell-derived hepatoblasts can be captured and stably maintained using conditions previously established for mouse hepatoblast self-renewal, which includes basal media supplemented with insulin, transferrin, sodium selenite, epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. The cells can stably retain hepatoblast phenotypes during prolonged culture and can differentiate into mature hepatocytes through in vitro provision of hepatocyte lineage developmental cues. After being embedded into three-dimensional Matrigel, these cells efficiently formed bile duct-like structures resembling native bile duct tissues. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases. Somatic stem cells have been proposed as promising candidates for cell-based therapy; however, isolation of somatic stem cells from adult tissues is usually invasive and technically challenging. In the present study, hepatoblasts from human embryonic stem cells were efficiently generated
Dickens, Bernard M
The International Society for Stem Cell Research (ISSCR) Guidelines for the Conduct of Human Embryonic Stem Cell Research, prepared by an international group of leading stem cell scientists, bioethicists and lawyers, build on the proposals previously drafted by agencies active in the conduct and funding of stem cell science. They aim not to duplicate existing codes of ethics in research with humans or animals, but to address special sensitivities about human embryo research and acquisition of ova, sperm, embryos and somatic tissues from often vulnerable or dependent donors. Guidelines concern the procurement of research materials, the derivation of stem cells, and banking , distribution and use of cells and cell lines derived from pre-implantation stages of human development. They categorize research that on ethical grounds requires special stem cell research oversight (SCRO), and indicate how that should be undertaken. They emphasize international collaboration in these activities, to maximize benefits to humanity.
Ezashi, T; Telugu, B P V L; Roberts, R M
Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the naïve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise. © 2012 Blackwell Verlag GmbH.
Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang
Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491
Chung, Henry; Sidhu, Kuldip S
Epigenetics is a growing field not only in the area of cancer research but recently in stem cells including human embryonic stem cell (hESC) research. The hallmark of profiling epigenetic changes in stem cells lies in maintaining pluripotency or multipotency and in attaining lineage specifications that are relevant for regenerative medicine. Epigenetic modifications including DNA methylation, histone acetylation and methylation, play important roles in regulating gene expressions. Other epigenetic modifications include X chromosome silencing, genomic stability and imprinting and mammalian development. This review attempts to elucidate the mechanism(s) behind epigenetic modifications and review techniques scientists use for identifying each modification. We also discuss some of the trends of epigenetic modifications in the fields of directed differentiation of embryonic stem cells and de-differentiation of somatic cells.
Wang, Han; Chen, Shuai; Cheng, Xiang; Dou, Zhongying; Wang, Huayan
To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.
Wu, Yi-Lin; Ma, Shi-Rong; Peng, Tao; Teng, Zeng-Hui; Liang, Xiang-Yan; Guo, Guo-Zhen; Zhang, Hai-Feng; Li, Kang-Chu
Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells. PMID:25196518
The National Institutes of Health (NIH) closure of the agency's Center for Regenerative Medicine (CRM), which focused on therapeutic development of induced pluripotent stem cells (iPS), was caused by the lack of progress in practical development of the iPSs for use in human therapies. As the NIH evaluates priorities in future stem cell therapeutic development, adult stem cell processes in the human body need to be prioritized for a number of key reasons, including (1) adult stem cells release many types of molecules that provide much of the therapeutic benefit of stem cells and (2) adult stem cells and somatic cells exist in a state of dynamic transition between different potency levels and can be naturally driven by the microenvironment to a state of pluripotency. Thus, the study and development of adult stems for therapeutic use can include naturally induced pluripotent stem cells (NiPSs) that lack the problematic genetic and epigenetic reprogramming errors found in iPSs.
Stem cell therapy in non-Western countries such as India has received a lot of attention. Apart from media reports, there are a number of social science analyses of stem cell policy, therapy, and research, their ethical implications, and impact of advertising on patients. Nevertheless, in the media reports as well as in academic studies, experiences of patients, who undertake overseas journeys for stem cell therapy, have largely been either ignored or presented reductively, often as a "false hope." In this article, I analyze the experiences of patients and their "journeys of hope" to NuTech Mediworld, an embryonic stem cell therapy clinic in New Delhi, India. My analysis, which draws on my observations in the clinic and patients' experiences, instead of seeking to adjudicate whether embryonic stem cell therapy in clinics such as NuTech is right or wrong, true or false, focuses on how patients navigate and contest these concerns. I utilize Gilles Deleuze and Felix Guattari's "concepts," lines of flight and deterritorialization, to highlight how embryonic stem cell therapy's "political economy of hope" embodies deterritorialization of several "regimes of truth" and how these deterritorializations impact patients' experiences. © The Author(s) 2014.
Kawanabe, Noriaki; Murata, Satoko; Fukushima, Hiroaki; Ishihara, Yoshihito; Yanagita, Takeshi; Yanagita, Emmy; Ono, Mitsuaki; Kurosaka, Hiroshi; Kamioka, Hiroshi; Itoh, Tomoo; Kuboki, Takuo; Yamashiro, Takashi
Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.
Wang, Yun; Yu, Xiaohua; Baker, Christopher; Murphy, William L; McDevitt, Todd C
Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications. Directing stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo. This hybrid biomaterial-ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi
Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).
Perera, Jonathan R; Jaiswal, Parag K; Khan, Wasim S; Adesida, Adetola
As our population changes osteoarthritis and cartilage defects are becoming more prevalent. The discovery of stems cells and their ability for indefinite regeneration has revolutionised the way cartilage problems are viewed. Tissue engineering has been shown to be the ideal way of repairing articular cartilage lesions, i.e. back to native tissue. The two main types of stem cells being investigated in chondrogenesis are embryological and mesenchymal stem cells. Research into embryological stem cells has been surrounded by controversy because of tumour formation and damaging embryos during the harvest of cells. We discuss the use of embryological and mesenchymal stem cells in cartilage repair and the various factors involved in the differentiation into chondrocytes.
To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...
Chester, Ronald; Sackstein, Robert
The breakneck speed of scientific developments in embryonic stem (ES) cell technologies is, commensurately, ushering forth new bioethical debate(s) regarding these cells. A framework of bioethical principles is presented here to guide biomedical scientists and others engaged in improving human welfare through the application of ES cell-based therapies.
Moller, Mark S
In 2003, Ruth Faden and eighteen other colleagues argued that a "problem of unequal biological access" is likely to arise in access to therapies resulting from human embryonic stem cell research. They showed that unless deliberate steps are taken in the United States to ensure that the human embryonic stem cell lines available to researchers mirrors the genetic diversity of the general population, white Americans will likely receive the benefits of these therapies to the relative exclusion of minority ethnic groups. Over the past five years the problem of unequal biological access has not received much attention from politicians, bioethicists and even many researchers in the United States, in spite of the widely held belief in the country that there is an obligation to prevent and correct ethnic disparities in access to medical care. The purpose of this paper is to increase awareness of the problem of unequal biological access and of the need to do more than is currently being done to ensure that ethnic disparities in access to human embryonic stem cell-based therapies do not arise. Specifically, this paper explains why the problem of unequal biological access will likely arise in the United States in such a way that white Americans will disproportionately receive most of the benefits of the therapies resulting from human embryonic stem cell research. It also argues for why there is an obligation to prevent these ethnic disparities in access from happening and outlines four steps that need to be taken towards meeting this obligation.
To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...
Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...
Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...
Moller, Mark S
In 2003, Ruth Faden and eighteen other colleagues argued that a "problem of unequal biological access" is likely to arise in access to therapies resulting from human embryonic stem cell research. They showed that unless deliberate steps are taken in the United States to ensure that the human embryonic stem cell lines available to researchers mirrors the genetic diversity of the general population, white Americans will likely receive the benefits of these therapies to the relative exclusion of minority ethnic groups. Over the past five years the problem of unequal biological access has not received much attention from politicians, bioethicists and even many researchers in the United States, in spite of the widely held belief in the country that there is an obligation to prevent and correct ethnic disparities in access to medical care. The purpose of this paper is to increase awareness of the problem of unequal biological access and of the need to do more than is currently being done to ensure that ethnic disparities in access to human embryonic stem cell-based therapies do not arise. Specifically, this paper explains why the problem of unequal biological access will likely arise in the United States in such a way that white Americans will disproportionately receive most of the benefits of the therapies resulting from human embryonic stem cell research. It also argues for why there is an obligation to prevent these ethnic disparities in access from happening and outlines four steps that need to be taken towards meeting this obligation. PMID:18823539
The developmental effects of xenobiotics on differentiation can be profiled using mouse embryonic stem cells (mESCs). The adherent cell differentiation and cytotoxicity (ACDC) technique was used to evaluate a library of 42 metal and metaloid salts. Jl mESCs were allowed to prolif...
Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience
Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.
Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie
In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374
The developmental effects of xenobiotics on differentiation can be profiled using mouse embryonic stem cells (mESCs). The adherent cell differentiation and cytotoxicity (ACDC) technique was used to evaluate a library of 42 metal and metaloid salts. Jl mESCs were allowed to prolif...
The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...
Sakamoto, Tatsunori; Nakagawa, Takayuki; Endo, Tsuyoshi; Kim, Tae-Soo; Iguchi, Fukuichiro; Naito, Yasushi; Sasai, Yoshiki; Ito, Juichi
The potential of embryonic stem (ES) cells to differentiate into inner ear hair cells was examined in this study. Undifferentiated mouse ES cells transplanted into neomycin-damaged mouse inner ears were evaluated by immunohistochemistry 4 weeks after transplantation. Some ES cells were positive for E-cadherin or NCAM, and most transplanted cells were positive for SSEA3 and Ki67. None were positive for Myosin VIIa or MF20. These results indicate that the damaged inner ear may have some activity inducing ES cells to develop into ectoderm cells, but the effect was insufficient to induce inner ear hair cells. Next, SDIA/BMP-treated ES cells were transplanted into embryonic chicken inner ear rudiments. Embryonic chickens were expected to share the same developmental systems as mice. SDIA/BMP treatment drove ES cells to the population including neural crest cells and probably placode cells ES colonies were found next to or in the otic vesicles but were not a part of vesicle walls, indicating that transplanted ES cells could not be expected to be the same kind of cells as chicken otic vesicle cells Some ES colonies were found at the vestibulo-cochlear ganglions. To induce inner ear hair cells in this system, the competency of ES cells and otic induction signals should be defined further.
Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein
Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process.
Heiskanen, Annamari; Satomaa, Tero; Tiitinen, Sari; Laitinen, Anita; Mannelin, Sirkka; Impola, Ulla; Mikkola, Milla; Olsson, Cia; Miller-Podraza, Halina; Blomqvist, Maria; Olonen, Anne; Salo, Hanna; Lehenkari, Petri; Tuuri, Timo; Otonkoski, Timo; Natunen, Jari; Saarinen, Juhani; Laine, Jarmo
Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi
Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.
Hong, Ki-Sung; Bae, Daekyeong; Choi, Youngsok; Kang, Sun-Woong; Moon, Sung-Hwan; Lee, Hoon Taek; Chung, Hyung-Min
Pluripotent human embryonic stem cells (hESCs) acquire mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process. Here, we report a simple and an efficient isolation method for mesenchymal stem cells (MSCs) from hESCs undergoing EMT using a commercialized porous membrane transwell culture insert. Suspension culture of hESC colonies results in the formation of embryoid bodies, which adhered on the upper compartment of 8 μm porous membrane in the presence of EMG2-MV media. The population migrating through the permeable membrane to the lower compartment not only exhibited EMT markers but also expressed high levels of a panel of typical MSC surface antigen markers, and demonstrated multipotent differentiation capability. In addition, they have a prolonged proliferation capacity without characteristics and chromosomal changes. Furthermore, the isolated MSCs significantly enhanced cardiac functions in a rat model of myocardial infarction (MI) as measured by the left ventricle wall thickness (MI control, 32.9%±3.2% vs. hESCs-MSCs, 38.7%±2.4%), scar length (MI control, 46.1%±2.5% vs. hESCs-MSCs, 41.8%±1.3%), fibrosis area (MI control, 34.3%±1.6% vs. hESCs-MSCs, 28.9%±3.5%), and capillary density. Our findings demonstrate an ease with which hESCs-MSCs can be effectively isolated using the porous membrane, which overcomes the lack of availability of MSCs for therapeutic applications in various diseased animal models.
Pluripotential embryonic stem (ES) cells have been derived very efficiently from spare human embryos produced by IVF and grown in culture to the nascent blastocyst stage. The inner cell mass (ICM) is isolated by immunosurgery and grown on selected embryonic fibroblast monolayer cultures. ICM cells lose their memory for axis during formation of ES cell colonies and are then unable to integrate tissue formation with a body plan. ES cells form teratomas in vivo with cells and tissues representative of the three major embryonic lineages (ectoderm, mesoderm, endoderm). The ES cells are continuously renewable and can be directed to differentiate into early progenitors of neural stem cells (Noggin cells) and from there into mature neurons and glia (astrocytes and oligodendrocytes). The neural stem cells formed from human ES cells repopulate the brains of newborn mice when injected into the lateral cerebral ventricles, forming astrocytes dominantly in the parenchyma. The human neural cells can be observed migrating from the subventricular areas along the rostral migratory stream. Human neurons can be found in the olfactory bulb. Human ES cells can also be directed into cardiomyocytes when co-cultured with visceral endoderm-like cells (END-2). These observations provide further scope to explore stem cell therapies, gene therapies and drug discovery. For compatible transplantation, ES may need to be derived with a range of HLA types or by nuclear transplantation or stem cell fusion.
Kumagai, Ayako; Suga, Mika; Yanagihara, Kana; Itoh, Yumi; Furue, Miho K.
Mitochondrial oxidative phosphorylation is a major source of cellular ATP. Its usage as an energy source varies, not only according to the extracellular environment, but also during development and differentiation, as indicated by the reported changes in the flux ratio of glycolysis to oxidative phosphorylation during embryonic stem (ES) cell differentiation. The fluorescent probe JC-1 allows visualization of changes in the mitochondrial membrane potential produced by oxidative phosphorylation. Strong JC-1 signals were localized in the differentiated cells located at the edge of H9 ES colonies that expressed vimentin, an early differentiation maker. The JC-1 signals were further intensified when individual adjacent colonies were in contact with each other. Time-lapse analyses revealed that JC-1-labeled H9 cells under an overconfluent condition were highly differentiated after subculture, suggesting that monitoring oxidative phosphorylation in live cells might facilitate the prediction of induced pluripotent stem cells, as well as ES cells, that are destined to lose their undifferentiated potency. Significance Skillful cell manipulation is a major factor in both maintaining and disrupting the undifferentiation potency of human embryonic stem (hES) cells. Staining with JC-1, a mitochondrial membrane potential probe, is a simple monitoring method that can be used to predict embryonic stem cell quality under live conditions, which might help ensure the future use of hES and human induced pluripotent stem cells after subculture. PMID:26819254
Schiele, Nathan R.; Marturano, Joseph E.; Kuo, Catherine K.
Tendons are connective tissues required for motion and are frequently injured. Poor healing and inadequate return to normal tissue structure and mechanical function make tendon a prime candidate for tissue engineering, however functional tendons have yet to be engineered. The physical environment, from substrate stiffness to dynamic mechanical loading, may regulate tenogenic stem cell differentiation. Tissue stiffness and loading parameters derived from embryonic development may enhance tenogenic stem cell differentiation and tendon tissue formation. We highlight current understanding of the mechanical environment experienced by embryonic tendons and how progenitor cells may sense and respond to physical inputs. We further discuss how mechanical factors have only recently been used to induce tenogenic fate in stem cells. PMID:23916867
Schiele, Nathan R; Marturano, Joseph E; Kuo, Catherine K
Tendons are connective tissues required for motion and are frequently injured. Poor healing and inadequate return to normal tissue structure and mechanical function make tendon a prime candidate for tissue engineering; however functional tendons have yet to be engineered. The physical environment, from substrate stiffness to dynamic mechanical loading, may regulate tenogenic stem cell differentiation. Tissue stiffness and loading parameters derived from embryonic development may enhance tenogenic stem cell differentiation and tendon tissue formation. We highlight the current understanding of the mechanical environment experienced by embryonic tendons and how progenitor cells may sense and respond to physical inputs. We further discuss how mechanical factors have only recently been used to induce tenogenic fate in stem cells.
Olynik, Brendan M.; Rastegar, Mojgan
Epigenetic changes occur throughout life from embryonic development into adulthood. This results in the timely expression of developmentally important genes, determining the morphology and identity of different cell types and tissues within the body. Epigenetics regulate gene expression and cellular morphology through multiple mechanisms without alteration in the underlying DNA sequences. Different epigenetic mechanisms include chromatin condensation, post-translational modification of histone proteins, DNA cytosine marks, and the activity of non-coding RNA molecules. Epigenetics play key roles in development, stem cell differentiation, and have high impact in human disease. In this review, we will discuss our current knowledge about these epigenetic mechanisms, with a focus on histone and DNA marks. We will then talk about the genetics and epigenetics of embryonic stem cell self-renewal and differentiation into neural stem cells, and further into specific neuronal cell types. PMID:22629283
Wang, Yun; Yu, Xiaohua; Baker, Christopher; Murphy, William L.; McDevitt, Todd C.
Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications. PMID:26597546
Tuano, Natasha K; Okabe, Jun; Ziemann, Mark; Cooper, Mark E; El-Osta, Assam
Histone methylation by lysine methyltransferase enzymes regulate the expression of genes implicated in lineage specificity and cellular differentiation. While it is known that Set7 catalyzes mono-methylation of histone and non-histone proteins, the functional importance of this enzyme in stem cell differentiation remains poorly understood. We show Set7 expression is increased during mouse embryonic stem cell (mESC) differentiation and is regulated by the pluripotency factors, Oct4 and Sox2. Transcriptional network analyses reveal smooth muscle (SM) associated genes are subject to Set7-mediated regulation. Furthermore, pharmacological inhibition of Set7 activity confirms this regulation. We observe Set7-mediated modification of serum response factor (SRF) and mono-methylation of histone H4 lysine 4 (H3K4me1) regulate gene expression. We conclude the broad substrate specificity of Set7 serves to control key transcriptional networks in embryonic stem cells.
Srivastava, Nishit; Venugopalan, Vijay; Divya, M.S.; Rasheed, V.A.
Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950
Soucie, Erinn L.; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J.-C.; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H.
Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. PMID:26797145
Glass, Carley; Singla, Reetu; Arora, Anshu; Singla, Dinender K
Embryonic stem (ES) cells are pluripotent stem cells capable of self-renewal and have broad differentiation potential yielding cell types from all three germ layers. In the absence of differentiation inhibitory factors, when cultured in suspension, ES cells spontaneously differentiate and form three-dimensional cell aggregates termed embryoid bodies (EBs). Although various methods exist for the generation of EBs, the hanging drop method offers reproducibility and homogeneity from a predetermined number of ES cells. Herein, we describe the in vitro differentiation of mouse embryonic stem cells into cardiac myocytes using the hanging drop method and immunocytochemistry to identify cardiomyogenic differentiation. In brief, ES cells, placed in droplets on the lid of culture dishes following a 2-day incubation, yield embryoid bodies, which are resuspended and plated. 1-2 weeks following plating of the EBs, spontaneous beating areas can be observed and staining for specific cardiac markers can be achieved.
Bhartiya, Deepa; Kasiviswananthan, Sandhya; Shaikh, Ambreen
It has been suggested that testicular germ stem cells represent the only adult body stem cells that dedifferentiate and reprogram into a pluripotent state without any genetic modification. Emerging debate about the authenticity of embryonic stem cell (ES)-like cells derived from adult testicular tissue has prompted us to put forth this letter. We wish to reinforce our findings that pluripotent very small embryonic-like stem cells (VSELs) exist as a small population in adult mammalian testis and may result in ES-like colonies. Because of their small size, it is felt that VSELs could be contaminating the initial cells used for seeding, although efforts were made to place a single germ cell per well in a 96-well plate for clonal expansion, or magnetic activated cell sorting (MACS)-sorted α6 integrin positive cells were used. On a similar note, it is felt that the presence of VSELs in various tissues along with mesenchymal stem cells (MSCs) may provide an alternative explanation to the transdifferentiation potential of MSCs. We conclude that like Oct-4 biology, presence of VSELs in adult body tissues has somewhat surprised stem cell biologists.
Snykers, Sarah; De Kock, Joery; Rogiers, Vera; Vanhaecke, Tamara
Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells. PMID:19056906
Sokolov, Mykyta V.; Neumann, Ronald D.
Human embryonic stem cells, which are derived from the inner cell mass of the blastocyst, have become an object of intense study over the last decade. They possess two unique properties that distinguish them from many other cell types: (i) the ability to self-renew indefinitely in culture under permissive conditions, and (ii) the pluripotency, defined as the capability of giving rise to all cell types of embryonic lineage under the guidance of the appropriate developmental cues. The focus of many recent efforts has been on the elucidating the signaling pathways and molecular networks operating in human embryonic stem cells. These cells hold great promise in cell-based regenerative therapies, disease modeling, drug screening and testing, assessing genotoxic and mutagenic risks associated with exposures to a variety of environmental factors, and so forth. Ionizing radiation is ubiquitous in nature, and it is widely used in diagnostic and therapeutic procedures in medicine. In this paper, our goal is to summarize the recent progress in understanding how human embryonic stem cells respond to ionizing radiation exposures, using novel methodologies based on “omics” approaches, and to provide a critical discussion of what remains unknown; thus proposing a roadmap for the future research in this area. PMID:22966236
The excitatory neurons of the mammalian cerebral cortex arise from asymmetric divisions of radial glial cells in the ventricular zone and symmetric division of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ) of the embryonic cortex. Little is known about the microenvironment in which IPCs divide or whether a stem cell niche exists in the SVZ of the embryonic cortex. Recent evidence suggests that vasculature may provide a niche for adult stem cells but its role in development is less clear. We have investigated the vasculature in the embryonic cortex during neurogenesis and find that IPCs are spatially and temporally associated with blood vessels during cortical development. Intermediate progenitors mimic the pattern of capillaries suggesting patterns of angiogenesis and neurogenesis are coordinated during development. More importantly, we find that IPCs divide near blood vessel branch points suggesting that cerebral vasculature establishes a stem cell niche for intermediate progenitors in the SVZ. These data provide novel evidence for the presence of a neurogenic niche for intermediate progenitors in the embryonic SVZ and suggest blood vessels are important for proper patterning of neurogenesis. PMID:19346271
Seiler, Andrea E M; Spielmann, Horst
In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.
Okech, William; Kuo, Catherine K
Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.
Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.
Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.
Hug, Kristina; Hermerén, Göran
While scientific community disagrees about similarities and differences between human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells, some politicians embrace translational hiPS cell research as a replacement for translational hES cell research. We examine the ethical relevance of the main differences between hES and hiPS cell-based therapies and discuss whether, given the current state of knowledge, certain differences are essential. We discuss whether well-founded preferences can be made in hypothetical scenarios with varying levels of patient safety, treatment efficacy, treatment accessibility and ethical controversy.
Behfar, Atta; Perez-Terzic, Carmen; Faustino, Randolph S; Arrell, D Kent; Hodgson, Denice M; Yamada, Satsuki; Puceat, Michel; Niederländer, Nicolas; Alekseev, Alexey E; Zingman, Leonid V; Terzic, Andre
Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-alpha-induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.
Research using human embryos and embryonic stem cells is viewed as important for various reasons. Apart from questions concerning legal regulations, numerous ethical objections are raised pertaining to the use of surplus embryos from reproductive medicine as well as the creation of embryos and stem cells through cloning. In the hopes of avoiding ethical problems, alternatives have been proposed including the extraction of egg cells from "dead" embryos derived from in vitro fertilization procedures, the extraction of pluripotent stem cells from blastocysts, technologies such as "altered nuclear transfer" (ANT) and "oocyte-assisted reprogramming" (ANT-OAR) as well as parthenogenesis. Initial ethical assessments show that certain questions pertaining to such strategies have remained unanswered. Furthermore, with the help of new or more differentiated biotechnological procedures, it is possible to create chimeras and hybrids in which human and non-human cells are combined. Human-animal chimeras, in which gametes or embryonic tissue have been mixed with embryonic or adult stem cells, demonstrate a different "quality" and "degree of penetration" from those produced in previous experiments. Not only does this have consequences regarding questions of patentability, this situation also raises fundamental questions concerning the human being's self image, the concept of person, identity and species and the moral rights and duties that are connected with such concepts. There is a need for legal regulation, on the national as well as the international level.
Sun, Fan; An, Shi-Min; Tang, Ya-Bin; Meng, Shuang; Wang, Cong-Hui; Shen, Ying; Chen, Hong-Zhuan; Zhu, Liang
Glutamate behaves as the principal excitatory neurotransmitter in the vertebrate central nervous system and recently demonstrates intercellular signaling activities in periphery cancer cells. How the glutamatergic transmission is organized and operated in cancer stem cells remains undefined. We have identified a glutamatergic transmission circuit in embryonal carcinoma stem cells. The circuit is organized and operated in an autocrine mechanism and suppresses the cell proliferation and motility. Biological analyses determined a repertoire of glutamatergic transmission components, glutaminase, vesicular glutamate transporter, glutamate NMDA receptor, and cell membrane excitatory amino-acid transporter, for glutamate biosynthesis, package for secretion, reaction, and reuptake in mouse and human embryonal carcinoma stem cells. The glutamatergic components were also identified in mouse transplanted teratocarcinoma and in human primary teratocarcinoma tissues. Released glutamate acting as the signal was directly quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Genetic and pharmacological abolishment of the endogenously released glutamate-induced tonic activation of the NMDA receptors increased the cell proliferation and motility. The finding suggests that embryonal carcinoma stem cells can be actively regulated by establishing a glutamatergic autocrine/paracrine niche via releasing and responding to the transmitter. PMID:27322683
Huntriss, John; Picton, Helen M
Imprinted genes are expressed predominantly or exclusively from one allele only. This mode of gene expression makes the regulation of imprinted genes susceptible to epigenetic insults, which may in turn lead to disease. There is compelling experimental evidence that certain aspects of assisted reproductive technology (ART) such as in vitro cell culture may have adverse effects on the regulation of epigenetic information in mammalian embryos, including the disruption of imprinted genes and epigenetic regulators. Moreover, in humans, disorders of genomic imprinting have been reported in children conceived by ART. The derivation and in vitro culture of embryonic stem (ES) cells are potential points of origin for epigenetic abnormalities. There is evidence that defects of genomic imprinting occur in mouse embryonic stem cells, with similar data now emerging in related studies in non-human primate and human ES cells. It is therefore pertinent to rigorously assess the epigenetic status of all stem cells and their derivatives prior to their therapeutic use in humans. Focusing on the stability of genomic imprinting, this review discusses the current evidence for epigenetic disruption in mammalian embryonic stem cells in light of the epigenetic disruption observed in ART-derived mammalian embryos.
Gámez Escalona, José Antonio
The possibility to isolate, cultivate, preserve, characterize and differentiate Human Embryonic Stem Cells (ES) discovered by James Thomson and his colleagues in 1998 was a milestone in the history of Stem Cell Research. Immediately after this discovery many speculations were made about the therapeutic possibilities of ES, motivated by ideological, political and economic aspects. The episode made clear the lack of scientific rationality and ethics when assessing realities as meaningful as those of human embryos obtained by in vitro fertilization techniques (IVF) or human eggs. Therapeutic Cloning as a promise to produce ″tailored″ Stem Cells reported by Hwang and his team in 2004, ended up being a scandal within the scientific community. The technical difficulties and ethical controversies that arose from obtaining ES were insurmountable. In 2010 only two clinical trials were reported using these cells. Those trials were abandoned in late 2011 arguing financial reasons. The discovery of Induced Pluripotent Stem Cell (iPS) in 2006 in mice and in 2007 in humans, represented the possibility of obtaining pluripotent stem cells without the need to destroy embryos. Today, the absence of clinical trials using ES, caused by financial difficulties as a result of its ineffectiveness, anticipates that the use of ES will be limited to certain experimental controls. Probably, the main contribution of Embryonic Stem Cells will be the understanding that biomedical research should follow an ethically and rationally based rigorous method that cannot be ignore.
Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y
Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.
Fernández, M; Pirondi, S; Chen, B L; Del Vecchio, G; Alessandri, M; Farnedi, A; Pession, A; Feki, A; Jaconi, M E E; Calzà, L
The establishment of rat embryonic stem cells constitutes a precious tool since rat has been extensively used in biomedical research, in particular for the generation of human neurodisease animal models. Up to now only a few studies have described the isolation of rat embryonic stem-like cells. One out of 9 isolated rat embryonic stem-like cell lines (B1-RESC) obtained from a 4.5-day post-coitum blastocyst were extensively characterized and kept in culture for up to 80 passages on feeders with LIF. The stable growth of these cells and the expression of pluripotent markers were confirmed up to a high number of passages in culture, also in the absence of feeders and LIF. B1-RESC expresses the three germ layers markers both in vitro, within differentiating embryoid bodies, and in vivo through teratoma formation. Collectively, the B1-RESC line with a stable near-diploid karyotype can be used as a highly sensitive tool for testing anti-proliferative molecules. Copyright © 2011 Wiley Periodicals, Inc.
Li, Guangnan; Fang, Li; Fernández, Gloria; Pleasure, Samuel J
Adult neurogenesis represents a unique form of plasticity in the dentate gyrus requiring the presence of long-lived neural stem cells (LL-NSCs). However, the embryonic origin of these LL-NSCs remains unclear. The prevailing model assumes that the dentate neuroepithelium throughout the longitudinal axis of the hippocampus generates both the LL-NSCs and embryonically produced granule neurons. Here we show that the NSCs initially originate from the ventral hippocampus during late gestation and then relocate into the dorsal hippocampus. The descendants of these cells are the source for the LL-NSCs in the subgranular zone (SGZ). Furthermore, we show that the origin of these cells and their maintenance in the dentate are controlled by distinct sources of Sonic Hedgehog (Shh). The revelation of the complexity of both the embryonic origin of hippocampal LL-NSCs and the sources of Shh has important implications for the functions of LL-NSCs in the adult hippocampus.
Chen, Aaron; Lieu, Deborah K; Freschauf, Lauren; Lew, Valerie; Sharma, Himanshu; Wang, Jiaxian; Nguyen, Diep; Karakikes, Ioannis; Hajjar, Roger J; Gopinathan, Ajay; Botvinick, Elliot; Fowlkes, Charless C; Li, Ronald A; Khine, Michelle
A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.
Tecirlioglu, R Tayfur; Trounson, Alan O
Reproductive technologies have made impressive advances since the 1950s owing to the development of new and innovative technologies. Most of these advances were driven largely by commercial opportunities and the potential improvement of farm livestock production and human health. Companion animals live long and healthy lives and the greatest expense for pet owners are services related to veterinary care and healthcare products. The recent development of embryonic stem cell and nuclear transfer technology in primates and mice has enabled the production of individual specific embryonic stem cell lines in a number of species for potential cell-replacement therapy. Stem cell technology is a fast-developing area in companion animals because many of the diseases and musculoskeletal injuries of cats, dogs and horses are similar to those in humans. Nuclear transfer-derived stem cells may also be selected and directed into differentiation pathways leading to the production of specific cell types, tissues and, eventually, even organs for research and transplantaton. Furthermore, investigations into the treatment of inherited or acquired pathologies have been performed mainly in mice. However, mouse models do not always faithfully represent the human disease. Naturally occurring diseases in companion animals can be more ideal as disease models of human genetic and acquired diseases and could help to define the potential therapeutic efficiency and safety of stem cell therapies. In the present review, we focus on the economic implications of companion animals in society, as well as recent biotechnological progress that has been made in horse, dog and cat embryonic stem cell derivation.
The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...
The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...
Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.
Sigova, Alla A.; Mullen, Alan C.; Molinie, Benoit; Gupta, Sumeet; Orlando, David A.; Guenther, Matthew G.; Almada, Albert E.; Lin, Charles; Sharp, Phillip A.; Giallourakis, Cosmas C.; Young, Richard A.
Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. PMID:23382218
Sperber, Henrik; Mathieu, Julie; Wang, Yuliang; Ferreccio, Amy; Hesson, Jennifer; Xu, Zhuojin; Fischer, Karin A; Devi, Arikketh; Detraux, Damien; Gu, Haiwei; Battle, Stephanie L; Showalter, Megan; Valensisi, Cristina; Bielas, Jason H; Ericson, Nolan G; Margaretha, Lilyana; Robitaille, Aaron M; Margineantu, Daciana; Fiehn, Oliver; Hockenbery, David; Blau, C Anthony; Raftery, Daniel; Margolin, Adam A; Hawkins, R David; Moon, Randall T; Ware, Carol B; Ruohola-Baker, Hannele
For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.
Ghanbarian, Hossein; Wagner, Nicole; Michiels, Jean-François; Cuzin, François; Wagner, Kay-Dietrich; Rassoulzadegan, Minoo
Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.
Bouzenita, Anke I
This paper gives insight into the Islamic bioethical discussion on harvesting and using human embryonic (hESC) and adult stem cells. It describes some of the Islamic legal mechanisms involved in the bioethical discourse among Muslims. As the contemporary Islamic bioethical discourse is very diverse, the paper focuses on the critical discussion of related resolutions of the Saudi-based Islamic Fiqh Academy due to the esteem in which the IFA is held in the Islamic world and the pertinence of their rulings on this issue. This study discusses the different sources of human adult and embryonic stem cells and their use from an Islamic perspective, while questioning some directions the Islamic bioethical discourse has taken. The paper invites interested parties to deliberate the use of some of the legal means resorted to in the ongoing Islamic bioethical discourse.
Mokhber Dezfouli, Mohammad Reza; Chaleshtori, Sirous Sadeghian; Dehghan, Mohammad Mehdi; Tavanaeimanesh, Hamid; Baharvand, Hossein; Tahamtani, Yaser
Lung diseases cause great morbidity and mortality. The choice of effective medical treatment is limited and the number of lung diseases are difficult to treat with current treatments. The embryonic stem cells (ESCs) have the potential to differentiate into cell types of all three germinal layers, including lung epithelial cells. So they can be a potential source for new cell therapies for hereditary or acquired diseases of the airways and lungs. One method for treatment of lung diseases is cell therapy and the use of ESCs that can replace the damaged epithelial and endothelial cells. Progress using ESCs has developed slowly for lung regeneration because differentiation of lung cells from ESCs is more difficult as compared to differentiation of other cells. The review studies the therapeutic effects of differentiated lung cells from embryonic stem cells in lung diseases. There are few studies of differentiation of ESCs into a lineage of respiratory and then investigation of this cell in experimental model of lung diseases.
Ghanbarian, Hossein; Wagner, Nicole; Michiels, Jean-François; Cuzin, François; Wagner, Kay-Dietrich; Rassoulzadegan, Minoo
Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine. PMID:28165496
Wichterle, Hynek; Peljto, Mirza; Nedelec, Stephane
A growing number of specific cell types have been successfully derived from embryonic stem cells (ES cells), including a variety of neural cells. In vitro generated cells need to be extensively characterized to establish functional equivalency with their in vivo counterparts. The ultimate test for the ability of ES cell-derived neurons to functionally integrate into neural networks is transplantation into the developing central nervous system, a challenging technique limited by the poor accessibility of mammalian embryos. Here we describe xenotransplantation of mouse embryonic stem cell-derived motor neurons into the developing chick neural tube as an alternative for testing the ability of in vitro generated neurons to survive, integrate, extend axons, and form appropriate synaptic contacts with functionally relevant targets in vivo. Similar methods can be adapted to study functionality of other mammalian cells, including derivatives of human ES cells.
Li, Xiang-Yun; Jia, Qing; Di, Ke-Qian; Gao, Shu-Min; Wen, Xiao-Hui; Zhou, Rong-Yan; Wei, Wei; Wang, Li-Ze
The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.
Li, Lin-Yan; Cao, Fen-Fang; Su, Zhi-Jian; Zhang, Qi-Hao; Dai, Xiao-Yong; Xiao, Xue; Huang, Ya-Dong; Zheng, Qing; Xu, Hua
Rhizoma Atractylodes macrocephala, Radix Isatidis, Coptis chinensis and Flos Genkwa are common herbal remedies used by pregnant woman in China. In this study, their potential embryotoxicity was assessed using the embryonic stem cell test (EST) and a prediction model. The potential embryotoxicity of the herbs was based on three endpoints: the concentrations of the compounds that inhibited the proliferation of 50% of embryonic stem cells (ESCs) (IC50ES), the concentrations that inhibited 50% of 3T3 cells (IC503T3), and the concentrations that inhibited the differentiation of 50% of ESCs (ID50ES). The results revealed that Rhizoma Atractylodes macrocephala and Radix Isatidis are non-embryotoxic compounds. Coptis chinensis extracts appeared to demonstrated weak embryotoxicity, and Flos Genkwa exhibited strong embryotoxicity. These results may be useful in guiding the clinical use of these herbs and in expanding the application of the EST to the field of traditional Chinese medicine.
Elstner, A; Damaschun, A; Kurtz, A; Stacey, G; Arán, B; Veiga, A; Borstlap, J
Legislation in individual member states of the European Union on human embryonic stem cell (hESC) research is as divergent as the different cultural, ethical, and religious views on the issue. On the occasion of the public launch of the European Human Embryonic Stem Cell Registry (hESCreg: www.hescreg.eu), a two-day symposium was held on 18 and 19 January 2008 in Berlin to offer participants an overview of state-of-the-art hESC research and legislation throughout Europe and in selected regions of the world. Thirty leading scientists from Europe as well as from the United States, Japan, and Australia reported on a range of aspects related to research on hESC and reviewed the key elements of the newly established hESCreg database of hESC lines. In this article we summarize and complete the information on the current status of international hESC regulation.
In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells.
Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia Mc; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez
Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration.
Denissova, Natalia G; Tereshchenko, Irina V; Cui, Eric; Stambrook, Peter J; Shao, Changshun; Tischfield, Jay A
Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5-10Gy of X-rays. This induction is 50-100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.
Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E
We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.
van Dartel, Dorien A M; Schulpen, Sjors H; Theunissen, Peter T; Bunschoten, Annelies; Piersma, Aldert H; Keijer, Jaap
Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
George, Sophia H. L.; Gertsenstein, Marina; Vintersten, Kristina; Korets-Smith, Ella; Murphy, John; Stevens, Mary E.; Haigh, Jody J.; Nagy, Andras
Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F1 hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F1 hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10–40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function. PMID:17360545
Wetsel, Rick A; Wang, Dachun; Calame, Daniel G
Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell-based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized.
Wetsel, Rick A.; Wang, Dachun; Calame, Daniel G.
Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell–based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized. PMID:21226612
Ma, Quanfu; Kuang, Anren
Patients with irreversible hypothyroidism require lifelong levo-thyroxin ( L-T4) replacement therapy, which makes them feel discomfortable. With the development of the thyroid tissues autotransplantation and embryonic stem cell (ESC), this would be a more physiological approach to the treatment of irreversible hypothyroidism. The animal experiments and human clinical trials on thyroid tissues autotransplantation have shown that the autograft can survive and function. The advanced researches have demonstrated that ESC can differentiate into thyroid follicular cells.
Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko
The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345799
Jacquet, Laureen; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko
The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345813
Devito, Liani; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko
The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345806
Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko
The KCL037 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345800
Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi
There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512
Read a case study to learn more about how Dr. Bradley Merrill of the University of Illinois at Chicago and his lab were able to perform their first gene expression array experiment comparing a mutant mouse embryonic stem cell line to a non-mutant control line using GenePattern, an application supported by the Molecular Analysis Tools Knowledge Center which provides bioinformatics tools for gene expression, proteomic and SNP analysis.
Akopian, Veronika; Andrews, Peter W; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; McKay, Ronald D G; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K W; Pera, Martin F; Rossant, Janet; Stacey, Glyn N; Suemori, Hirofumi
There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.
Lefort, Nathalie; Feyeux, Maxime; Bas, Cécile; Féraud, Olivier; Bennaceur-Griscelli, Annelise; Tachdjian, Gerard; Peschanski, Marc; Perrier, Anselme L
By analyzing five human embryonic stem (hES) cell lines over long-term culture, we identified a recurrent genomic instability in the human genome. An amplification of 2.5-4.6 Mb at 20q11.21, encompassing approximately 23 genes in common, was detected in four cell lines of different origins. This amplification, which has been associated with oncogenic transformation, may provide a selective advantage to hES cells in culture.
In this work, we explore how embryonic stem (ES) cell differentiation patterns are affected by surface interactions with plasma-processed materials. We hypothesize that mouse embryonic stem-cell exposure to certain plasma-polymerized tetraglyme surfaces will direct their differentiation into endothelial cells. R1 mouse embryonic stem (ES) cells were plated on surfaces onto which tetraglyme was deposited by plasma polymerization. In addition, tissue-treated polystyrene and control glass cover slips were also examined. Some samples were fixed three days after plating and immunofluorescence stained with platelet endothelial-cell adhesion molecule, while the others were fixed seven days after plating and immunofluorescence stained with von Willebrand Factor. Positive results seen by ES cell derivatives precociously expressing the vWF and PECAM genetic markers on the plasma-polymerized tetraglyme treated surfaces suggest that the plasma-polymerized surfaces direct differentiation of ES cells into endothelial cells. Research goals of this dissertation include: characterization of the material properties of the plasma-polymerized tetraglyme surfaces that induce directed differentiation of ES cells into endothelial cells, optimization of the plasma-polymerization process to maximize the number of endothelial cells derived from R1 ES cells, and biological experimentation to characterize properties of the mechanism of directed differentiation. A potential application of this work is in the design and construction of an artificial blood vessel. Current small-scale arterial substitutes have proved inadequate because of thrombogenicity and infection. Moreover, the lower blood flow velocities of smaller vessels pose a different set of design criteria and introduce new problems not encountered in large arterial substitutes. By utilizing a tissue engineering approach that incorporates embryonic stem cell-derived endothelial cells, the longevity of the prosthesis can be ensured.
Wang, Zhi-qiang; Chen, Kevin; Ying, Qi-long; Li, Ping
Monoamine oxidase (MAO) A is the major metabolizing enzyme of serotonin (5-hydroxytryptamine, 5-HT) which regulates early brain development. In this study, wild-type (WT) and MAO Aneo embryonic stem (ES) cell lines were established from the inner cell mass of murine blastocysts and their characteristics during ES and differentiating stages were studied. Our results show that the differentiation to neural cells in MAO Aneo ES cells was reduced compared to WT, suggesting MAO A played a regulatory role in stem cells neural differentiation. PMID:21607742
Farber, Debora B; Katsman, Diana
Mouse embryonic stem cells (mESCs) release into the medium in which they are cultured heterogeneous populations of microvesicles (mESMVs), important components of cell-cell communication, that transfer their contents not only to other stem cells but also to cells of other origins. The purpose of these studies was to demonstrate that ESMVs could be the signals that lead the retinal progenitor Müller cells to de-differentiate and re-entry the cell cycle, followed by differentiation along retinal lineages. Indeed, we found that ESMVs induce these processes and change Müller cells' microenvironment towards a more permissive state for tissue regeneration.
Waite, Lee; Nindl, Gabi
Stem cells refer to a broad class of cells with potential for prolonged self-renewal and the potential to differentiate. Human embryonic stem (ES) cells are derived from a fertilized human egg, before it has been implanted into a womb. Recently published research has used human embryos, produced by in-vitro fertilization for clinical purposes, to develop lines of pluripotent human ES cells. This review discusses the legal status and ethical implications of ES cell research with special attention paid to the situation in the USA and Germany.
Langerova, Alena; Fulka, Helena; Fulka, Josef
The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.
Giachino, Claudia; Orlando, Luca; Turinetto, Valentina
Recent studies have shown that mouse embryonic stem cells (mESCs) rely on a distinctive genome caretaking network. In this review, we will discuss how mESCs functionally respond to DNA damage and describe several modifications in mESC DNA damage response, which accommodate dynamic cycling and preservation of genetic information. Subsequently, we will discuss how the transition from mESCs to adult stem/progenitor cells can be involved in the decline of tissue integrity and function in the elderly. PMID:23358251
Hopkinson, Branden M.; Kalisz, Mark; Vestentoft, Peter Siig; Juel Rasmussen, Lene; Bisgaard, Hanne Cathrine
Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless, a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation, oxidative phosphorylation is essential at all stages of differentiation. PMID:28265337
The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.
sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene expression profiles of...AD_____________ Award Number: W81XWH-12-1-0106 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making Improve
Conclusion. We have used FAC sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene...AD_____________ Award Number: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...4. TITLE AND SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic
satellites ” resulted in reversion of the cells to an epithelial state, re-entry into the cell cycle, and restoration of their ability to generate both...1 AWARD NUMBER: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer...W81XWH-12-1-0107 Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making
Gasimli, Leyla; Hickey, Anne Marie; Yang, Bo; Li, Guoyun; dela Rosa, Mitche; Nairn, Alison V; Kulik, Michael J; Dordick, Jonathan S; Moremen, Kelley W; Dalton, Stephen; Linhardt, Robert J
Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Differentiation of embryonic stem cells markedly changes the proteoglycanome. The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. Copyright © 2013 Elsevier B.V. All rights reserved.
Gao, Qian; Zhao, Lili; Song, Ziyi; Yang, Gongshe
Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.
Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta
The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785
Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian
The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).
Yang, Yang; Liu, Bei; Xu, Jun; Wang, Jinlin; Wu, Jun; Shi, Cheng; Xu, Yaxing; Dong, Jiebin; Wang, Chengyan; Lai, Weifeng; Zhu, Jialiang; Xiong, Liang; Zhu, Dicong; Li, Xiang; Yang, Weifeng; Yamauchi, Takayoshi; Sugawara, Atsushi; Li, Zhongwei; Sun, Fangyuan; Li, Xiangyun; Li, Chen; He, Aibin; Du, Yaqin; Wang, Ting; Zhao, Chaoran; Li, Haibo; Chi, Xiaochun; Zhang, Hongquan; Liu, Yifang; Li, Cheng; Duo, Shuguang; Yin, Ming; Shen, Huan; Belmonte, Juan Carlos Izpisua; Deng, Hongkui
Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.
Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer; Sharov, Alexei A; Migueles, Rosa Portero; Ko, Minoru S H; Brickman, Joshua M
Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.
Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang
In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.
Morgani, Sophie M.; Canham, Maurice A.; Nichols, Jennifer; Sharov, Alexei A.; Migueles, Rosa Portero; Ko, Minoru S.H.; Brickman, Joshua M.
Summary Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants. PMID:23746443
Suri, Shalu; Singh, Ankur; Nguyen, Anh H.; Bratt-Leal, Andres M.; McDevitt, Todd C.
In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments. PMID:24113509
Pieters, Tim; van Roy, Frans
Pluripotent embryonic stem cells (ESCs) can self-renew or differentiate into any cell type within an organism. Here, we focus on the roles of cadherins and catenins - their cytoplasmic scaffold proteins - in the fate, maintenance and differentiation of mammalian ESCs. E-cadherin is a master stem cell regulator that is required for both mouse ESC (mESC) maintenance and differentiation. E-cadherin interacts with key components of the naive stemness pathway and ablating it prevents stem cells from forming well-differentiated teratomas or contributing to chimeric animals. In addition, depleting E-cadherin converts naive mouse ESCs into primed epiblast-like stem cells (EpiSCs). In line with this, a mesenchymal-to-epithelial transition (MET) occurs during reprogramming of somatic cells towards induced pluripotent stem cells (iPSCs), leading to downregulation of N-cadherin and acquisition of high E-cadherin levels. β-catenin exerts a dual function; it acts in cadherin-based adhesion and in WNT signaling and, although WNT signaling is important for stemness, the adhesive function of β-catenin might be crucial for maintaining the naive state of stem cells. In addition, evidence is rising that other junctional proteins are also important in ESC biology. Thus, precisely regulated levels and activities of several junctional proteins, in particular E-cadherin, safeguard naive pluripotency and are a prerequisite for complete somatic cell reprogramming.
Mthunzi, Patience; Dholakia, Kishan; Gunn-Moore, Frank
Owing to their self renewal and pluripotency properties, stem cells can efficiently advance current therapies in tissue regeneration and/or engineering. Under appropriate culture conditions in vitro, pluripotent stem cells can be primed to differentiate into any cell type some examples including neural, cardiac and blood cells. However, there still remains a pressing necessity to answer the biological questions concerning how stem cell renewal and how differentiation programs are operated and regulated at the genetic level. In stem cell research, an urgent requirement on experimental procedures allowing non-invasive, marker-free observation of growth, proliferation and stability of living stem cells under physiological conditions exists. Femtosecond (fs) laser pulses have been reported to non-invasively deliver exogenous materials, including foreign genetic species into both multipotent and pluripotent stem cells successfully. Through this multi-photon facilitated technique, directly administering fs laser pulses onto the cell plasma membrane induces transient submicrometer holes, thereby promoting cytosolic uptake of the surrounding extracellular matter. To display a chemical-free cell transfection procedure that utilises micro-litre scale volumes of reagents, we report for the first time on 70 % transfection efficiency in ES-E14TG2a cells using the enhanced green fluorescing protein (EGFP) DNA plasmid. We also show how varying the average power output during optical transfection influences cell viability, proliferation and cytotoxicity in embryonic stem cells. The impact of utilizing objective lenses of different numerical aperture (NA) on the optical transfection efficiency in ES-E14TG2a cells is presented. Finally, we report on embryonic and mesenchymal stem cell differentiation. The produced specialized cell types could thereafter be characterized and used for cell based therapies.
Faravelli, Irene; Bucchia, Monica; Rinchetti, Paola; Nizzardo, Monica; Simone, Chiara; Frattini, Emanuele; Corti, Stefania
Motor neurons are cells located in specific areas of the central nervous system, such as brain cortex (upper motor neurons), brain stem, and spinal cord (lower motor neurons), which maintain control over voluntary actions. Motor neurons are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs): these disorders share symptoms related to muscular atrophy and paralysis leading to death. No effective treatments are currently available. Stem cell-derived motor neurons represent a promising research tool in disease modeling, drug screening, and development of therapeutic approaches for MNDs and spinal cord injuries. Directed differentiation of human pluripotent stem cells - human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) - toward specific lineages is the first crucial step in order to extensively employ these cells in early human development investigation and potential clinical applications. Induced pluripotent stem cells (iPSCs) can be generated from patients' own somatic cells (for example, fibroblasts) by reprogramming them with specific factors. They can be considered embryonic stem cell-like cells, which express stem cell markers and have the ability to give rise to all three germ layers, bypassing the ethical concerns. Thus, hiPSCs constitute an appealing alternative source of motor neurons. These motor neurons might be a great research tool, creating a model for investigating the cellular and molecular interactions underlying early human brain development and pathologies during neurodegeneration. Patient-specific iPSCs may also provide the premises for autologous cell replacement therapies without related risks of immune rejection. Here, we review the most recent reported methods by which hESCs or iPSCs can be differentiated toward functional motor neurons with an overview on the potential clinical applications.
Correia, Marcelo; Rodrigues, Ana S; Perestrelo, Tânia; Pereira, Sandro L; Ribeiro, Marcelo F; Sousa, Maria I; Ramalho-Santos, João
Kaempferol (3,4',5,7-tetrahydroxyflavone) is a natural flavonoid with several beneficial and protective effects. It has been demonstrated that kaempferol has anticancer properties, particularly due to its effects on proliferation, apoptosis and the cell cycle. However, possible effects on pluripotent embryonic stem cell function have not yet been addressed. Embryonic stem cells have the ability to self-renew and to differentiate into all three germ layers with potential applications in regenerative medicine and in vitro toxicology. We show that exposure of murine embryonic stem cells (mESC) to high concentrations of kaempferol (200 μM) leads to decreased cell numbers, although the resulting smaller cell colonies remain pluripotent. However, lower concentrations of this compound (20 μM) increase the expression of pluripotency markers in mESCs. Mitochondrial membrane potential and mitochondrial mass are not affected, but a dose-dependent increase in apoptosis takes place. Moreover, mESC differentiation is impaired by kaempferol, which was not related to apoptosis induction. Our results show that low concentrations of kaempferol can be beneficial for pluripotency, but inhibit proper differentiation of mESCs. Additionally, high concentrations induce apoptosis and increase mitochondrial reactive oxygen species (ROS).
Chaerkady, Raghothama; Letzen, Brian; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Kumar, Praveen; All, Angelo H.; Thakor, Nitish V.; Delanghe, Bernard; Gearhart, John D.; Pandey, Akhilesh; Kerr, Candace L.
Oligodendrocytes (OLs) are glial cells of the central nervous system which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs), as well as providing a model to study human oligodendrocyte development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during oligodendrocyte differentiation including neural precursors (NPCs), glial precursors (GPCs), and oligodendrocyte progenitors (OPCs) compared to undifferentiated embryonic stem cells. Using a 1% false discovery rate cutoff, ~3,145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as TF, NCAM1, APOE, and WNT5A showed increased expression from the NPC to OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including FABP4, THBS1, BMP1, CRYAB, TF, TNC, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human embryonic stem cell differentiation into oligodendrocyte progenitor cells, this study provides many novel proteins that are potentially involved in OL development. PMID:21770034
Hu, Deqing; Garruss, Alexander S; Gao, Xin; Morgan, Marc A; Cook, Malcolm; Smith, Edwin R; Shilatifard, Ali
Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalentlymarked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation.
Kunafina, E R; Chaplina, M V; Filiasova, E I; Gibanova, N V; Khodarovich, Iu M; Larionov, O A; Zatsepina, O V
We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.
Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative Fundamental Cryobiology of Multiple Mouse Embryonic Stem Cell Lines and the Implications for Embryonic Stem Cell Cryopreservation Protocols
Kashuba, Corinna M.; Benson, James D.; Critser, John K.
The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols. PMID:24384367
Akyash, Fatemeh; Sadeghian-Nodoushan, Fatemeh; Tahajjodi, Somayyeh Sadat; Nikukar, Habib; Farashahi Yazd, Ehsan; Azimzadeh, Mostafa; D Moore, Harry; Aflatoonian, Behrouz
This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27(th) April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.
Centanin, Lázaro; Ander, Janina-J; Hoeckendorf, Burkhard; Lust, Katharina; Kellner, Tanja; Kraemer, Isabel; Urbany, Cedric; Hasel, Eva; Harris, William A; Simons, Benjamin D; Wittbrodt, Joachim
The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.
Zhao, Zhu-Ran; Yu, Wei-Dong; Shi, Cheng; Liang, Rong; Chen, Xi; Feng, Xiao; Zhang, Xue; Mu, Qing; Shen, Huan; Guo, Jing-Zhu
Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. Thus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.
Qin, Sisi; Liao, Wenbin; Ma, Yupo; Simon, Marcia; Rafailovich, Miriam; Stony Brook Medical Center Collaboration; Stony Brook Dental Schoo Collaboration
Currently, proliferation and differentiation of stem cell is usually accomplished either in vivo, or on chemical coated tissue culture petri dish with the presence of feeder cells. Here we investigated whether they can be directly cultured on polymeric substrates, in the absence of additional factors. We found that mouse embryonic stem cells did not require gelatin and could remain in the undifferentiated state without feeder cells at least for four passages on partially sulfonated polystyrene. The modulii of cells was measured and found to be higher for cells plated directly on the polymer surface than for those on the same surface covered with gelatin and feeder cells. When plated with feeder cells, the modulii was not sensitive to gelatin. Whereas the differentiation properties of human bone marrow stem cells, which are not adherent, are less dependent on either chemical or mechanical properties of the substrate. However, they behave differently on different toughness hydrogels as oppose to on polymer coated thin films.
Zhao, Zhu-ran; Yu, Wei-dong; Shi, Cheng; Liang, Rong; Chen, Xi; Feng, Xiao; Zhang, Xue; Mu, Qing; Shen, Huan; Guo, Jing-zhu
Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. Thus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced. PMID:28250757
Tsagias, Nikos; Kouzi-Koliakos, Kokkona; Karagiannis, Vasileios; Tsikouras, P; Koliakos, George G
Recently the matrix of umbilical cord began to use as an alternative source of stem cells additionally to the blood of umbilical cord. Umbilical cord has been used mainly for mesenchymal stem cell banking. The immunological characteristics of mesenchymal stem cells in combination with their ability to avoid rejection make them an attractive biological material for transplantations. In this study the isolation of small in size pluripotent stem cells from umbilical cord expressing early transcription factors with characteristics that resemble to embryonic stem cells is investigated. Pluripotent stem cells were isolated from human umbilical cords, by a new strategy method based on unique characteristics such as the small size and the positivity on early transcription factors OCT and Nanog. An enriched population of CXCR4(+) OCT(+) Nanog(+) CD45(-) small stem cells from the cord was isolated. This fraction was able to create alkaline phosphatase positive like spheres forms in a mesenchymal layer with multilineage differentiation capacity. Our results were assessed by RT PCR and electophoresis for the pluripotent genes. These data suggest that umbilical cord provides an attractive source not only of mesenchymal stem cells but moreover of pluripotent stem cells. The method described herein should be applied in the field of stem cell banking in addition to the classical umbilical cord harvesting method. Isolation of a population of cells with pluripotent characteristics from umbilical cord. Adoption of a second centrifugation step for the pluripotent stem isolation. Increasing the value of the cord and explaining the pluripotency. This work will enhance the value of umbilical cord harvesting.
Hurlbut, William B
Ethical controversy in stem cell research arises because current methods to produce embryonic stem cell lines require the destruction of living human embryos. For this reason, there is increasing interest in developing alternative, non-embryonic sources of pluripotent stem cells. This effort is especially important in the US due to the prevailing policy against federal funding of embryo-destructive research. Altered nuclear transfer (ANT) is one of several potential methods to develop alternative sources of pluripotent stem cells. This approach employs the technique of somatic cell nuclear transfer, but the somatic cell nucleus or egg cytoplasm (or both) are first altered before the somatic cell nucleus is transferred into the oocyte. This alteration precludes the coordinated organization and developmental potential that is necessary for the resulting biological entity to be an embryo, but it still allows the entity to generate pluripotent stem cells. Proof-of-principle for one variant of ANT has been established in mice by silencing the functional expression of the gene Cdx2 in the somatic cell nucleus prior to its transfer into an enucleated egg. From the resulting non-embryonic laboratory construct, fully functional pluripotent stem cells were procured. Other more recent studies have suggested the possibility of achieving the same results by preemptively silencing maternally derived Cdx2 messenger RNA in the egg before the act of nuclear transfer. The procedure would produce the equivalent of a tissue culture of pluripotent stem cells. In contrast to the use of embryos 'left over' from clinical in vitro fertilization, ANT could produce pluripotent stem cell lines with an unlimited range of specifically selected and controlled genotypes. Such flexibility would greatly facilitate the study of disease, drug development, and toxicology testing, and may allow the production of therapeutically useful pluripotent stem cells that are immune-compatible. If developed to
Gamie, Zakareya; MacFarlane, Robert J; Tomkinson, Alicia; Moniakis, Alexandros; Tran, Gui Tong; Gamie, Yehya; Mantalaris, Athanasios; Tsiridis, Eleftherios
Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources. In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases. Enhanced results have been found when combining bone marrow-derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue-derived stem cells and umbilical cord tissue-derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.
Lombaert, Isabelle. M.A.; Hoffman, Matthew. P.
Salivary gland organogenesis involves the specification, maintenance, lineage commitment, and differentiation of epithelial stem/progenitor cells. Identifying how stem/progenitor cells are directed along a series of cell fate decisions to form a functional salivary gland will be necessary for future stem cell regenerative therapy. The identification of stem/progenitor cells within the salivary gland has focused on their role in postnatal glands and little is known about them in embryonic glands. Here, we have reviewed the information available for other developing organ systems and used it to determine whether similar cell populations exist in the mouse submandibular gland. Additionally, using growth factors that influence salivary gland epithelial morphogenesis during development, we have taken a simple experimental approach asking whether any of these growth factors influence early developmental lineages within the salivary epithelium on a transcriptional level. These preliminary findings show that salivary epithelial stem/progenitor populations exist within the gland, and that growth factors that are reported to control epithelial morphogenesis may also impact cell fate decisions. Further investigation of the signaling networks that influence stem/progenitor cell behavior will allow us to hypothesize how we might induce autologous stem cells to regenerate damaged salivary tissue in a therapeutic context. PMID:20428013
Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen
Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.
Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.
SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168
Kucia, Magda; Zuba-Surma, Ewa K; Wysoczynski, Marcin; Wu, Wan; Ratajczak, Janina; Machalinski, Boguslaw; Ratajczak, Mariusz Z
A population of CXCR4(+) lin(-) CD45(-) cells that express SSEA, Oct-4 and Nanog has been identified in adult bone marrow. These cells are very small and display several features typical for primary embryonic stem cells such as: i) a large nuclei surrounded by a narrow rim of cytoplasm; ii) open-type chromatin (euchromatin); and iii) high telomerase activity. These cells were named very small embryonic-like stem cells (VSEL-SC). The authors hypothesized that they are direct descendants of the germ lineage. Germ lineage, in order to pass genes on to the next generation, has to create soma and thus becomes a 'mother lineage' for all somatic cell lineages present in the adult body. Germ potential is established after conception in a totipotent zygote and retained subsequently during development in blastomers of morula, cells form the inner cell mass of blastocyst, epiblast and population of primordial germ cells. The authors envision that VSEL-SC are epiblast-derived pluripotent stem cells and could potentially become a less-controversial source of stem cells for regeneration.
A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors.
Zhao Yong . E-mail: firstname.lastname@example.org; Wang Honglan; Mazzone, Theodore
We identified stem cells from the umbilical cord blood, designated cord blood-stem cells (CB-SC). CB-SC displayed important embryonic stem (ES) cell characteristics including expression of ES-cell-specific molecular markers including transcription factors OCT-4 and Nanog, along with stage-specific embryonic antigen (SSEA)-3 and SSEA-4. CB-SC also expressed hematopoietic cell antigens including CD9, CD45 and CD117, but were negative for CD34. CB-SC displayed very low immunogenicity as indicated by expression of a very low level of major histocompatibility complex (MHC) antigens and failure to stimulate the proliferation of allogeneic lymphocytes. CB-SC could give rise to cells with endothelial-like and neuronal-like characteristics in vitro, as demonstrated by expression of lineage-associated markers. Notably, CB-SC could be stimulated to differentiate into functional insulin-producing cells in vivo and eliminated hyperglycemia after transplantation into a streptozotocin-induced diabetic mouse model. These findings may have significant potential to advance stem-cell-based therapeutics.
Xiong, Anqi; Kundu, Soumi; Forsberg, Maud; Xiong, Yuyuan; Bergström, Tobias; Paavilainen, Tanja; Kjellén, Lena; Li, Jin-Ping; Forsberg-Nilsson, Karin
Heparan sulfate proteoglycans (HSPGs), ubiquitous components of mammalian cells, play important roles in development and homeostasis. These molecules are located primarily on the cell surface and in the pericellular matrix, where they interact with a multitude of macromolecules, including many growth factors. Manipulation of the enzymes involved in biosynthesis and modification of HSPG structures alters the properties of stem cells. Here, we focus on the involvement of heparanase (HPSE), the sole endo-glucuronidase capable of cleaving of HS, in differentiation of embryonic stem cells into the cells of the neural lineage. Embryonic stem (ES) cells overexpressing HPSE (Hpse-Tg) proliferated more rapidly than WT ES cells in culture and formed larger teratomas in vivo. In addition, differentiating Hpse-Tg ES cells also had a higher growth rate, and overexpression of HPSE in NSPCs enhanced Erk and Akt phosphorylation. Employing a two-step, monolayer differentiation, we observed an increase in HPSE as wild-type (WT) ES cells differentiated into neural stem and progenitor cells followed by down-regulation of HPSE as these NSPCs differentiated into mature cells of the neural lineage. Furthermore, NSPCs overexpressing HPSE gave rise to more oligodendrocytes than WT cultures, with a concomitant reduction in the number of neurons. Our present findings emphasize the importance of HS, in neural differentiation and suggest that by regulating the availability of growth factors and, or other macromolecules, HPSE promotes differentiation into oligodendrocytes. Copyright © 2016 Elsevier B.V. All rights reserved.
Ilkilic, Ilhan; Ertin, Hakan
Rapid technological developments in human embryonic stem cell research are holding promises of future new medical treatment for a range of currently incurable chronic diseases. At the same time, stem cell research using human embryos raises radically new, previously unimaginable ethical issues posing a dramatic challenge to humankind. By analysing the discourses on these ethical issues we can show that the cultural values and religious convictions of all stakeholders involved play a decisive role in formulating ethical positions. In the Islamic world, too, stem cell research using human embryos provokes new discussions about the moral status of the embryo according to Islamic ethical norms. In our paper we describe the theological and philosophical criteria used in this debate and discuss some ethical positions vis-à-vis embryonic stem cell research formulated in the Islamic world, including official regulations existing in some Muslim countries. While most of the existing literature in this field is primarily descriptive, the present paper endeavours to examine not only the arguments and their historical conditions as such; in addition, we will for the first time provide a critical reflection on the methodology underlying commonly held positions. In our view, this reflection is of paramount importance in establishing a straightforward constructive dialogue between different cultures and academic disciplines.
Gokhale, Paul J; Au-Young, Janice K; Dadi, SriVidya; Keys, David N; Harrison, Neil J; Jones, Mark; Soneji, Shamit; Enver, Tariq; Sherlock, Jon K; Andrews, Peter W
We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.
A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors. PMID:28018144
Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang
Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411
Li, Shuping; Lan, He; Men, Hongsheng; Wu, Yuanyuan; Li, Ning; Capecchi, Mario R; Bryda, Elizabeth C; Wu, Sen
Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. Stem Cells Translational Medicine 2017;6:340-351. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.
Fujii, Yoichi R.
Embryonic stem cells (ESCs) are capable of undergoing self-renewal, and their developmental ability is known as the stemness. Recently, microRNAs (miRNAs) as regulators have been isolated from ESCs. Although Dicer and DiGeorge syndrome critical region gene 8 (DGCR8) are essential factors for the biogeneration of miRNA, Dicer-knockout (KO) ESCs have showed to fail to express differentiation markers and DGCR8-KO ESCs have showed to be arrest in the G1 phase. Furthermore, Dicer-KO ESCs lost the ability to epigenetically silence retroelemtns (REs). REs are expressed and transposed in ESCs, whose transcripts control expression of miRNAs, and their transposable retroelement (TE) expression is, therefore related to ESC proliferation and differentiation, suggesting that the interplay between miRNAs and REs may have a deep responsibility for the stemness including a short G1/S transition and for RE regulation in ESCs. PMID:20835360
Bollerot, Karine; Pouget, Claire; Jaffredo, Thierry
The developmental origin of hematopoietic stem cells has been for decades the subject of great interest. Once thought to emerge from the yolk sac, hematopoietic stem cells have now been shown to originate from the embryonic aorta. Increasing evidence suggests that hematopoietic stem cells are produced from an endothelial intermediate designated by the authors as hemangioblast or hemogenic endothelium. Recently, the allantois in the avian embryo and the placenta in the mouse embryo were shown to be a site of hematopoietic cell production/expansion and thus appear to play a critical role in the formation of the hematopoietic system. In this review we shall give an overview of the data obtained from human, mouse and avian models on the cellular origins of the hematopoietic system and discuss some aspects of the molecular mechanisms controlling hematopoietic cell production.
Holm, Frida; Hellqvist, Eva; Mason, Cayla N.; Ali, Shawn A.; Delos-Santos, Nathaniel; Barrett, Christian L.; Chun, Hye-Jung; Minden, Mark D.; Moore, Richard A.; Marra, Marco A.; Runza, Valeria; Frazer, Kelly A.; Sadarangani, Anil; Jamieson, Catriona H. M.
Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination. PMID:26621726
Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J
Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.
Desai, Nina; Rambhia, Pooja; Gishto, Arsela
Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.
Banda, Erin; McKinsey, Anna; Germain, Noelle; Carter, James; Anderson, Nickesha Camille; Grabel, Laura
Embryonic stem cells (ESCs) undergoing neural differentiation form radial arrays of neural stem cells, termed neural rosettes. These structures manifest many of the properties associated with embryonic and adult neurogenesis, including cell polarization, interkinetic nuclear migration (INM), and a gradient of neuronal differentiation. We now identify novel rosette structural features that serve to localize key regulators of neurogenesis. Cells within neural rosettes have specialized basal as well as apical surfaces, based on localization of the extracellular matrix receptor β1 integrin. Apical processes of cells in mature rosettes terminate at the lumen, where adherens junctions are apparent. Primary cilia are randomly distributed in immature rosettes and tightly associated with the neural stem cell's apical domain as rosettes mature. Components of two signaling pathways known to regulate neurogenesis in vivo and in rosettes, Hedgehog and Notch, are apically localized, with the Hedgehog effector Smoothened (Smo) associated with primary cilia and the Notch pathway γ-secretase subunit Presenilin 2 associated with the adherens junction. Increased neuron production upon treatment with the Notch inhibitor DAPT suggests a major role for Notch signaling in maintaining the neural stem cell state, as previously described. A less robust outcome was observed with manipulation of Hedgehog levels, though consistent with a role in neural stem cell survival or proliferation. Inhibition of both pathways resulted in an additive effect. These data support a model by which cells extending a process to the rosette lumen maintain neural stem cell identity whereas release from this association, either through asymmetric cell division or apical abscission, promotes neuronal differentiation.
Lebkowski, J S; Gold, J; Xu, C; Funk, W; Chiu, C P; Carpenter, M K
Human embryonic stem (hES) cells can proliferate extensively in culture and can differentiate into representatives of all three embryonic germ layers in vitro and in vivo. The undifferentiated hES cells have now been cultured for more than 50 passages in vitro, yet maintain a normal karyotype. The hES cells express a series of specific surface antigens, as well as OCT-4 and human telomerase, proteins associated with a pluripotent and immortal phenotype. On differentiation, OCT-4 and human telomerase expression decreases with the emergence of a maturing population of cells. During hES cell differentiation, modulation of the expression of many genes has been evaluated using microarray analysis. To improve the ease, reproducibility, and scalability of hES culture, methods have been developed to propagate the cells in the absence of mouse embryonic cell feeders. hES cells maintained in culture using extracellular matrix factors together with mouse embryonic cell conditioned medium proliferate indefinitely while maintaining a normal karyotype, proliferation rate, and complement of undifferentiated cell markers. hES cells cultured without feeder layers retain their capacity to differentiate into cells of all three germ layers in vitro and in teratomas. The hES cells can also be genetically modified transiently or stably using both plasmid and viral gene transfer agents. These analyses and technological developments will aid in the realization of the full potential of hES cells for both research and therapeutic applications.
Gauthaman, K; Manasi, N; Bongso, A
Background and purpose: Statins inhibit proliferation of various human cancer cell lines in vitro. As human embryonic stem cells (hESCs) possess neoplastic-like properties we have evaluated the role of various statins on karyotypically normal hESCs (HES3 and BG01), abnormal hESCs (BG01V) and breast adenocarcinoma cells (MCF-7) to evaluate whether the mode of action of the statins was via a stemness pathway. Experimental approach: All cell lines were treated with simvastatin, pravastatin, lovastatin and mevastatin (1 µmol·L−1 to 20 µmol·L−1) up to 7 days and their effects on cell proliferation, cell cycle, apoptosis and pluripotency studied. Key results: All four statins did not inhibit HES3 and BG01 proliferation, but BG01V and MCF-7 were inhibited by simvastatin, lovastatin and mevastatin. These inhibitory effects were reversed by the endogenous isoprenoids, farnesylpyrophosphate and geranylgeranylpyrophosphate. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and cell cycle assay confirmed apoptosis in BG01V and MCF-7. Stem cell surface markers [stage-specific embryonic antigen-4, tumour rejection antigen-1-81, octamer-4 (OCT-4)] were expressed in HES3 and BG01, but not in BG01V cells, even after prolonged treatment with simvastatin. In BG01V and MCF-7, the pro-apoptotic Bcl-2-associated X protein genes were up-regulated, while the antiapoptotic BCL2 and SURVIVIN genes were down-regulated. Expression of the stemness-related genes namely, the growth differentiation factor-3, NANOG and OCT-4 was decreased in BG01V compared with BG01 and HES3. Conclusions and implications: Normal hESCs were resistant to prolonged exposure to statins over a range of doses, compared with BG01V and MCF-7, probably because of genetic and behavioural differences. The statins not only have anti-cancer properties but can suppress abnormal hESCs thus promoting growth of normal hESCs in vitro. PMID:19438511
Needham, Karina; Hyakumura, Tomoko; Gunewardene, Niliksha; Dottori, Mirella; Nayagam, Bryony A
In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.
Blaber, Elizabeth A.; Finkelstein, Hayley; Dvorochkin, Natalya; Sato, Kevin Y.; Yousuf, Rukhsana; Burns, Brendan P.; Globus, Ruth K.
Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth's gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth's gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth. PMID:26414276
Blaber, Elizabeth A; Finkelstein, Hayley; Dvorochkin, Natalya; Sato, Kevin Y; Yousuf, Rukhsana; Burns, Brendan P; Globus, Ruth K; Almeida, Eduardo A C
Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth's gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth's gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth.
Sauerzweig, Steven Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger
The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.
Bhartiya, Deepa; Hinduja, Indira; Patel, Hiren; Bhilawadikar, Rashmi
The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing
Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph
A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.
Krais, Annette M.; Mühlbauer, Karl-Rudolf; Kucab, Jill E.; Chinbuah, Helena; Cornelius, Michael G.; Wei, Quan-Xiang; Hollstein, Monica; Phillips, David H.; Arlt, Volker M.; Schmeiser, Heinz H.
We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by 32P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells. PMID:25230394
Feng, Guowei; Cui, Jian; Zheng, Yizhou; Han, Zhongchao; Xu, Yong; Li, Zongjin
The progress of stem cell research, along with technological innovation, has brought researchers to focus on the potential role of stem cells in regenerative medicine. Ethical and technological issues have limited the applications of human embryonic stem cells (hESCs) in this field. As a promising candidate, very small embryonic-like stem cells (VSELs) express a multitude of pluripotent stem cell markers and demonstrate the ability to differentiate into three germ-layer lineages in vitro. Optimized methods for isolation and expansion of VSELs have aroused the scientific community's interest in use of this kind of cells for regenerative purposes. In this review, we will focus on the biological characteristics, as well as the potentiality and remaining challenges in clinical application of VSELs. Moreover, a comparison among VSELs and the other pluripotent stem cells will be illustrated to highlight the unique advantages of VSELs.
Deglincerti, Alessia; Etoc, Fred; Ozair, M. Zeeshan; Brivanlou, Ali H.
The developing embryo is a remarkable example of self-organization, where functional units are created in a complex spatio-temporal choreography. Recently, human embryonic stem cells (ESCs) have been used to recapitulate in vitro the self-organization programs that are executed in the embryo in vivo. This represents a unique opportunity to address self-organization in humans that is otherwise not addressable with current technologies. In this essay, we review the recent literature on self-organization of human ESCs, with a particular focus on two examples: formation of embryonic germ layers and neural rosettes. Intriguingly, both activation and elimination of TGFβ signaling can initiate self-organization, albeit with different molecular underpinnings. We discuss the mechanisms underlying the formation of these structures in vitro and explore future challenges in the field. PMID:26970615
Amit, Michal; Itskovitz-Eldor, Joseph
The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.
Deglincerti, Alessia; Etoc, Fred; Ozair, M Zeeshan; Brivanlou, Ali H
The developing embryo is a remarkable example of self-organization, where functional units are created in a complex spatiotemporal choreography. Recently, human embryonic stem cells (ESCs) have been used to recapitulate in vitro the self-organization programs that are executed in the embryo in vivo. This represents an unique opportunity to address self-organization in humans that is otherwise not addressable with current technologies. In this chapter, we review the recent literature on self-organization of human ESCs, with a particular focus on two examples: formation of embryonic germ layers and neural rosettes. Intriguingly, both activation and elimination of TGFβ signaling can initiate self-organization, albeit with different molecular underpinnings. We discuss the mechanisms underlying the formation of these structures in vitro and explore future challenges in the field.
Williams, Matthew J.; Hamazaki, Takashi; Terada, Naohiro; Clapp, William L.; Adin, Christopher; Ellison, Gary W.; Jorgensen, Marda; Batich, Christopher D.
The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways. PMID:19729441
Li, Victor C.; Kirschner, Marc W.
Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells. PMID:24979803
Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru
Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.
Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T
Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.
Li, Victor C; Kirschner, Marc W
Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells.
Hwang, Woo Suk; Ryu, Young June; Park, Jong Hyuk; Park, Eul Soon; Lee, Eu Gene; Koo, Ja Min; Jeon, Hyun Yong; Lee, Byeong Chun; Kang, Sung Keun; Kim, Sun Jong; Ahn, Curie; Hwang, Jung Hye; Park, Ky Young; Cibelli, Jose B; Moon, Shin Yong
Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.
Bodak, Maxime; Cirera-Salinas, Daniel; Yu, Jian; Ngondo, Richard P; Ciaudo, Constance
A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer-deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE-1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self-renewal and genome integrity by controlling L1s regulation.
Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen
We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366
Lee, Han; Park, Jung; Forget, Bernard G; Gaines, Peter
Human embryonic stem cells (ESCs) can be induced to differentiate into a wide range of tissues that soon could be used for therapeutic applications in regenerative medicine. Despite their developmental potential, sources used to generate human ESC lines raise serious ethical concerns, which recently prompted efforts to reprogram somatic cells back to a pluripotent state. These efforts resulted in the generation of induced pluripotent stem (iPS) cells that are functionally similar to ESCs. However, the genetic manipulations required to generate iPS cells may complicate their growth and developmental characteristics, which poses serious problems in predicting how they will behave when used for tissue-regenerative purposes. In this article we summarize the recently developed methodologies used to generate iPS cells, including those that minimize their genetic manipulation, and discuss several important complicating features of iPS cells that may compromise their future use for therapies in regenerative medicine.
Li, Shuping; Lan, He; Men, Hongsheng; Wu, Yuanyuan; Li, Ning; Capecchi, Mario R; Bryda, Elizabeth C; Wu, Sen
: Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. To date, no species other than the mouse have derived transgene-free induced pluripotent stem (iPS) cells that can truly mimic ES cell quality. In the current study, episomal vectors were used to obtain rat transgene-free iPS cells, which contributed to chimeric
Apáti, Agota; Orbán, Tamás I; Varga, Nóra; Németh, Andrea; Schamberger, Anita; Krizsik, Virág; Erdélyi-Belle, Boglárka; Homolya, László; Várady, György; Padányi, Rita; Karászi, Eva; Kemna, Evelien W M; Német, Katalin; Sarkadi, Balázs
Expression of multidrug resistance ABC transporters has been suggested as a functional marker and chemoprotective element in early human progenitor cell types. In this study we examined the expression and function of the key multidrug-ABC transporters, ABCB1, ABCC1 and ABCG2 in two human embryonic stem (HuES) cell lines. We detected a high level ABCG2 expression in the undifferentiated HuES cells, while the expression of this protein significantly decreased during early cell differentiation. ABCG2 in HuES cells provided protection against mitoxantrone toxicity, with a drug-stimulated overexpression of the transporter. No significant expression of ABCB1/ABCC1 was found either in the undifferentiated or partially differentiated HuES cells. Examination of the ABCG2 mRNA in HuES cells indicated the use of selected promoter sites and a truncated 3' untranslated region, suggesting a functionally distinct regulation of this transporter in undifferentiated stem cells. The selective expression of the ABCG2 multidrug transporter indicates that ABCG2 can be applied as a marker for undifferentiated HuES cells. Moreover, protection of embryonic stem cells against xenobiotics and endobiotics may depend on ABCG2 expression and regulation.
Sawan, Carla; Hernandez-Vargas, Hector; Murr, Rabih; Lopez, Fabrice; Vaissière, Thomas; Ghantous, Akram Y; Cuenin, Cyrille; Imbert, Jean; Wang, Zhao-Qi; Ren, Bing; Herceg, Zdenko
Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs. Copyright © 2013 AlphaMed Press.
Lakshmipathy, Uma; Love, Brad; Goff, Loyal A.; Jörnsten, Rebecka; Graichen, Ralph; Hart, Ronald P.; Chesnut, Jonathan D.
Many of the currently established human embryonic stem cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that miRNA, a class of non-coding small RNA that participate in the regulation of gene expression, may play a key role in stem cell self renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated human embryonic stem cells (hESC) and their corresponding differentiated cells that underwent differentiation in vitro over a period of two weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hESCs. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically-interpretable mRNAs. We identify patterns of expression in the progression from hESC to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hESC “miRNA-ome” provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis and human disease states. PMID:18004940
Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.
Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly-cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from twelve species of ethnomedically utilized plants, we found fractions from three species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. PMID:20955699
Reynertson, Kurt A; Charlson, Mary E; Gudas, Lorraine J
Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. Copyright © 2010 Elsevier Inc. All rights reserved.
Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.
Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.
El-Badawy, Ahmed; El-Badri, Nagwa
The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.
Liu, W; Stein, P; Cheng, X; Yang, W; Shao, N-Y; Morrisey, E E; Schultz, R M; You, J
Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant-negative approach suppresses Nanog expression, and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)), a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.
Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit
The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.
Azevedo-Pereira, Ricardo Luiz; Morrot, Alexandre; Machado, Gabriele Santos; Paredes, Bruno Diaz; Rodrigues, Deivid de Carvalho; de Carvalho, Antonio Carlos Campos; Mendez-Otero, Rosalia
Embryonic stem cells (ES cells) express a transient and heterogeneous pattern of molecules, which suggests a notable mechanism to control self-renewal avoid the differentiation into germ layers. We show that 9-O-acetyl GD3 (9OacGD3), a highly expressed b-series ganglioside in neural stem (NS) cells, is expressed in undifferentiated mouse ES cells in a heterogeneous fashion. After sorting, undifferentiated 9OacGD3(+) ES cell population had higher levels of nestin and Sox2 mRNA than the 9OacGD3(-) cells. Even with elevated expression of these neural transcription factors, 9OacGD3(+) cells did not give rise to more neural progenitors than 9OacGD3(-) cells. Expression of 9OacGD3 was recovered from 9OacGD3(-) cell population, demonstrating that expression of this ganglioside in mouse embryonic stem cells is transient, and does not reflect cell fate. Our findings show that the ganglioside 9OacGD3 is expressed heterogeneously and transiently in ES cells, and this expression corresponds to higher levels of Sox2 and Nestin transcripts. © 2014 International Federation for Cell Biology.
Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.
Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023
Kim, Heechul; Walczak, Piotr; Kerr, Candace; Galpoththawela, Chulani; Gilad, Assaf A.; Muja, Naser; Bulte, Jeff W.M.
Transplantation of embryonic stem cells and their neural derivatives can lead to amelioration of the disease symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Oligodendroglial progenitors (OPs), derived from human embryonic stem cells (hESC, HES-1), were labeled with superparamagnetic iron oxide and transduced with luciferase. At 7 days following induction of EAE in C57/BL6 mice, 1 × 106 cells were transplanted in the ventricles of C57/BL6 mice and noninvasively monitored by magnetic resonance and bioluminescence imaging. Cells were found to remain within the cerebroventricular system and did not survive for more than 10 days. However, EAE mice that received hESC-OPs showed a significant improvement in neurological disability scores (0.9 ± 0.2; n = 12) compared to that of control animals (3.3 ± 0.4; n = 12) at day 15 post-transplantation. Histopathologically, transplanted hESC-OPs generated TREM2-positive CD45 cells, increased TIMP-1 expression, confined inflammatory cells within the subarachnoid space, and gave rise to higher numbers of Foxp3-positive regulatory T cells in the spinal cord and spleen. Our results suggest that transplantation of hESC-OPs can alter the pathogenesis of EAE through immunomodulation, potentially providing new avenues for stem cell-based treatment of MS. PMID:22949039
Hou, Shaoping; Tom, Veronica J.; Graham, Lori
High-level spinal cord injury can lead to cardiovascular dysfunction, including disordered hemodynamics at rest and autonomic dysreflexia during noxious stimulation. To restore supraspinal control of sympathetic preganglionic neurons (SPNs), we grafted embryonic brainstem-derived neural stem cells (BS-NSCs) or spinal cord-derived neural stem cells (SC-NSCs) expressing green fluorescent protein into the T4 complete transection site of adult rats. Animals with injury alone served as controls. Implanting of BS-NSCs but not SC-NSCs resulted in recovery of basal cardiovascular parameters, whereas both cell grafts alleviated autonomic dysreflexia. Subsequent spinal cord retransection above the graft abolished the recovery of basal hemodynamics and reflexic response. BS-NSC graft-derived catecholaminergic and serotonergic neurons showed remarkable long-distance axon growth and topographical innervation of caudal SPNs. Anterograde tracing indicated growth of medullar axons into stem cell grafts and formation of synapses. Thus, grafted embryonic brainstem-derived neurons can act as functional relays to restore supraspinal regulation of denervated SPNs, thereby contributing to cardiovascular functional improvement. PMID:24155317
Scott, Christopher Thomas; Reijo Pera, Renee A
The controversies surrounding embryonic stem cell research have prompted scientists to invent beyond restrictive national policy and moral concerns. The impetus behind these reports comes from different sources, including individually held moral beliefs, societal pressures and resource constraints, both biological and financial. Along with other contributions to public policy such as advocacy or public testimony, experimentation and scientific curiosity are perhaps more natural responses scientists use to surmount impediments to research. In a research context, we review the history of the first stem cell discoveries, and describe scientific efforts leading up to recent reports of pluripotent lines made without the use of human embryos and eggs. We argue that despite the promise of these new lines, we must not lose sight of fundamental questions remaining at the frontiers of embryology and early human development. The answers to these questions will impact studies of genetics, cell biology and diseases such as cancer, autoimmunity and disorders of development. Human embryonic stem cell research is barely a decade old. The recent pace of discovery--in spite of federal restrictions--is testament to the potential of these cells to uncover some of biology's most intractable mysteries.
Background Embryonic stem cells (ESCs) have been implicated to have tremendous impact in regenerative therapeutics of various diseases, including Type 1 Diabetes. Upon generation of functionally mature ESC derived islet-like cells, they need to be implanted into diabetic patients to restore the loss of islet activity. Encapsulation in alginate microcapsules is a promising route of implantation, which can protect the cells from the recipient’s immune system. While there has been a significant investigation into islet encapsulation over the past decade, the feasibility of encapsulation and differentiation of ESCs has been less explored. Research over the past few years has identified the cellular mechanical microenvironment to play a central role in phenotype commitment of stem cells. Therefore it will be important to design the encapsulation material to be supportive to cellular functionality and maturation. Results This work investigated the effect of stiffness of alginate substrate on initial differentiation and phenotype commitment of murine ESCs. ESCs grown on alginate substrates tuned to similar biomechanical properties of native pancreatic tissue elicited both an enhanced and incrementally responsive differentiation towards endodermal lineage traits. Conclusions The insight into these biophysical phenomena found in this study can be used along with other cues to enhance the differentiation of embryonic stem cells toward a specific lineage fate. PMID:23570553
Feng, Shumei; Mo, Lijuan; Wu, Rongrong; Chen, Xiaopan; Zhang, Ming
Mouse embryonic stem cells (mESCs) have played a key role in the newly emerging fields of stem cell research. The traditional derivation and culture of mESCs have been based on the use of mouse embryonic fibroblasts (MEFs) treated with exogenous leukemia inhibitory factor (LIF). However, the rapid senescence of MEFs, coupled with the high cost of LIF, has significantly hampered the widespread use of mESCs in stem cell research. Thus, we present a novel exogenous LIF-free culture system for general mESCs applications, comprising fibroblast-like cells derived from the rabbit spleen (RSFs). We demonstrated that mESCs cultured on RSFs (mESCs-RSFs) maintained all mESC features after prolonged LIF-free culture, including alkaline phosphatase, cell surface markers (SSEA-1), molecular markers (OCT-4, NANOG, TERT, REX-1), karyotype, and pluripotency. The high expression level of both LIF and WNT3A in the RSFs may account for their ability to maintain mESCs without exogenous LIF. Moreover, this exogenous LIF-free culture system was verified to be of microbiological quality through analysis with electron transmission microscopy.
Lo, Bernard; Chou, Vicki; Cedars, Marcelle I; Gates, Elena; Taylor, Robert N; Wagner, Richard M; Wolf, Leslie; Yamamoto, Keith R
Research with human oocytes, embryos, and additional embryonic stem cell lines is needed to address important scientific questions and to fulfill the promise of stem cell transplantation for degenerative diseases. Proponents need to develop guidelines for the appropriate conduct of embryonic stem cell research. Such guidelines will help build public trust and acceptance for this research. In this article, we offer recommendations for informed consent, discussing who should give consent, what the consent process should cover, when consent should be obtained, and who should obtain consent. Consent to use embryos for research should be obtained from oocyte and sperm donors as well as from the woman or couple undergoing infertility treatment. The consent discussion must cover information that donors need to know to make an informed decision about various types of research. Donations for research should be discussed at the initiation of advanced infertility treatment and reconfirmed if possible at the time of actual donation for research. Treating assisted reproduction technology physicians can help with the consent process, provided that they are not involved in the research.
Salewski, Ryan P.; Mitchell, Robert A.; Shen, Carl
The pathology of spinal cord injury (SCI) makes it appropriate for cell-based therapies. Treatments using neural stem cells (NSCs) in animal models of SCI have shown positive outcomes, although uncertainty remains regarding the optimal cell source. Pluripotent cell sources such as embryonic stem cells (ESCs) provide a limitless supply of therapeutic cells. NSCs derived using embryoid bodies (EB) from ESCs have shown tumorigenic potential. Clonal neurosphere generation is an alternative method to generate safer and more clinically relevant NSCs without the use of an EB stage for use in cell-based therapies. We generated clonally derived definitive NSCs (dNSCs) from ESC. These cells were transplanted into a mouse thoracic SCI model. Embryonic stem cell-derived definitive neural stem cell (ES-dNSC)-transplanted mice were compared with controls using behavioral measures and histopathological analysis of tissue. In addition, the role of remyelination in injury recovery was investigated using transmission electron microscopy. The SCI group that received ES-dNSC transplantation showed significant improvements in locomotor function compared with controls in open field and gait analysis. The cell treatment group had a significant enhancement of spared neural tissue. Immunohistological assessments showed that dNSCs differentiated primarily to oligodendrocytes. These cells were shown to express myelin basic protein, associate with axons, and support nodal architecture as well as display proper compact, multilayer myelination in electron microscopic analysis. This study provides strong evidence that dNSCs clonally derived from pluripotent cells using the default pathway of neuralization improve motor function after SCI and enhance sparing of neural tissue, while remaining safe and clinically relevant. PMID:25119334
Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu
Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.
Fridley, Krista M.; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B.
Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem