Parthenogenesis-derived Multipotent Stem Cells Adapted for Tissue Engineering Applications

PubMed Central

Koh, Chester J.; Delo, Dawn M.; Lee, Jang Won; Siddiqui, M. Minhaj; Lanza, Robert P.; Soker, Shay; Yoo, James J.; Atala, Anthony

2009-01-01

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications. PMID:18799133

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127

YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Dasol; Byun, Sung-Hyun; Park, Soojeong

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 sizemore » 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.« less

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

PubMed

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

[Cell therapy for Parkinson's disease: III. Neonatal, fetal and embryonic stem cell-based applications].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Isolation and characterization of the trophectoderm from the Arabian camel (Camelus dromedarius).

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Elsafadi, Mona; Moumen, Abdullah F; Alzahrani, Faisal A; Mahmood, Amer; Alfayez, Musaad; Alowaimer, Abdullah N

2017-09-01

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developing predictions of in vivo developmental toxicity of ToxCast chemicals using mouse embryonic stem cells.

EPA Science Inventory

Developing predictions of in vivo developmental toxicity of ToxCast chemicals using mouse embryonic stem cells S. Hunter, M. Rosen, M. Hoopes, H. Nichols, S. Jeffay, K. Chandler1, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Labor...

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Human embryonic stem cell-derived pancreatic endoderm alleviates diabetic pathology and improves reproductive outcome in C57BL/KsJ-Lep(db/+) gestational diabetes mellitus mice.

PubMed

Xing, Baoheng; Wang, Lili; Li, Qin; Cao, Yalei; Dong, Xiujuan; Liang, Jun; Wu, Xiaohua

2015-07-01

Gestational diabetes mellitus is a condition commonly encountered during mid to late pregnancy with pathologic manifestations including hyperglycemia, hyperinsulinemia, insulin resistance, and fetal maldevelopment. The cause of gestational diabetes mellitus can be attributed to both genetic and environmental factors, hence complicating its diagnosis and treatment. Pancreatic progenitors derived from human embryonic stem cells were shown to be able to effectively treat diabetes in mice. In this study, we have developed a system of treating diabetes using human embryonic stem cell-derived pancreatic endoderm in a mouse model of gestational diabetes mellitus. Human embryonic stem cells were differentiated in vitro into pancreatic endoderm, which were then transplanted into db/+ mice suffering from gestational diabetes mellitus. The transplant greatly improved glucose metabolism and reproductive outcome of the females compared with the control groups. Our findings support the feasibility of using differentiated human embryonic stem cells for treating gestational diabetes mellitus patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Integration of immunological aspects in the European Human Embryonic Stem Cell Registry.

PubMed

Borstlap, Joeri; Kurtz, Andreas

2008-05-01

The immunological properties of stem cells are of increasing importance in regenerative medicine. Immunomodulatory mechanisms seem to play an important role not only with respect to the understanding of underlying mechanisms of autologous versus allogenic therapeutic approaches, but also for endogeneous tissue regeneration. The newly established European human embryonic stem cell registry (hESCreg) offers an international database for the registration, documentation and characterisation of human embryonic stem cells (hESC) and their use. By doing so, hESCreg aims to develop a model procedure for further standardisation efforts in the field of stem cell research and regenerative medicine, and eventually the registry may lead to a repository of therapy-related information. Currently the stem cell characterisation data acquired by the registry are divided into several categories such as cell derivation, culture conditions, genetic constitution, stem cell marker expression and degree of modification. This article describes immunological aspects of stem cell characterisation and explores the layout and relevance of a possible additional section to the hESCreg repository to include immunological characteristics of human embryonic stem cells.

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

NASA Astrophysics Data System (ADS)

Shinde, Vaibhav; Brungs, Sonja; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios

2016-06-01

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic 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 those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

PubMed

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.

Stem cell research as innovation: expanding the ethical and policy conversation.

PubMed

Dresser, Rebecca

2010-01-01

Research using human embryonic stem cells raises an array of complex ethical issues, including, but by no means limited to, the moral status of developing human life. Unfortunately much of the public discussion fails to take into account this complexity. Advocacy for liberal and conservative positions on human embryonic stem cell research can be simplistic and misleading. Ethical concepts such as truth-telling, scientific integrity, and social justice should be part of the debate over federal support for human embryonic stem cell research. Moreover, the debate should be conducted in accord with principles of deliberative democracy, including respect for people holding competing views.

Cell differentiation: therapeutical challenges in diabetes.

PubMed

Roche, Enrique; Vicente-Salar, Nestor; Arribas, Maribel; Paredes, Beatriz

2012-01-01

Stem cells, derived from either embryonic or adult tissues, are considered to be potential sources of insulin-secreting cells to be transplanted into type 1 and advanced stages of type 2 diabetic patients. Many laboratories have considered this possibility, resulting in a large amount of published protocols, with a wide degree of complexity among them. Our group was the first to report that it was possible to obtain insulin-secreting cells from mouse embryonic stem cells, proving the feasibility of this new challenge. The same observation was immediately reported using human embryonic stem cells. However, the resulting cell product was not properly characterised, affecting the reproducibility of the protocol by other groups. A more elaborated protocol was developed by Lumelsky and co-workers, demonstrating that neuroectodermal cells could be an alternative source for insulin-producing cells. However, the resulting cells of this protocol produced low amounts of the hormone. This aimed other groups to perform key changes in order to improve the insulin content of the resulting cells. Recently, Baetge's group has published a new protocol based on the knowledge accumulated in pancreatic development. In this protocol, human embryonic stem cells were differentiated into islet-like structures through a five step protocol, emulating the key steps during embryonic development of the endocrine pancreas. The final cell product, however, seemed to be in an immature state, thus further improvement is required. Despite this drawback, the protocol represents the culmination of work performed by different groups and offers new research challenges for the investigators in this exciting field. Concerning adult stem cells, the possibility of identifying pancreatic precursors or of reprogramming extrapancreatic derived cells are key possibilities that may circumvent the problems that appear when using embryonic stem cells, such as immune rejection and tumour formation.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

PubMed

Lee, Seung Tae; Choi, Mun Hwan; Lee, Eun Ju; Gong, Seung Pyo; Jang, Mi; Park, Sang Hyun; Jee, Hyang; Kim, Dae Yong; Han, Jae Yong; Lim, Jeong Mook

2008-11-01

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Generation of stomach tissue from mouse embryonic stem cells.

PubMed

Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2015-08-01

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.

[Cell therapy for Parkinson's disease: IV. Risks and future trends].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Stem Cells

MedlinePlus

Stem cells are cells with the potential to develop into many different types of cells in the body. They serve as a repair ... body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

PubMed

Bürgin, M T; Bürkli, P

2002-11-01

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development. PMID:23451281

A Tense Situation: Forcing Tumour Progression

DTIC Science & Technology

2009-02-01

Orthopaedic Surgery, University of California at San Francisco, San Francisco, California 94143-0512, USA § Institute for Regenerative Medicine ...growth factor signalling and force from the ECM8,9 (BOX 2). Force and embryogenesis Force has a fundamental role in directing stem cell fate and in...dictating embryonic development10–12. For instance, embryonic stem cells progressively stiffen as cells differentiate13, whereas stem cell shape and

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

PubMed

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

The role of nanotechnology in induced pluripotent and embryonic stem cells research.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen

2014-12-01

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.

Human pluripotent stem cells: an emerging model in developmental biology.

PubMed

Zhu, Zengrong; Huangfu, Danwei

2013-02-01

Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development 'in a dish'. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development.

Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions*

PubMed Central

Barone, Angela; Benktander, John; Ångström, Jonas; Aspegren, Anders; Björquist, Petter; Teneberg, Susann; Breimer, Michael. E.

2013-01-01

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 109 cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Lex pentaosylceramide, and the Ley hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated. PMID:23404501

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

PubMed

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

[Stem cells--cloning, plasticity, bioethic].

PubMed

Pflegerl, Pamina; Keller, Thomas; Hantusch, Brigitte; Hoffmann, Thomas Sören; Kenner, Lukas

2008-01-01

Stem cells with certain characteristics have become promising tools for molecular medicine. They have the potential to self-regenerate and to differentiate into specific tissues. Besides their great potential, embryonic stem cells (ESC) run the risk of enhanced tumorigenesis. The use of human embryonic stem cells (hESC) is ethically problematic because their isolation involves the destruction of human embryos. Recently developed methods generate are able to pluripotent stem cells from fibroblasts. Alternatives for ESC are adult stem cells (ASC) derived from bone marrow, cord blood, amniotic fluid and other tissues. The following article is on the basis of testimony of Lukas Kenner for the German Bundestag about the use of ESC for research, therapy and drug development. Ethical aspects are taken into consideration.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

PubMed

Ju, Jin Young; Park, Chun Young; Gupta, Mukesh Kumar; Uhm, Sang Jun; Paik, Eun Chan; Ryoo, Zae Young; Cho, Youl Hee; Chung, Kil Saeng; Lee, Hoon Taek

2008-05-01

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Which bank? A guardian model for regulation of embryonic stem cell research in Australia.

PubMed

McLennan, A

2007-08-01

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.

Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

PubMed

Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

2007-10-01

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.

Human pluripotent stem cells: an emerging model in developmental biology

PubMed Central

Zhu, Zengrong; Huangfu, Danwei

2013-01-01

Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development ‘in a dish’. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development. PMID:23362344

Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

PubMed

Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

2015-07-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

Stem cells: sources and applications.

PubMed

Vats, A; Tolley, N S; Polak, J M; Buttery, L D K

2002-08-01

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Autophagy in Human Embryonic Stem Cells

PubMed Central

Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

2011-01-01

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

[Low expression of activin A in mouse and human embryonic teratocarcinoma cells].

PubMed

Gordeeva, O F

2014-01-01

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

PubMed

Ramalho-Santos, João; Varum, Sandra; Amaral, Sandra; Mota, Paula C; Sousa, Ana Paula; Amaral, Alexandra

2009-01-01

Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction. The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells. ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates. Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-29

...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

PubMed

Gertow, Karin; Cedervall, Jessica; Jamil, Seema; Ali, Rouknuddin; Imreh, Marta P; Gulyas, Miklos; Sandstedt, Bengt; Ahrlund-Richter, Lars

2011-01-01

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

Ethical questions to ponder in the European stem cell patent debate.

PubMed

Curley, Duncan; Sharples, Andrew

2006-01-01

Patents may be refused in Europe on ethical grounds. Whereas in the past this issue has arisen only infrequently, recent developments in human embryonic stem cell research have given rise to conflicting opinions in Europe as to the approach that should be adopted in relation to patents. The United Kingdom Patent Office has adopted a positive policy towards inventions involving human embryonic stem cells, but the European Patent Office has to date refused to grant patent applications involving similar subject-matter. A series of legal questions on the role of ethics in granting European patents is now to be considered for clarification by the European Patent Office. The answers to these questions should eventually resolve the debate on the patenting of human embryonic stem cells throughout Europe.

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

PubMed

Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

PubMed

Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

2016-05-06

Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

PubMed

Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian

2012-03-01

The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

Beyond the permissibility of embryonic and stem cell research: substantive requirements and procedural safeguards.

PubMed

Isasi, Rosario M; Knoppers, Bartha M

2006-10-01

This report provides a comparative analysis of the regulation of embryonic stem cells and cloning research in 50 countries. The development of international stem cell consortia involving the exchange of materials, data and knowledge presumes 'policy know-how' on the varying positions and governing regulations of the various partners; knowledge is essential for the feasibility of such international collaborative projects. Across the spectrum of restrictive-to-liberal policies, requirements regarding the justification for or the setting of substantive limits on (i) embryo use and/or (ii) destruction in research are often present. These goals justify the regulation, the control and even the prohibition of embryonic stem cell and cloning research. Moreover, irrespective of whether a country adopts a restrictive or a liberal approach, there is significant symmetry in both the substantive and the procedural requirements. Procedural safeguards provide another layer of protection and control over the research. In reality, such safeguards may have a greater systemic impact than the substantive requirements. They can be subdivided into three broad categories: (i) safeguards relating to the stage of embryonic development, (ii) safeguards relating to the donors of blastocysts, gametes, embryos and somatic cells and (iii) requirements for research governance.

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

PubMed

Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

2015-01-01

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

In vitro spatially organizing the differentiation in individual multicellular stem cell aggregates.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yu Long; Lin, Wang; Li, Xiujun; Wang, Shuqi; Lu, Tian Jian; Xu, Feng

2016-01-01

With significant potential as a robust source to produce specific somatic cells for regenerative medicine, stem cells have attracted increasing attention from both academia and government. In vivo, stem cell differentiation is a process under complicated regulations to precisely build tissue with unique spatial structures. Since multicellular spheroidal aggregates of stem cells, commonly called as embryoid bodies (EBs), are considered to be capable of recapitulating the events in early stage of embryonic development, a variety of methods have been developed to form EBs in vitro for studying differentiation of embryonic stem cells. The regulation of stem cell differentiation is crucial in directing stem cells to build tissue with the correct spatial architecture for specific functions. However, stem cells within the three-dimensional multicellular aggregates undergo differentiation in a less unpredictable and spatially controlled manner in vitro than in vivo. Recently, various microengineering technologies have been developed to manipulate stem cells in vitro in a spatially controlled manner. Herein, we take the spotlight on these technologies and researches that bring us the new potential for manipulation of stem cells for specific purposes.

Intermolecular Interactions of Homologs of Germ Plasm Components in Mammalian Germ Cells

PubMed Central

Fox, Mark S.; Clark, Amander T.; El Majdoubi, Mohammed; Vigne, Jean-Louis; Urano, Jun; Hostetler, Chris E.; Griswold, Michael D.; Weiner, Richard I.; Pera, Renee A. Reijo

2007-01-01

In some species such as flies, worms, frogs, and fish the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically-distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration, that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells. PMID:16996493

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells

PubMed Central

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2012-01-01

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 plays an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that histone demethylase LSD1, a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development. PMID:22068596

Cardiac Progenitor Cells and Bone Marrow-Derived Very Small Embryonic-Like Stem Cells for Cardiac Repair After Myocardial Infarction

PubMed Central

Tang, Xian-Liang; Rokosh, D. Gregg; Guo, Yiru; Bolli, Roberto

2010-01-01

Heart failure after myocardial infarction (MI) continues to be the most prevalent cause of morbidity and mortality worldwide. Although pharmaceutical agents and interventional strategies have contributed greatly to therapy, new and superior treatment modalities are urgently needed given the overall disease burden. Stem cell-based therapy is potentially a promising strategy to lead to cardiac repair after MI. An array of cell types has been explored in this respect, including skeletal myoblasts, bone marrow (BM)-derived stem cells, embryonic stem cells, and more recently, cardiac progenitor cells (CPCs). Recently studies have obtained evidence that transplantation of CPCs or BM-derived very small embryonic-like stem cells can improve cardiac function and alleviate cardiac remodeling, supporting the potential therapeutic utility of these cells for cardiac repair. This report summarizes the current data from those studies and discusses the potential implication of these cells in developing clinically-relevant stem cell-based therapeutic strategies for cardiac regeneration. PMID:20081317

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

PubMed

Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

PubMed

Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

2014-01-01

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

Embryonic stem cells: testing the germ-cell theory.

PubMed

Hochedlinger, Konrad

2011-10-25

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. Copyright © 2011 Elsevier Ltd. All rights reserved.

In vitro fertilization, the Nobel Prize, and human embryonic stem cells.

PubMed

Gearhart, John; Coutifaris, Christos

2011-01-07

Robert Edwards was awarded the 2010 Nobel Prize in Physiology or Medicine for the development of human in vitro fertilization. His work not only provided the means to overcome many forms of infertility, but it also enabled research on early stages of human embryos and the derivation of human embryonic stem cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in the development of neural tissues, but has not been well studied as a bioactive signaling element in neural tissue engineering matrices. The present study uses a systematic continuous gradient approach to identify concentration dependent effects of n-cadherin derived peptide, HAVDI, on the survival and neural differentiation of murine embryonic stem cells. This work underscores the need for greater use to combinatorial strategies to understand the effect complex bioactive signaling, such as n-cadherin, and the need to optimize the concentration of such bioactive signaling within tissue engineering matrices for maximal cellular response. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Formation of gut-like structures in vitro from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko

2006-01-01

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.

Developments in stem cell research and therapeutic cloning: Islamic ethical positions, a review.

PubMed

Fadel, Hossam E

2012-03-01

Stem cell research is very promising. The use of human embryos has been confronted with objections based on ethical and religious positions. The recent production of reprogrammed adult (induced pluripotent) cells does not - in the opinion of scientists - reduce the need to continue human embryonic stem cell research. So the debate continues. Islam always encouraged scientific research, particularly research directed toward finding cures for human disease. Based on the expectation of potential benefits, Islamic teachings permit and support human embryonic stem cell research. The majority of Muslim scholars also support therapeutic cloning. This permissibility is conditional on the use of supernumerary early pre-embryos which are obtained during infertility treatment in vitro fertilization (IVF) clinics. The early pre-embryos are considered in Islamic jurisprudence as worthy of respect but do not have the full sanctity offered to the embryo after implantation in the uterus and especially after ensoulment. In this paper the Islamic positions regarding human embryonic stem cell research and therapeutic cloning are reviewed in some detail, whereas positions in other religious traditions are mentioned only briefly. The status of human embryonic stem cell research and therapeutic cloning in different countries, including the USA and especially in Muslim countries, is discussed. © 2010 Blackwell Publishing Ltd.

X-chromosome dosage as a modulator of pluripotency, signalling and differentiation?

PubMed

Schulz, Edda G

2017-11-05

Already during early embryogenesis, before sex-specific hormone production is initiated, sex differences in embryonic development have been observed in several mammalian species. Typically, female embryos develop more slowly than their male siblings. A similar phenotype has recently been described in differentiating murine embryonic stem cells, where a double dose of the X-chromosome halts differentiation until dosage-compensation has been achieved through X-chromosome inactivation. On the molecular level, several processes associated with early differentiation of embryonic stem cells have been found to be affected by X-chromosome dosage, such as the transcriptional state of the pluripotency network, the activity pattern of several signal transduction pathways and global levels of DNA-methylation. This review provides an overview of the sex differences described in embryonic stem cells from mice and discusses a series of X-linked genes that are associated with pluripotency, signalling and differentiation and their potential involvement in mediating the observed X-dosage-dependent effects.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Stem cells to gametes: how far should we go?

PubMed

Whittaker, Peter

2007-03-01

Murine embryonic stem cells have recently been shown to be capable of differentiating in vitro into oocytes or sperm. Should these findings be duplicated using human embryonic stem cells, this would raise a number of social and ethical concerns, some specific to these particular developments, others shared with other aspects of stem cell research. This review outlines the properties of stem cells and their conversion to gametes. Concerns raised include embryo destruction, quality of gametes derived in this way, possibility for children with two male biological parents, movement towards germ line gene therapy and 'designer babies', and the future impacts on health service provisions. It is important that public discussion of some of these issues should take place.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells.

PubMed

Hirano, Mariko; Yamamoto, Akitsugu; Yoshimura, Naoko; Tokunaga, Tomoyuki; Motohashi, Tsutomu; Ishizaki, Katsuhiko; Yoshida, Hisahiro; Okazaki, Kenji; Yamazaki, Hidetoshi; Hayashi, Shin-Ichi; Kunisada, Takahiro

2003-12-01

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye-like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye-like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye. Copyright 2003 Wiley-Liss, Inc.

Eliminating Cancer Stem Cells by Targeting Embryonic Signaling Pathways.

PubMed

Oren, Ohad; Smith, B Douglas

2017-02-01

Dramatic advances have been made in the understanding of cancer over the past decade. Prime among those are better appreciation of the biology of cancer and the development of targeted therapies. Despite these improvements, however, most tumors remain refractory to anti-cancer medications and frequently recur. Cancer Stem Cells (CSCs), which in some cases express markers of pluripotency (e.g., Oct-4), share many of the molecular features of normal stem cells. These cells have been hypothesised to play a role in tumor resistance and relapse. They exhibit dependence on many primitive regulatory pathways and may be best viewed in the context of embryonic signaling pathways. In this article, we review important embryonic signaling cascades and their differential expression in CSCs. We also discuss these pathways as actionable targets for novel therapies in hopes that eliminating cancer stem cells will lead to an improvement in overall survival for patients.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

PubMed

Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

2018-06-01

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Establishment of mouse expanded potential stem cells

PubMed Central

Gao, Xuefei; Antunes, Liliana; Yu, Yong; Zhu, Zhexin; Wang, Juexuan; Kolodziejczyk, Aleksandra A.; Campos, Lia S.; Wang, Cui; Yang, Fengtang; Zhong, Zhen; Fu, Beiyuan; Eckersley-Maslin, Melanie A.; Woods, Michael; Tanaka, Yosuke; Chen, Xi; Wilkinson, Adam C.; Bussell, James; White, Jacqui; Ramirez-Solis, Ramiro; Reik, Wolf; Göttgens, Berthold; Teichmann, Sarah A.; Tam, Patrick P. L.; Nakauchi, Hiromitsu; Zou, Xiangang; Lu, Liming; Liu, Pentao

2018-01-01

Mouse embryonic stem cells derived from the epiblast1 contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm2 upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species. PMID:29019987

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells.

PubMed

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2011-11-08

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 has an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that the histone lysine-specific demethylase 1 (LSD1), a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development.

Scaffolding for Three-Dimensional Embryonic Vasculogenesis

NASA Astrophysics Data System (ADS)

Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

Recent Advances towards the Clinical Application of Stem Cells for Retinal Regeneration

PubMed Central

Becker, Silke; Jayaram, Hari; Limb, G. Astrid

2012-01-01

Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE) affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice. PMID:24710533

Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

EPA Science Inventory

Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

Will embryonic stem cells change health policy?

PubMed

Sage, William M

2010-01-01

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.

Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids

EPA Science Inventory

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 ...

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

PubMed

Duncan, S A

2005-12-01

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

PubMed

Kuwahara, M; Ogaeri, T; Matsuura, R; Kogo, H; Fujimoto, T; Torihashi, S

2004-04-01

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.

PubMed

Deutsch, Marcus-André; Doppler, Stefanie A; Li, Xinghai; Lahm, Harald; Santamaria, Gianluca; Cuda, Giovanni; Eichhorn, Stefan; Ratschiller, Thomas; Dzilic, Elda; Dreßen, Martina; Eckart, Annekathrin; Stark, Konstantin; Massberg, Steffen; Bartels, Anna; Rischpler, Christoph; Gilsbach, Ralf; Hein, Lutz; Fleischmann, Bernd K; Wu, Sean M; Lange, Rüdiger; Krane, Markus

2018-03-20

The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells during embryonic development. We hypothesized that these MI induced cells (MICs) harbor cardiomyogenic properties similar to their embryonic counterparts. MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs one week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic cardiac progenitor cells and fibroblasts and in vitro culture of MICs unveiled that (myo-) fibroblastic features predominated and that cardiac transcription factors were only expressed at background levels. Adult injury induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.

miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification.

PubMed

Nissan, Xavier; Denis, Jérôme Alexandre; Saidani, Manoubia; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

2011-08-15

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ. Copyright © 2011 Elsevier Inc. All rights reserved.

Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

PubMed

Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

2015-08-01

We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

Human Embryonic Stem Cell Therapy in Crohn’s Disease: A Case Report

PubMed Central

Shroff, Geeta

2016-01-01

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

Adult bone marrow-derived stem cells for organ regeneration and repair.

PubMed

Tögel, Florian; Westenfelder, Christof

2007-12-01

Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow-derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine. 2007 Wiley-Liss, Inc

GENOMIC ADAPTATION OF THE EMBRYONIC STEM CELL TEST (EST) FOR A TOXICOLOGICAL STUDY OF DRINKING WATER DISINFECTION BY-PRODUCTS

EPA Science Inventory

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...

Derivation of porcine pluripotent stem cells for biomedical research.

PubMed

Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

2016-07-01

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

Cortex shatters the glass ceiling.

PubMed

Au, Edmund; Fishell, Gord

2008-11-06

Recreating developmental structures in vitro has been a primary challenge for stem cell biologists. Recent studies in Cell Stem Cell (Eiraku et al., 2008) and Nature (Gaspard et al., 2008) demonstrate that embryonic stem cells can recapitulate early cortical development, enabling them to generate specific cortical subtypes.

Epigenomic Analysis of Multi-lineage Differentiation of Human Embryonic Stem Cells

PubMed Central

Xie, Wei; Schultz, Matthew D.; Lister, Ryan; Hou, Zhonggang; Rajagopal, Nisha; Ray, Pradipta; Whitaker, John W.; Tian, Shulan; Hawkins, R. David; Leung, Danny; Yang, Hongbo; Wang, Tao; Lee, Ah Young; Swanson, Scott A.; Zhang, Jiuchun; Zhu, Yun; Kim, Audrey; Nery, Joseph R.; Urich, Mark A.; Kuan, Samantha; Yen, Chia-an; Klugman, Sarit; Yu, Pengzhi; Suknuntha, Kran; Propson, Nicholas E.; Chen, Huaming; Edsall, Lee E.; Wagner, Ulrich; Li, Yan; Ye, Zhen; Kulkarni, Ashwinikumar; Xuan, Zhenyu; Chung, Wen-Yu; Chi, Neil C.; Antosiewicz-Bourget, Jessica E.; Slukvin, Igor; Stewart, Ron; Zhang, Michael Q.; Wang, Wei; Thomson, James A.; Ecker, Joseph R.; Ren, Bing

2013-01-01

SUMMARY Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells, and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in non-expressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation. PMID:23664764

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; 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 exposuremore » 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.« less

Embryonic Stem Cell Patents and Human Dignity

PubMed Central

Resnik, David B.

2009-01-01

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

Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells.

PubMed

Matveeva, Natalia M; Kizilova, Elena A; Serov, Oleg L

2015-01-01

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.

Embryonic death and the creation of human embryonic stem cells.

PubMed

Landry, Donald W; Zucker, Howard A

2004-11-01

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.

Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

PubMed

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

2015-03-01

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.

[Breakthrough in research on pluripotent stem cells and their application in medicine].

PubMed

Valdimarsdóttir, Guðrún; Richter, Anne

2015-12-01

Embryonic stem cells are, as the name indicates, isolated from embryos. They are pluripotent cells which can be maintained undifferentiated or induced to differentiate into any cell type of the body. In 1998 the first isolation of human embryonic stem cells was successful and they became an interesting source for stem cell regenerative medicine. Only 8 years later pluripotent stem cells were generated by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This was a revolution in the way people thought of cell commitment during development. Since then, a lot of research has been done in understanding the molecular biology of pluripotent stem cells. iPSCs can be generated from somatic cells of a patient and therefore have the same genome. Hence, iPSCs have great potential application in medicine, as they can be utilized in disease modelling, drug screening and cell replacement therapy.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells

PubMed Central

Bayarsaihan, Dashzeveg

2016-01-01

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

[The use of embryonic stem cells for medical-therapeutical purposes: a study of attitudes among Icelandic physicians, lawyers and clergymen.].

PubMed

Oskarsson, Trausti; Guðmundsson, Flóki; Sigurðsson, Jóhann Agúst; Getz, Linn; Arnason, Vilhjálmur

2003-06-01

To study the bioethical standpoints among three groups of Icelandic professionals in relation to the use of embryonic stem cells for medical-therapeutical purposes. In June 2002, a questionnaire was sent by mail to a random sample of 284 doctors and 293 lawyers, as well as all 168 practicing clergymen in Iceland. The participants' position in relation to the use of embryonic stem cells for therapeutical purposes was elicited through general questions as well as case examples. 290 questionnaires (39%) were returned. 62% of participants believed the embryo to have an ethical status superior to that of biologically comparable life forms. 20% of respondents considered its status as equal to that of a grown human being, whilst 18% considered it equal to biologically comparable primitive life forms. There was a difference between the respondent groups (p<0,05). A vast majority believed the use of embryonic stem cells for therapeutical purposes to be justifiable, although the origin of the stem cells appeared to make a difference to many respondents. 8% of participants took an unconditional position against the use of embryonic stem cells. Among those who considered the use of embryonic stem cells with a therapeutic aim to be justifiable, 71% believed that embryonic stem cells should only be utilized to treat diseases of a severe nature. 64% of participants defended the idea of therapeutic cloning with the intention to treat a patient with Parkinson's disease, but the case history elicited considerable difference between professional groups. Clergymen and lawyers tended to hold firmer attitudes, clergymen against and lawyers for the use of stem cells, whilst medical doctors as a group positioned themselves more towards the middle. Female respondents generally took a more modest stand whilst males were more likely to take a firmer stand in both directions. A vast majority (87%) of the participants believed there to be a need for public debate in relation to the use of embryonic stem cells for therapeutical purposes. Overall, participants views in relation to the use of embryonic stem cells for medical purposes were rather liberal. There were however significant differences between professional groups. The relatively high tolerance in regard to therapeutic cloning is interesting in view of the considerable controversy over this topic in many countries. There appears to be fertile ground for a public debate about the use of embryonic stem cells for medical purposes in Iceland.

Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids In Vitro and its Perturbation by Low-Dose Bisphenol A Exposure.

PubMed

Calderon-Gierszal, Esther L; Prins, Gail S

2015-01-01

Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA) can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC) into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20-30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by in utero BPA exposures.

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

PubMed

Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application.

PubMed

De Sousa, P A; Downie, J M; Tye, B J; Bruce, K; Dand, P; Dhanjal, S; Serhal, P; Harper, J; Turner, M; Bateman, M

2016-09-01

From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources. Copyright © 2016. Published by Elsevier B.V.

Placenta-an alternative source of stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matikainen, Tiina; Laine, Jarmo

2005-09-01

The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst ismore » destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications.« less

Human embryonic stem cell lines: socio-legal concerns and therapeutic promise.

PubMed

McLaren, Anne

2002-10-01

Stem cell lines would be very valuable for the repair of diseased or damaged organs. Stem cells derived from adult tissues raise few ethical problems, and would not be rejected if derived from the patient. They show considerable plasticity and might be appropriate for some clinical conditions, but they tend not to grow well in culture. Stem cells derived from the early human embryo proliferate indefinitely in culture and can give rise to many different tissues, but their derivation requires destruction of the embryo, which is not ethically acceptable in some countries. Other countries allow strictly regulated destructive research on human embryos, usually those that have been produced for infertile couples in infertility clinics. Embryos that are no longer required for the couple's own reproductive project could be donated for research rather than just discarded. Different approaches are being developed to avoid immunological rejection of embryonic stem cells used for therapy. Derivation of embryonic stem cell lines by somatic cell nuclear transfer ('cloning') from the patients themselves might be one possible approach, but is unlikely to be used in routine clinical practice if more cost-effective methods are available.

Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

PubMed

Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

2013-12-07

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.

Microfluidic-based patterning of embryonic stem cells for in vitro development studies

PubMed Central

Suri, Shalu; Singh, Ankur; Nguyen, Anh H.; Bratt-Leal, Andres M.; McDevitt, Todd C.

2013-01-01

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

Redox environment in stem and differentiated cells: A quantitative approach.

PubMed

Lyublinskaya, O G; Ivanova, Ju S; Pugovkina, N A; Kozhukharova, I V; Kovaleva, Z V; Shatrova, A N; Aksenov, N D; Zenin, V V; Kaulin, Yu A; Gamaley, I A; Nikolsky, N N

2017-08-01

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H 2 DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Somatic cell nuclear transfer in Oregon: expanding the pluripotent space and informing research ethics.

PubMed

Lomax, Geoffrey P; DeWitt, Natalie D

2013-12-01

In May, Oregon Health and Science University (OHSU) announced the successful derivation, by the Mitalipov laboratory, of embryonic stem cells by somatic cell nuclear transfer. This experiment was recognized as a "formidable technical feat" and potentially a key step toward developing cell-based therapies. The OHSU report is also an example of how a scientific breakthrough can inform research ethics. This article suggests ways that nuclear transfer embryonic stem cell lines may contribute to research ethics by adding rigor to studies addressing pressing research questions important to the development of cell-based therapies.

Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro.

PubMed

Gerlach, Jörg C; Hout, Mariah; Edsbagge, Josefina; Björquist, Petter; Lübberstedt, Marc; Miki, Toshio; Stachelscheid, Harald; Schmelzer, Eva; Schatten, Gerald; Zeilinger, Katrin

2010-02-01

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

The effects of triclosan on pluripotency factors and development of mouse embryonic stem cells and zebrafish.

PubMed

Chen, Xiaojiao; Xu, Bo; Han, Xiumei; Mao, Zhilei; Chen, Minjian; Du, Guizhen; Talbot, Prue; Wang, Xinru; Xia, Yankai

2015-04-01

Triclosan (TCS) poses potential risks to reproduction and development due to its endocrine-disrupting properties. However, the mechanism of TCS's effects on early embryonic development is little known. Embryonic stem cells (ESC) and zebrafish embryos provide valuable models for testing the toxic effects of environmental chemicals on early embryogenesis. In this study, mouse embryonic stem cells (mESC) were acutely exposed to TCS for 24 h, and general cytotoxicity and the effect of TCS on pluripotency were then evaluated. In addition, zebrafish embryos were exposed to TCS from 2- to 24-h post-fertilization (hpf), and their morphology was evaluated. In mESC, alkaline phosphatase staining was significantly decreased after treatment with the highest concentration of TCS (50 μM). Although the expression levels of Sox2 mRNA were not changed, the mRNA levels of Oct4 and Nanog in TCS-treated groups were significantly decreased compared to controls. In addition, the protein levels of Oct4, Sox2 and Nanog were significantly reduced in response to TCS treatment. MicroRNA (miR)-134, an expression inhibitor of pluripotency markers, was significantly increased in TCS-treated mESC. In zebrafish experiments, after 24 hpf of treatment, the controls had developed to the late stage of somitogenesis, while embryos exposed to 300 μg/L of TCS were still at the early stage of somitogenesis, and three genes (Oct4, Sox2 and Nanog) were upregulated in treated groups when compared with the controls. The two models demonstrated that TCS may affect early embryonic development by disturbing the expression of the pluripotency markers (Oct4, Sox2 and Nanog).

78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-28

... Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research... Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research. OMB No. 0925-0601-- Expiration Date... cell lines be approved for use in NIH funded research. Applicants may submit applications at any time...

Patently controversial: markets, morals, and the President's proposal for embryonic stem cell research.

PubMed

Fins, Joseph J; Schachter, Madeleine

2002-09-01

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.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

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

2010-01-01

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

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A new Postdoctoral Fellow position is immediately available in the laboratory of Dr. Terry Yamaguchi at the National Cancer Institute. Dr.Yamaguchi's lab investigates how secreted growth factors regulate the gene regulatory networks that control the fate of embryonic and adult stem cells. Current projects focus on understanding how Wnts and Fgfs regulate the formation and differentiation of the neuromesodermal progenitor (NMP), a multipotent embryonic cell that generates the spinal cord neurons and musculoskeletal system of the body. Using a combination of mouse genetics, mouse and human embryonic stem cell in vitro differentiation, and genomic, proteomic and biochemical approaches, Dr. Yamaguchi’s lab is investigating the molecular mechanisms underlying the activity of key transcriptional determinants of NMP development.

Zika Virus Selectively Kills Aggressive Human Embryonal CNS Tumor Cells In Vitro and In Vivo.

PubMed

Kaid, Carolini; Goulart, Ernesto; Caires-Júnior, Luiz C; Araujo, Bruno H S; Soares-Schanoski, Alessandra; Bueno, Heloisa M S; Telles-Silva, Kayque A; Astray, Renato M; Assoni, Amanda F; Júnior, Antônio F R; Ventini, Daniella C; Puglia, Ana L P; Gomes, Roselane P; Zatz, Mayana; Okamoto, Oswaldo K

2018-06-15

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKV BR ) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKV BR was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKV BR in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKV BR Furthermore, modulation of Wnt signaling pathway significantly affected ZIKV BR -induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKV BR could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects. Significance: Brazilian Zika virus strain kills aggressive metastatic forms of human CNS tumors and could be a potential oncolytic agent for cancer therapy. Cancer Res; 78(12); 3363-74. ©2018 AACR . ©2018 American Association for Cancer Research.

Embryonic and Induced Pluripotent Stem Cells: Understanding, Creating, and Exploiting the Nano-Niche for Regenerative Medicine

PubMed Central

2013-01-01

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the capacity to differentiate into any specialized cell type of the human body, and therefore, ESC/iPSC-derived cell types offer great potential for regenerative medicine. However, key to realizing this potential requires a strong understanding of stem cell biology, techniques to maintain stem cells, and strategies to manipulate cells to efficiently direct cell differentiation toward a desired cell type. As nanoscale science and engineering continues to produce novel nanotechnology platforms, which inform, infiltrate, and impinge on many aspects of everyday life, it is no surprise that stem cell research is turning toward developments in nanotechnology to answer research questions and to overcome obstacles in regenerative medicine. Here we discuss recent advances in ESC and iPSC manipulation using nanomaterials and highlight future challenges within this area of research. PMID:23414366

In vitro developmental model of the gastrointestinal tract from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko; Kuwahara, Masaki; Kurahashi, Masaaki

2007-10-01

Mouse embryonic stem (ES) cells are pluripotent and retain their potential to form cells, tissues and organs originated from three embryonic germ layers. Recently, we developed in vitro organ--gut-like structures--from mouse ES cells. They had basically similar morphological features to a mouse gastrointestinal tract in vivo composed of three distinct layers (i.e., epithelium, connective tissue and musculature). Gut-like structures showed spontaneous contractions derived from pacemaker cells (interstitial cells of Cajal) in the musculature. We also examined their formation process and expression pattern of transcription factors crucial for gut organogenesis such as Id2, Sox17, HNF3beta/Foxa2 and GATA4. We found that they mimic the development of embryonic gut in vivo and showed a similar expression pattern of common transcription factors. They also maintain their developmental potential after transplantation to a renal capsule. Therefore, gut-like structures are suitable for in vitro models of gastrointestinal tracts and their development. In addition, we pointed out several unique features different from gut in vivo that provide useful and advantageous tools to investigate the developmental mechanism of the gastrointestinal tract.

Response to: Dittrich et al.: Non-Embryo-Destructive Extraction of Pluripotent Embryonic Stem Cells – Overlooked Legal Prohibitions, Professional Legal Consequences and Inconsistencies in Patent Law

PubMed Central

Faltus, T.; Storz, U.

2016-01-01

The publication of “Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine” by Dittrich et al. in Geburtshilfe und Frauenheilkunde 2015; 75: 1239–1242 1 describes various possibilities which could result from the non-embryo-destructive extraction of embryonic stem cells from human blastocysts. But implementing this method is more problematic, both legally and ethically, than the authors have represented it to be and is illegal in Germany. German patent DE 10 2004 062 184 on the non-embryo-destructive extraction of embryonic stem cells referred to by Dittrich et al. contravenes the higher-ranking case-law of the European Court of Justice. Ultimately, the non-embryo-destructive harvesting of embryonic stem cells with the aim of storing these cells for use in potential therapies as proposed by Dittrich et al. is prohibited in Germany and could lead to criminal prosecution. PMID:28094826

Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

PubMed

Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

2016-07-01

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events. Copyright © 2016. Published by Elsevier B.V.

Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

PubMed

Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

2010-04-29

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

2017-03-09

Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells

PubMed Central

West, Michael D.; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B.; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-01

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1, encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro-derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition. PMID:29487692

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

PubMed

West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

Molecular cloning and functional analysis of ESGP, an embryonic stem cell and germ cell specific protein.

PubMed

Chen, Yan-Mei; Du, Zhong-Wei; Yao, Zhen

2005-12-01

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends. ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG) (SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic caoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression, forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.

Effects of long-term endocrine disrupting compound exposure on Macaca mulatta embryonic stem cells

PubMed Central

Midic, Uros; Vincent, Kailey A.; VandeVoort, Catherine A; Latham, Keith E.

2016-01-01

Endocrine disrupting chemicals (EDCs) exert significant effects on health and physiology, many traceable to effects on stem cell programming underlying development. Understanding risk of low-level, chronic EDC exposure will be enhanced by knowledge of effects on stem cells. We exposed rhesus monkey embryonic stem cells to low levels of five EDCs [bisphenol A (BPA), atrazine (ATR), tributyltin (TBT), perfluorooctanoic acid (PFOA), and di-(2-ethylhexyl) phthalate (DEHP)] for 28 days, and evaluated effects on gene expression by RNAseq transcriptome profiling. We observed little effect of BPA, and small numbers of affected genes (≤119) with other EDCs. There was substantial overlap in effects across two, three, or four treatments. Ingenuity Pathway analysis indicated suppression of cell survival genes and genes downstream of several stress response mediators, activation of cell death genes, and modulations in several genes regulating pluripotency, differentiation, and germ layer development. Potential adverse effects of these changes on development are discussed. PMID:27614199

Effects of long-term endocrine disrupting compound exposure on Macaca mulatta embryonic stem cells.

PubMed

Midic, Uros; Vincent, Kailey A; VandeVoort, Catherine A; Latham, Keith E

2016-10-01

Endocrine disrupting chemicals (EDCs) exert significant effects on health and physiology, many traceable to effects on stem cell programming underlying development. Understanding risk of low-level, chronic EDC exposure will be enhanced by knowledge of effects on stem cells. We exposed rhesus monkey embryonic stem cells to low levels of five EDCs [bisphenol A (BPA), atrazine (ATR), tributyltin (TBT), perfluorooctanoic acid (PFOA), and di-(2-ethylhexyl) phthalate (DEHP)] for 28days, and evaluated effects on gene expression by RNAseq transcriptome profiling. We observed little effect of BPA, and small numbers of affected genes (≤119) with other EDCs. There was substantial overlap in effects across two, three, or four treatments. Ingenuity Pathway analysis indicated suppression of cell survival genes and genes downstream of several stress response mediators, activation of cell death genes, and modulations in several genes regulating pluripotency, differentiation, and germ layer development. Potential adverse effects of these changes on development are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data.

PubMed

Gamie, Zakareya; MacFarlane, Robert J; Tomkinson, Alicia; Moniakis, Alexandros; Tran, Gui Tong; Gamie, Yehya; Mantalaris, Athanasios; Tsiridis, Eleftherios

2014-11-01

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.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

PubMed

Akyash, Fatemeh; Sadeghian-Nodoushan, Fatemeh; Tahajjodi, Somayyeh Sadat; Nikukar, Habib; Farashahi Yazd, Ehsan; Azimzadeh, Mostafa; D Moore, Harry; Aflatoonian, Behrouz

2017-05-01

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.

Diversity and Complexity in Chromatin Recognition by TFII-I Transcription Factors in Pluripotent Embryonic Stem Cells and Embryonic Tissues

PubMed Central

Makeyev, Aleksandr V.; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a “feed-forward model” of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells. PMID:22970219

Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues.

PubMed

Makeyev, Aleksandr V; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a "feed-forward model" of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells.

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

PubMed

Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

2018-04-01

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

PubMed

Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

2012-12-01

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

3 CFR 13505 - Executive Order 13505 of March 9, 2009. Removing Barriers to Responsible Scientific Research...

Code of Federal Regulations, 2010 CFR

2010-01-01

... Barriers to Responsible Scientific Research Involving Human Stem Cells 13505 Order 13505 Presidential... Scientific Research Involving Human Stem Cells By the authority vested in me as President by the Constitution.... Research involving human embryonic stem cells and human non-embryonic stem cells has the potential to lead...

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

EPA Science Inventory

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...

Generation of Viable Mice from Induced Pluripotent Stem Cells (iPSCs) Through Tetraploid Complementation.

PubMed

Kang, Lan; Gao, Shaorong

2015-01-01

Tetraploid complementation assay is the most rigorous criteria for pluripotency characterization of pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells could complement the developmental deficiency of tetraploid embryos and thus support the full-term mice development. Here we describe the protocol for tetraploid complementation using iPSCs to produce viable all-iPSC mice.

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

2010-02-01

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate

PubMed Central

Ratajczak, M Z; Zuba-Surma, E; Wojakowski, W; Suszynska, M; Mierzejewska, K; Liu, R; Ratajczak, J; Shin, D M; Kucia, M

2014-01-01

The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation. PMID:24018851

Many layers of embryonic hematopoiesis: new insights into B-cell ontogeny and the origin of hematopoietic stem cells.

PubMed

Hadland, Brandon; Yoshimoto, Momoko

2018-04-01

In adult hematopoiesis, the hematopoietic stem cell (HSC) sits at the top of a hierarchy of hematopoietic progenitors responsible for generating the diverse repertoire of blood and immune cells. During embryonic development, however, the initial waves of hematopoiesis provide the first functioning blood cells of the developing embryo, such as primitive erythrocytes arising in the yolk sac, independently of HSCs. In the field of developmental immunology, it has been recognized that some components of the immune system, such as B-1a lymphocytes, are uniquely produced during the embryonic and neonatal period, suggesting a "layered" development of immunity. Several recent studies have shed new light on the developmental origin of the layered immune system, suggesting complex and sometimes multiple contributions to unique populations of innate-like immune cells from both fetal HSCs and earlier HSC-independent progenitors. In this review, we will attempt to synthesize these studies to provide an integrated model of developmental hematopoiesis and layered immunity that may offer new insights into the origin of HSCs. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Synergistic use of adult and embryonic stem cells to study human hematopoiesis.

PubMed

Martin, Colin H; Kaufman, Dan S

2005-10-01

Embryonic stem cells (ESCs) and adult stem cells both provide important resources to define the mechanisms of hematopoietic cell development. To date, studies that utilize hematopoietic stem cells (HSCs) isolated from sites such as bone marrow or umbilical cord blood have been the primary means to identify molecular and phenotypic characteristics of blood cell populations able to mediate long-term hematopoietic engraftment. Although these HSCs are very useful clinically, they are difficult to expand in culture. Now, basic research on human ESCs provides opportunities for novel investigations into the mechanisms of HSC self-renewal. Eventually, the long history of basic and clinical research with adult hematopoietic cell transplantation could translate to establish human ESCs as a suitable alternative starting cell source for clinical hematopoietic reconstitution.

Developmental effects of tobacco smoke exposure during human embryonic stem cell differentiation are mediated through the transforming growth factor-β superfamily member, Nodal

PubMed Central

Liszewski, Walter; Ritner, Carissa; Aurigui, Julian; Wong, Sharon S. Y.; Hussain, Naveed; Krueger, Winfried; Oncken, Cheryl; Bernstein, Harold S.

2012-01-01

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs. PMID:22381624

Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

PubMed Central

Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

2012-01-01

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

PubMed

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

The ethics of cloning and human embryo research.

PubMed

Saran, Madeleine

2002-01-01

The successful cloning experiments that led to Dolly in 1997 have raised many ethical and policy questions. This paper will focus on cloning research in human embryonic cells. The possible gains of the research will be judged against the moral issues of doing research on a person. This paper concludes that while the embryo has some moral status, its moral status is outweighed by the multitude of benefits that embryonic stem cell research will bring to humanity. Policy suggestions are given for dealing with this new and developing field of stem cell research.

Human embryonic stem cells and therapeutic cloning.

PubMed

Hwang, Woo Suk; Lee, Byeong Chun; Lee, Chang Kyu; Kang, Sung Keun

2005-06-01

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.

Human Embryonic Stem Cell Therapy in Crohn's Disease: A Case Report.

PubMed

Shroff, Geeta

2016-02-29

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. 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. Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn's disease.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

PubMed

Roche, E; Sepulcre, M P; Enseñat-Waser, R; Maestre, I; Reig, J A; Soria, B

2003-07-01

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

Conference Scene: Induced pluripotent cells: a new path for regenerative medicine. 7 October 2010, BioPark, Welwyn Garden City, Hertfordshire, UK.

PubMed

Crutzen, Hélène S G

2011-01-01

Embryonic stem cells and induced pluripotent stem (iPS) cells, which are embryonic stem-like cells derived from adult tissues, have the broadest differentiation potential. These cells are unique in their ability to self-renew, to be maintained in an undifferentiated state for long periods of culturing and to give rise to many different cell lineages including germ-line cells. They therefore represent an invaluable tool for facilitating research towards the realization of regenerative medicine. The recent developments in embryonic stem cell and iPS cell technology have allowed human cell models to be developed that will hopefully provide novel platforms for disease analysis not only at the basic science level, but also for drug discovery and screening, and other clinical applications. This 1-day conference, chaired by Professor Peter Andrews from the University of Sheffield, UK, and Dr Chris Denning from the University of Nottingham, UK, focused on generation of iPS cells, their differentiation into specific fates and applications to disease modeling. It consisted of 11 talks by UK-based and international researchers, and three posters; Ms Azra Fatima from Cologne University, Germany, won the competition for her poster on the derivation of iPS cells from a patient with arrhythmogenic right ventricular cardiomyopathy.

Neural stem cells induce the formation of their physical niche during organogenesis

PubMed Central

Riebesehl, Bea F; Ambrosio, Elizabeth M; Stolper, Julian S; Lischik, Colin Q; Dross, Nicolas

2017-01-01

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches. PMID:28950935

An alternative pluripotent state confers interspecies chimaeric competency

PubMed Central

Wu, Jun; Okamura, Daiji; Li, Mo; Suzuki, Keiichiro; Luo, Chongyuan; Ma, Li; He, Yupeng; Li, Zhongwei; Benner, Chris; Tamura, Isao; Krause, Marie N.; Nery, Joseph R.; Du, Tingting; Zhang, Zhuzhu; Hishida, Tomoaki; Takahashi, Yuta; Aizawa, Emi; Kim, Na Young; Lajara, Jeronimo; Guillen, Pedro; Campistol, Josep M.; Esteban, Concepcion Rodriguez; Ross, Pablo J.; Saghatelian, Alan; Ren, Bing; Ecker, Joseph R.; Belmonte, Juan Carlos Izpisua

2017-01-01

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution. PMID:25945737

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313

The retinoblastoma tumor suppressor and stem cell biology.

PubMed

Sage, Julien

2012-07-01

Stem cells play a critical role during embryonic development and in the maintenance of homeostasis in adult individuals. A better understanding of stem cell biology, including embryonic and adult stem cells, will allow the scientific community to better comprehend a number of pathologies and possibly design novel approaches to treat patients with a variety of diseases. The retinoblastoma tumor suppressor RB controls the proliferation, differentiation, and survival of cells, and accumulating evidence points to a central role for RB activity in the biology of stem and progenitor cells. In some contexts, loss of RB function in stem or progenitor cells is a key event in the initiation of cancer and determines the subtype of cancer arising from these pluripotent cells by altering their fate. In other cases, RB inactivation is often not sufficient to initiate cancer but may still lead to some stem cell expansion, raising the possibility that strategies aimed at transiently inactivating RB might provide a novel way to expand functional stem cell populations. Future experiments dedicated to better understanding how RB and the RB pathway control a stem cell's decisions to divide, self-renew, or give rise to differentiated progeny may eventually increase our capacity to control these decisions to enhance regeneration or help prevent cancer development.

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.

HIGH INFORMATION CONTENT TOXICITY SCREENING USING MOUSE AND HUMAN STEM CELL MODELS OF ENDOCRINE DEVELOPMENT AND FUNCTION

EPA Science Inventory

The project will result in the rapid assessment of chemicals for adverse effects on the development of gametes, adipocytes, and islet B-cells; and on the adipocyte and B-cell endocrine signaling function in human and murine embryonic stem cells. Based on the data, hierarchical...

Asynchronous Replication and Autosome-Pair Non-Equivalence in Human Embryonic Stem Cells

PubMed Central

Dutta, Devkanya; Ensminger, Alexander W.; Zucker, Jacob P.; Chess, Andrew

2009-01-01

A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s) that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells. PMID:19325893

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

PubMed Central

Xu, Zijian; Wang, Wenjie; Jiang, Kaiju; Yu, Zhou; Huang, Huanwei; Wang, Fengchao; Zhou, Bin; Chen, Ting

2015-01-01

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. DOI: http://dx.doi.org/10.7554/eLife.10567.001 PMID:26653852

ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

EPA Science Inventory

ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

The ethics of patenting human embryonic stem cells.

PubMed

Chapman, Audrey R

2009-09-01

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.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs

PubMed Central

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A.

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial–mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues. PMID:22539926

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs.

PubMed

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.

The ‘Ventral Organs’ of Pycnogonida (Arthropoda) Are Neurogenic Niches of Late Embryonic and Post-Embryonic Nervous System Development

PubMed Central

Brenneis, Georg; Scholtz, Gerhard

2014-01-01

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions – traditionally designated as ‘ventral organs’ – detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons – as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient posterior ganglia in the ventral nerve cord of Pseudopallene sp. and evaluate this finding in light of the often discussed reduction of a segmented ‘opisthosoma’ during pycnogonid evolution. PMID:24736377

Modeling TSC and LAM Using Patient Derived Induced Pluripotent Stem Cells

DTIC Science & Technology

2016-10-01

lentiviral knockdown, and CRISPR /Cas9 genome editing in embryonic stem cells (ESCs). We have characterized the iPSCs extensively and found that they display...induced pluripotent stem cells (iPSCs) embryonic stem cells (ESCs) reprogramming CRISPR /Cas9 genome editing neural stem cells (NSCs) neural crest... CRISPR /cas9 in two additional human pluripotent stem cell lines (WA07 (H7) – female cell line registry #0061; and a control male iPSC lines generated

Self-organization phenomena in embryonic stem cell-derived embryoid bodies: axis formation and breaking of symmetry during cardiomyogenesis.

PubMed

Fuchs, Christiane; Scheinast, Matthias; Pasteiner, Waltraud; Lagger, Sabine; Hofner, Manuela; Hoellrigl, Alexandra; Schultheis, Martina; Weitzer, Georg

2012-01-01

Aggregation of embryonic stem cells gives rise to embryoid bodies (EBs) which undergo developmental processes reminiscent of early eutherian embryonic development. Development of the three germ layers suggests that gastrulation takes place. In vivo, gastrulation is a highly ordered process but in EBs only few data support the hypothesis that self-organization of differentiating cells leads to morphology, reminiscent of the early gastrula. Here we demonstrate that a timely implantation-like process is a prerequisite for the breaking of the radial symmetry of suspended EBs. Attached to a surface, EBs develop a bilateral symmetry and presumptive mesodermal cells emerge between the center of the EBs and a horseshoe-shaped ridge of cells. The development of an epithelial sheet of cells on one side of the EBs allows us to define an 'anterior' and a 'posterior' end of the EBs. In the mesodermal area, first cardiomyocytes (CMCs) develop mainly next to this epithelial sheet of cells. Development of twice as many CMCs at the 'left' side of the EBs breaks the bilateral symmetry and suggests that cardiomyogenesis reflects a local or temporal asymmetry in EBs. The asymmetric appearance of CMCs but not the development of mesoderm can be disturbed by ectopic expression of the muscle-specific protein Desmin. Later, the bilateral morphology becomes blurred by an apparently chaotic differentiation of many cell types. The absence of comparable structures in aggregates of cardiovascular progenitor cells isolated from the heart demonstrates that the self-organization of cells during a gastrulation-like process is a unique feature of embryonic stem cells. Copyright © 2011 S. Karger AG, Basel.

Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids In Vitro and its Perturbation by Low-Dose Bisphenol A Exposure

PubMed Central

Calderon-Gierszal, Esther L.; Prins, Gail S.

2015-01-01

Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA) can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC) into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20–30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by in utero BPA exposures. PMID:26222054

Neurogenesis in the embryonic and adult brain: same regulators, different roles

PubMed Central

Urbán, Noelia; Guillemot, François

2014-01-01

Neurogenesis persists in adult mammals in specific brain areas, known as neurogenic niches. Adult neurogenesis is highly dynamic and is modulated by multiple physiological stimuli and pathological states. There is a strong interest in understanding how this process is regulated, particularly since active neuronal production has been demonstrated in both the hippocampus and the subventricular zone (SVZ) of adult humans. The molecular mechanisms that control neurogenesis have been extensively studied during embryonic development. Therefore, we have a broad knowledge of the intrinsic factors and extracellular signaling pathways driving proliferation and differentiation of embryonic neural precursors. Many of these factors also play important roles during adult neurogenesis, but essential differences exist in the biological responses of neural precursors in the embryonic and adult contexts. Because adult neural stem cells (NSCs) are normally found in a quiescent state, regulatory pathways can affect adult neurogenesis in ways that have no clear counterpart during embryogenesis. BMP signaling, for instance, regulates NSC behavior both during embryonic and adult neurogenesis. However, this pathway maintains stem cell proliferation in the embryo, while it promotes quiescence to prevent stem cell exhaustion in the adult brain. In this review, we will compare and contrast the functions of transcription factors (TFs) and other regulatory molecules in the embryonic brain and in adult neurogenic regions of the adult brain in the mouse, with a special focus on the hippocampal niche and on the regulation of the balance between quiescence and activation of adult NSCs in this region. PMID:25505873

[Ethical aspects of human embryonic stem cell use and commercial umbilical cord blood stem cell banking. Ethical reflections on the occasion of the regulation of the European Council and Parliament on advanced therapy medicinal products].

PubMed

Virt, G

2010-01-01

The regulation of the European Council and Parliament on advanced therapy medicinal products also includes therapies with human embryonic stem cells. The use of these stem cells is controversially and heavily discussed. Contrary to the use of adult stem cells, medical and ethical problems concerning the use of human embryonic stem cells persists, because this use is based on the destruction of human life at the very beginning. The regulation foresees, therefore, subsidiarity within the European Member States. Although there are no ethical problems in principle with the use of stem cells from the umbilical cord blood, there are social ethical doubts with the banking of these stem cells for autologous use without any currently foreseeable medical advantage by commercial blood banks. Also in this case subsidiarity is valid.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

Are You Teaching Your Students about Stem Cells?

ERIC Educational Resources Information Center

Concannon, James; Brown, Patrick L.; Brandt, Trisha

2009-01-01

This activity targets students' misconceptions about embryonic and adult stem cells while also addressing an important grades 9-12 science content standard. The authors designed the activity to provide students an opportunity to explore differences between embryonic and adult stem cells prior to formal explanation. The overarching goal of this…

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation*

PubMed Central

Meade, Kate A.; White, Kathryn J.; Pickford, Claire E.; Holley, Rebecca J.; Marson, Andrew; Tillotson, Donna; van Kuppevelt, Toin H.; Whittle, Jason D.; Day, Anthony J.; Merry, Catherine L. R.

2013-01-01

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells. PMID:23235146

Immobilization of heparan sulfate on electrospun meshes to support embryonic stem cell culture and differentiation.

PubMed

Meade, Kate A; White, Kathryn J; Pickford, Claire E; Holley, Rebecca J; Marson, Andrew; Tillotson, Donna; van Kuppevelt, Toin H; Whittle, Jason D; Day, Anthony J; Merry, Catherine L R

2013-02-22

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

PubMed

Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

2007-11-22

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

High-Throughput Screening Assay for Embryoid Body Differentiation of Human Embryonic Stem Cells

PubMed Central

Outten, Joel T.; Gadue, Paul; French, Deborah L.; Diamond, Scott L.

2012-01-01

Serum-free human pluripotent stem cell media offer the potential to develop reproducible clinically applicable differentiation strategies and protocols. The vast array of possible growth factor and cytokine combinations for media formulations makes differentiation protocol optimization both labor and cost-intensive. This unit describes a 96-well plate, 4-color flow cytometry-based screening assay to optimize pluripotent stem cell differentiation protocols. We provide conditions both to differentiate human embryonic stem cells (hESCs) to the three primary germ layers, ectoderm, endoderm, and mesoderm, and to utilize flow cytometry to distinguish between them. This assay exhibits low inter-well variability and can be utilized to efficiently screen a variety of media formulations, reducing cost, incubator space, and labor. Protocols can be adapted to a variety of differentiation stages and lineages. PMID:22415836

Human induced pluripotent stem cells: a review of the US patent landscape.

PubMed

Georgieva, Bilyana P; Love, Jane M

2010-07-01

Human induced pluripotent stem (iPS) cells and human embryonic stem cells are cells that have the ability to differentiate into a variety of cell types. Embryonic stem cells are derived from human embryos; however, by contrast, human iPS cells can be obtained from somatic cells that have undergone a process of 'reprogramming' via genetic manipulation such that they develop pluripotency. Since iPS cells are not derived from human embryos, they are a less complicated source of human pluripotent cells and are considered valuable research tools and potentially useful in therapeutic applications in regenerative medicine. Worldwide, there are only three issued patents concerning iPS cells. Therefore, the patent landscape in this field is largely undefined. This article provides an overview of the issued patents as well as the pending published patent applications in the field.

Control of Cellular Structural Networks Through Unstructured Protein Domains

DTIC Science & Technology

2016-07-01

stem cells (hPSCs), including embryonic and induced pluripotent stem cells . We had a third paper accepted to Scientific Reports in which we showed...2012 Stem Cells Young Investigator Award. We then had a followup paper accepted to Integrative Biology extending these ideas to human pluripotent ...morphology, mechanics, and neurogenesis in neural stem cells ; (3) To develop and use multiscale computational 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

PubMed

Pierpont, Timothy M; Lyndaker, Amy M; Anderson, Claire M; Jin, Qiming; Moore, Elizabeth S; Roden, Jamie L; Braxton, Alicia; Bagepalli, Lina; Kataria, Nandita; Hu, Hilary Zhaoxu; Garness, Jason; Cook, Matthew S; Capel, Blanche; Schlafer, Donald H; Southard, Teresa; Weiss, Robert S

2017-11-14

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Modeling Niemann Pick type C1 using human embryonic and induced pluripotent stem cells.

PubMed

Ordoñez, M Paulina; Steele, John W

2017-02-01

Data generated in Niemann Pick type C1 (NPC1) human embryonic and human induced pluripotent stem cell derived neurons complement on-going studies in animal models and provide the first example, in disease-relevant human cells, of processes that underlie preferential neuronal defects in a NPC1. Our work and that of other investigators in human neurons derived from stem cells highlight the importance of performing rigorous mechanistic studies in relevant cell types to guide drug discovery and therapeutic development, alongside of existing animal models. Through the use of human stem cell-derived models of disease, we can identify and discover or repurpose drugs that revert early events that lead to neuronal failure in NPC1. Together with the study of disease pathogenesis and efficacy of therapies in animal models, these strategies will fulfill the promise of stem cell technology in the development of new treatments for human diseases. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors.

PubMed

Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu

2010-11-01

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.

Comparative Chondrogenesis of Human Cell Sources in 3D Scaffolds

PubMed Central

Tıg̑lı, R. Seda; Ghosh, Sourabh; Laha, Michael M.; Shevde, Nirupama K.; Daheron, Laurence; Gimble, Jeffrey; Gümüşdereliog̑lu, Menemşe; Kaplan, David L.

2009-01-01

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP-6) along with the standard chondrogenic differentiating factors. Embryonic stem cells derived MSCs showed unique characteristics with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopic analyses. After 4 weeks of cultivation, embryonic stem cells derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP-6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes and with the variables addressed here, the human embryonic stem cells derived MSCs were the preferred cell source. PMID:19382119

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

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.

Self-organization of spatial patterning in human embryonic stem cells

PubMed Central

Deglincerti, Alessia; Etoc, Fred; Ozair, M. Zeeshan; Brivanlou, Ali H.

2017-01-01

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

The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

PubMed Central

Nguyen, Duong Thi Thuy; Richter, Daniel; Michel, Geert; Mitschka, Sibylle; Kolanus, Waldemar; Cuevas, Elisa; Gregory Wulczyn, F

2017-01-01

Rapidity and specificity are characteristic features of proteolysis mediated by the ubiquitin-proteasome system. Therefore, the UPS is ideally suited for the remodeling of the embryonic stem cell proteome during the transition from pluripotent to differentiated states and its inverse, the generation of inducible pluripotent stem cells. The Trim-NHL family member LIN41 is among the first E3 ubiquitin ligases to be linked to stem cell pluripotency and reprogramming. Initially discovered in C. elegans as a downstream target of the let-7 miRNA, LIN41 is now recognized as a critical regulator of stem cell fates as well as the timing of neurogenesis. Despite being indispensable for embryonic development and neural tube closure in mice, the underlying mechanisms for LIN41 function in these processes are poorly understood. To better understand the specific contributions of the E3 ligase activity for the stem cell functions of LIN41, we characterized global changes in ubiquitin or ubiquitin-like modifications using Lin41-inducible mouse embryonic stem cells. The tumor suppressor protein p53 was among the five most strongly affected proteins in cells undergoing neural differentiation in response to LIN41 induction. We show that LIN41 interacts with p53, controls its abundance by ubiquitination and antagonizes p53-dependent pro-apoptotic and pro-differentiation responses. In vivo, the lack of LIN41 is associated with upregulation of Grhl3 and widespread caspase-3 activation, two downstream effectors of p53 with essential roles in neural tube closure. As Lin41-deficient mice display neural tube closure defects, we conclude that LIN41 is critical for the regulation of p53 functions in cell fate specification and survival during early brain development. PMID:28430184

A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development

PubMed Central

Paige, Sharon L.; Thomas, Sean; Stoick-Cooper, Cristi L.; Wang, Hao; Maves, Lisa; Sandstrom, Richard; Pabon, Lil; Reinecke, Hans; Pratt, Gabriel; Keller, Gordon; Moon, Randall T.; Stamatoyannopoulos, John; Murry, Charles E.

2012-01-01

Summary Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. PMID:22981225

Induced Pluripotent Stem Cells in Dermatology: Potentials, Advances, and Limitations

PubMed Central

Bilousova, Ganna; Roop, Dennis R.

2014-01-01

The discovery of methods for reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) has raised the possibility of producing truly personalized treatment options for numerous diseases. Similar to embryonic stem cells (ESCs), iPSCs can give rise to any cell type in the body and are amenable to genetic correction by homologous recombination. These ESC properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders, including inherited skin diseases, without using embryonic tissues or oocytes. Here, we review recent progress and limitations of iPSC research with a focus on clinical applications of iPSCs and using iPSCs to model human diseases for drug discovery in the field of dermatology. PMID:25368014

Stem cells - biological update and cell therapy progress

PubMed Central

GIRLOVANU, MIHAI; SUSMAN, SERGIU; SORITAU, OLGA; RUS-CIUCA, DAN; MELINCOVICI, CARMEN; CONSTANTIN, ANNE-MARIE; MIHU, CARMEN MIHAELA

2015-01-01

In recent years, the advances in stem cell research have suggested that the human body may have a higher plasticity than it was originally expected. Until now, four categories of stem cells were isolated and cultured in vivo: embryonic stem cells, fetal stem cells, adult stem cells and induced pluripotent stem cells (hiPSCs). Although multiple studies were published, several issues concerning the stem cells are still debated, such as: the molecular mechanisms of differentiation, the methods to prevent teratoma formation or the ethical and religious issues regarding especially the embryonic stem cell research. The direct differentiation of stem cells into specialized cells: cardiac myocytes, neural cells, pancreatic islets cells, may represent an option in treating incurable diseases such as: neurodegenerative diseases, type I diabetes, hematologic or cardiac diseases. Nevertheless, stem cell-based therapies, based on stem cell transplantation, remain mainly at the experimental stages and their major limitation is the development of teratoma and cancer after transplantation. The induced pluripotent stem cells (hiPSCs) represent a prime candidate for future cell therapy research because of their significant self-renewal and differentiation potential and the lack of ethical issues. This article presents an overview of the biological advances in the study of stem cells and the current progress made in the field of regenerative medicine. PMID:26609255

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

PubMed

Oberlin, Estelle; Fleury, Maud; Clay, Denis; Petit-Cocault, Laurence; Candelier, Jean-Jacques; Mennesson, Benoît; Jaffredo, Thierry; Souyri, Michèle

2010-11-25

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Ethics and policy in embryonic stem cell research.

PubMed

Robertson, John A

1999-06-01

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.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

PubMed

Ji, Junfeng; Risueño, Ruth M; Hong, Seokho; Allan, David; Rosten, Patty; Humphries, Keith; Bhatia, Mickie

2011-04-01

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. Copyright © 2011 AlphaMed Press.

Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans

PubMed Central

Kowalski, Madzia P.; Baylis, Howard A.; Krude, Torsten

2015-01-01

ABSTRACT Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates. PMID:25908866

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

In situ electrochemical detection of embryonic stem cell differentiation.

PubMed

Yea, Cheol-Heon; An, Jeung Hee; Kim, Jungho; Choi, Jeong-Woo

2013-06-20

Stem cell sensors have emerged as a promising technique to electrochemically monitor the functional status and viability of stem cells. However, efficient electrochemical analysis techniques are required for the development of effective electrochemical stem cell sensors. In the current study, we report a newly developed electrochemical cyclic voltammetry (CV) system to determine the status of mouse embryonic stem (ES) cells. 1-Naphthly phosphate (1-NP), which was dephosphorylated by alkaline phosphatase into a 1-naphthol on an undifferentiated mouse ES cell, was used as a substrate to electrochemically monitor the differentiation status of mouse ES cells. The peak current in the cyclic voltammetry of 1-NP increased linearly with the concentration of pure 1-NP (R(2)=0.9623). On the other hand, the peak current in the electrochemical responses of 1-NP decreased as the number of undifferentiated ES cells increased. The increased dephosphorylation of 1-NP to 1-naphthol made a decreased electrochemical signal. Non-toxicity of 1-NP was confirmed. In conclusion, the proposed electrochemical analysis system can be applied to an electrical stem cell chip for diagnosis, drug detection and on-site monitoring. Copyright © 2013 Elsevier B.V. All rights reserved.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

PubMed

Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A

2010-08-15

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.

PubMed

Du, Vicard; Luciani, Nathalie; Richard, Sophie; Mary, Gaëtan; Gay, Cyprien; Mazuel, François; Reffay, Myriam; Menasché, Philippe; Agbulut, Onnik; Wilhelm, Claire

2017-09-12

The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

PubMed

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

2012-01-01

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Ambivalent journeys of hope: embryonic stem cell therapy in a clinic in India.

PubMed

Prasad, Amit

2015-03-01

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.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

PubMed

Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

2015-05-01

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

PubMed

Acuna-Mendoza, Soledad; Martin, Sabrina; Kuchler-Bopp, Sabine; Ribes, Sandy; Thalgott, Jérémy; Chaussain, Catherine; Creuzet, Sophie; Lesot, Hervé; Lebrin, Franck; Poliard, Anne

2017-12-01

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update

PubMed Central

Miele, Lucio; Harris, Pamela Jo; Jeong, Woondong; Bando, Hideaki; Kahn, Michael; Yang, Sherry X.

2015-01-01

During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents. PMID:25850553

Generation of diverse neuronal subtypes in cloned populations of stem-like cells

PubMed Central

Varga, Balázs V; Hádinger, Nóra; Gócza, Elen; Dulberg, Vered; Demeter, Kornél; Madarász, Emília; Herberth, Balázs

2008-01-01

Background The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. Results In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. Conclusion The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification. PMID:18808670

Present state and future perspectives of using pluripotent stem cells in toxicology research

PubMed Central

Löser, Peter

2011-01-01

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed. PMID:21225242

Pluripotent stem cells and reprogrammed cells in farm animals.

PubMed

Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

2011-08-01

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

A scale out approach towards neural induction of human induced pluripotent stem cells for neurodevelopmental toxicity studies.

PubMed

Miranda, Cláudia C; Fernandes, Tiago G; Pinto, Sandra N; Prieto, Manuel; Diogo, M Margarida; Cabral, Joaquim M S

2018-05-21

Stem cell's unique properties confer them a multitude of potential applications in the fields of cellular therapy, disease modelling and drug screening fields. In particular, the ability to differentiate neural progenitors (NP) from human induced pluripotent stem cells (hiPSCs) using chemically-defined conditions provides an opportunity to create a simple and straightforward culture platform for application in these fields. Here, we demonstrated that hiPSCs are capable of undergoing neural commitment inside microwells, forming characteristic neural structures resembling neural rosettes and further give rise to glial and neuronal cells. Furthermore, this platform can be applied towards the study of the effect of neurotoxic molecules that impair normal embryonic development. As a proof of concept, the neural teratogenic potential of the antiepileptic drug valproic acid (VPA) was analyzed. It was verified that exposure to VPA, close to typical dosage values (0.3 to 0.75 mM), led to a prevalence of NP structures over neuronal differentiation, as confirmed by analysis of the expression of neural cell adhesion molecule, as well as neural rosette number and morphology assessment. The methodology proposed herein for the generation and neural differentiation of hiPSC aggregates can potentially complement current toxicity tests such as the humanized embryonic stem cell test for the detection of teratogenic compounds that can interfere with normal embryonic development. Copyright © 2018 Elsevier B.V. All rights reserved.

Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

PubMed Central

Kretsovali, Androniki; Hadjimichael, Christiana; Charmpilas, Nikolaos

2012-01-01

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. PMID:22550500

Clinical potentials of human pluripotent stem cells in lung diseases

PubMed Central

2014-01-01

Lung possesses very limited regenerative capacity. Failure to maintain homeostasis of lung epithelial cell populations has been implicated in the development of many life-threatening pulmonary diseases leading to substantial morbidity and mortality worldwide, and currently there is no known cure for these end-stage pulmonary diseases. Embryonic stem cells (ESCs) and somatic cell-derived induced pluripotent stem cells (iPSCs) possess unlimited self-renewal capacity and great potential to differentiate to various cell types of three embryonic germ layers (ectodermal, mesodermal, and endodermal). Therapeutic use of human ESC/iPSC-derived lung progenitor cells for regeneration of injured or diseased lungs will have an enormous clinical impact. This article provides an overview of recent advances in research on pluripotent stem cells in lung tissue regeneration and discusses technical challenges that must be overcome for their clinical applications in the future. PMID:24995122

The transcriptional landscape of hematopoietic stem cell ontogeny

PubMed Central

McKinney-Freeman, Shannon; Cahan, Patrick; Li, Hu; Lacadie, Scott A.; Huang, Hsuan-Ting; Curran, Matthew; Loewer, Sabine; Naveiras, Olaia; Kathrein, Katie L.; Konantz, Martina; Langdon, Erin M.; Lengerke, Claudia; Zon, Leonard I.; Collins, James J.; Daley, George Q.

2012-01-01

Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSC purified from >2500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSC, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource (http://hsc.hms.harvard.edu) will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification. PMID:23122293

Selective blockade of microRNA processing by Lin-28

PubMed Central

Viswanathan, Srinivas R.; Daley, George Q.; Gregory, Richard I.

2012-01-01

MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked post-transcriptionally in embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and primary tumors. Here we show that Lin-28, a developmentally regulated RNA-binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we demonstrate that Lin-28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin-28 as a negative regulator of miRNA biogenesis and suggest that Lin-28 may play a central role in blocking miRNA-mediated differentiation in stem cells and certain cancers. PMID:18292307

Self-Organization of Spatial Patterning in Human Embryonic Stem Cells.

PubMed

Deglincerti, Alessia; Etoc, Fred; Ozair, M Zeeshan; Brivanlou, Ali H

2016-01-01

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. © 2016 Elsevier Inc. All rights reserved.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

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

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

PubMed

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Delivery of Differentiation Factors by Mesoporous Silica Particles Assists Advanced Differentiation of Transplanted Murine Embryonic Stem Cells

PubMed Central

Kozhevnikova, Mariya; König, Niclas; Zhou, Chunfang; Leao, Richardson; Knöpfel, Thomas; Pankratova, Stanislava; Trolle, Carl; Berezin, Vladimir; Bock, Elisabeth; Aldskogius, Håkan

2013-01-01

Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long-term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application. Here, we report the development of a novel technological approach for the local delivery of exogenous trophic factor mimetics to transplanted cells using specifically designed silica nanoporous particles. We demonstrated that delivering Cintrofin and Gliafin, established peptide mimetics of the ciliary neurotrophic factor and glial cell line-derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long-term survival in vivo. We propose that the delivery of growth factors by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation. PMID:24089415

Functional Human Podocytes Generated in Organoids from Amniotic Fluid Stem Cells

PubMed Central

Benedetti, Valentina; Novelli, Rubina; Abbate, Mauro; Rizzo, Paola; Conti, Sara; Tomasoni, Susanna; Corna, Daniela; Pozzobon, Michela; Cavallotti, Daniela; Yokoo, Takashi; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe

2016-01-01

Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research. PMID:26516208

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

PubMed Central

Yvernogeau, Laurent

2017-01-01

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo. PMID:28526756

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

PubMed Central

Hartman, Matthew E.; Librande, Jason R.; Medvedev, Ivan O.; Ahmad, Rabiah N.; Moussavi-Harami, Farid; Gupta, Pritha P.; Chien, Wei-Ming; Chin, Michael T.

2014-01-01

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation. PMID:24667642

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

PubMed

Zhong, Xuefei; Hao, Ling; Lu, Jianfeng; Ye, Hui; Zhang, Su-Chun; Li, Lingjun

2016-04-01

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Selection and dynamics of embryonic stem cell integration into early mouse embryos

PubMed Central

Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

2016-01-01

The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases.

PubMed

Li, Yan; He, Lingjuan; Huang, Xiuzhen; Issa Bhaloo, Shirin; Zhao, Huan; Zhang, Shaohua; Pu, Wenjuan; Tian, Xueying; Li, Yi; Liu, Qiaozhen; Yu, Wei; Zhang, Libo; Liu, Xiuxiu; Liu, Kuo; Tang, Juan; Zhang, Hui; Cai, Dongqing; Adams, Ralf H; Xu, Qingbo; Lui, Kathy O; Zhou, Bin

2018-04-26

Background -Whether the adult mammalian heart harbors cardiac stem cells (CSCs) for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers such as the cardiosphere-derived cells or with markers such as Sca1 + , Bmi1 + , Isl1 + or Abcg2 + CSCs have been reported. Moreover, it remains unclear whether putative CSCs with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods -To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all non-myocyte populations that contain putative CSCs. Using dual lineage tracing system, we assessed if non-myocytes generated any new myocytes during embryonic development, adult homeostasis and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocytes generation from non-myocytes. Results -By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from non-myocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from non-myocytes in the skeletal muscle after injury. Conclusions -This study provides in vivo genetic evidence for non-myocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Stem cell sources for regenerative medicine.

PubMed

Riazi, Ali M; Kwon, Sarah Y; Stanford, William L

2009-01-01

Tissue-resident stem cells or primitive progenitors play an integral role in homeostasis of most organ systems. Recent developments in methodologies to isolate and culture embryonic and somatic stem cells have many new applications poised for clinical and preclinical trials, which will enable the potential of regenerative medicine to be realized. Here, we overview the current progress in therapeutic applications of various stem cells and discuss technical and social hurdles that must be overcome for their potential to be realized.

LASP-01: Distribution of Mouse Embryonic Stem Cells Expressing MicroRNAs | Frederick National Laboratory for Cancer Research

Cancer.gov

The Laboratory Animal Sciences Program manages the expansion, processing, and distribution of1,501 genetically engineered mouse embryonic stem cell (mESC) linesharboring conditional microRNA transgenes. The Laboratory Animal Sciences Prog

Chromatin in embryonic stem cell neuronal differentiation.

PubMed

Meshorer, E

2007-03-01

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

Human embryonic stem cell research: an intercultural perspective.

PubMed

Walters, LeRoy

2004-03-01

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.

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

PubMed

Bayarsaihan, Dashzeveg; Makeyev, Aleksandr V; Enkhmandakh, Badam

2012-10-01

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

NASA Astrophysics Data System (ADS)

Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

2017-02-01

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.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

PubMed

Niclis, Jonathan C; Gantner, Carlos W; Hunt, Cameron P J; Kauhausen, Jessica A; Durnall, Jennifer C; Haynes, John M; Pouton, Colin W; Parish, Clare L; Thompson, Lachlan H

2017-09-12

Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Transcriptional control of stem cell fate by E2Fs and pocket proteins

PubMed Central

Julian, Lisa M.; Blais, Alexandre

2015-01-01

E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. PMID:25972892

The Science and Ethics of Induced Pluripotency: What Will Become of Embryonic Stem Cells?

PubMed Central

Zacharias, David G.; Nelson, Timothy J.; Mueller, Paul S.; Hook, C. Christopher

2011-01-01

For over a decade, the field of stem cell research has advanced tremendously and gained new attention in light of novel insights and emerging developments for regenerative medicine. Invariably, multiple considerations come into play, and clinicians and researchers must weigh the benefits of certain stem cell platforms against the costs they incur. Notably, human embryonic stem (hES) cell research has been a source of continued debate, leading to differing policies and regulations worldwide. This article briefly reviews current stem cell platforms, looking specifically at the two existing pluripotent lines available for potential therapeutic applications: hES cells and induced pluripotent stem (iPS) cells. We submit iPS technology as a viable and possibly superior alternative for future medical and research endeavors as it obviates many ethical and resource-related concerns posed by hES cells while prospectively matching their potential for scientific use. However, while the clinical realities of iPS cells appear promising, we must recognize the current limitations of this technology, avoid hype, and articulate ethically acceptable medical and scientific goals. PMID:21719620

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

PubMed Central

JIN, Li-Fang; LI, Jin-Song

2016-01-01

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm.

PubMed

Francis, Natalie; Moore, Melanie; Asan, Simona G; Rutter, Guy A; Burns, Chris

2015-01-01

Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Regeneration of Solanum nigrum by somatic embryogenesis, involving frog egg-like body, a novel structure.

PubMed

Xu, Kedong; Chang, Yunxia; Liu, Kun; Wang, Feige; Liu, Zhongyuan; Zhang, Ting; Li, Tong; Zhang, Yi; Zhang, Fuli; Zhang, Ju; Wang, Yan; Niu, Wei; Jia, Shuzhao; Xie, Hengchang; Tan, Guangxuan; Li, Chengwei

2014-01-01

A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

PubMed

Ratcliffe, Elizabeth; Hourd, Paul; Guijarro-Leach, Juan; Rayment, Erin; Williams, David J; Thomas, Robert J

2013-01-01

Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

PubMed

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspension Bioreactor.

PubMed

Lei, Xiaohua; Deng, Zhili; Duan, Enkui

2016-01-01

Embryonic stem cells (ESCs) are capable of differentiating into almost all cell types in vitro and hold great promise for drug screening, developmental studies and have a huge potential in many therapeutic areas. ESCs can aggregate to form embryoid body (EB) in static suspension culture by spontaneous differentiation, which resembles an intact embryo; while static suspension culture cannot prevent agglomeration of cells and offers little control over the size and shape of EBs, it results in aggregation of EBs into large, irregular masses, which prejudice the efficiency of differentiation of cells. Recently, bioreactor-based platforms have been shown to not only offer a beneficial effect on increasing diffusion of nutrients and oxygen which promotes cell viability and proliferation but also display local biomechanical properties (e.g., low fluid shear stresses and hydrodynamic force) in tissue development and organogenesis. This chapter describes a protocol for using a rotary suspension bioreactor to produce embryoid bodies and process the differentiation of mouse embryonic stem cells (mESCs), and to assess the efficiency of EB differentiation in the bioreactor by real-time PCR and immunostaining.

Copine1 regulates neural stem cell functions during brain development.

PubMed

Kim, Tae Hwan; Sung, Soo-Eun; Cheal Yoo, Jae; Park, Jae-Yong; Yi, Gwan-Su; Heo, Jun Young; Lee, Jae-Ran; Kim, Nam-Soon; Lee, Da Yong

2018-01-01

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

PubMed

Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

2015-01-01

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

[Therapeutic cloning: far from application at this stage].

PubMed

De Both, N J

2001-11-03

Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.

Immuno- and gene expression analysis of EGFR and Nestin during mice skin development.

PubMed

Falodah, Fawaz Adnan; Al-Karim, Saleh

2016-06-01

Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. The main objectives of the present study were to identify the precise anatomical localization of stem cells in mice during skin developing; and to determine the expression levels by using immuno- and gene expression analysis. In this comparative cross sectional study, six ages been chosen and divided into: embryonic days (E12.5, E14.5 and E19.5) and litter days (L7, L14 and L19). Skin were removed from the back side and processed to assess both immuno- and gene-expression of EGFR and Nestin surface antigen markers. Data of the different studied age groups was compared using the SPSS software. EGFR was mainly expressed in the outer root sheath (ORS), in basal and, to a lesser extent, in suprabasal keratinocytes and tend to lie where the dermis comes closest to the skin surface, while Nestin expressed throughout the dermis in the early embryo, but it is subsequently restricted to the follicular connective tissue sheaths later in development and to hair follicles after birth. Immunoexpression analysis showed a strong EGFR expression in all group ages except E12.5 which recorded as moderate, while Nestin showed strong expression level for all embryonic stages, while in the litters it was moderate. The qRT-PCR results were consistent with those of the immunohistochemical study. The Pearson correlation analyze present a correlation between the cases of study with age (p≤0.01), which indicated to the effect of age to mice development. EGFR and Nestin showed to have vital role during mice development, and considered to be suitable markers for the study of skin stem cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gene targeting in embryonic stem cells, II: conditional technologies

USDA-ARS?s Scientific Manuscript database

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...

Stem cells: science, policy, and ethics

PubMed Central

Fischbach, Gerald D.; Fischbach, Ruth L.

2004-01-01

Human embryonic stem cells offer the promise of a new regenerative medicine in which damaged adult cells can be replaced with new cells. Research is needed to determine the most viable stem cell lines and reliable ways to promote the differentiation of pluripotent stem cells into specific cell types (neurons, muscle cells, etc.). To create new cell lines, it is necessary to destroy preimplantation blastocysts. This has led to an intense debate that threatens to limit embryonic stem cell research. The profound ethical issues raised call for informed, dispassionate debate. PMID:15545983

In vitro differentiation of embryonic stem cells into hepatocytes induced by fibroblast growth factors and bone morphological protein-4.

PubMed

Zhou, Qing-Jun; Huang, Yan-Dan; Xiang, Li-Xin; Shao, Jian-Zhong; Zhou, Guo-Shun; Yao, Hang; Dai, Li-Cheng; Lu, Yong-Liang

2007-01-01

The feasibility of transforming embryonic endoderm into different cell types is tightly controlled by mesodermal and septum transversumal signalings during early embryonic development. Here, an induction protocol tracing embryonic liver development was designed, in which, three growth factors, acid fibroblast growth factor, basic fibroblast growth factor and bone morphological protein-4 that secreted from pre-cardiac mesoderm and septum transversum mesenchyme, respectively, were employed to investigate their specific potency of modulating the mature hepatocyte proportion during the differentiation process. Results showed that hepatic differentiation took place spontaneously at a low level, however, supplements of the three growth factors gave rise to a significant up-regulation of mature hepatocytes. Bone morphological protein-4 highlighted the differentiation ratio to 40-55%, showing the most effective promotion, and also exhibited a synergistic effect with the other two fibroblast factors, whereas no similar phenomenon was observed between the other two factors, which was reported for the first time. Our study not only provides a high-performance system of embryonic stem cells differentiating into hepatocytes, which would supply a sufficient hepatic population for related studies, but also make it clear of the inductive effects of three important growth factors, which could support for further investigation on the mechanisms of mesodermal and septumal derived signalings that regulate hepatic differentiation.

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

PubMed

Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

Stem Cell Banking: A Global View.

PubMed

Stacey, Glyn

2017-01-01

Stem cell banking has been a topic of discussion and debate for more than a decade since the first public services to supply human embryonic stem cells (hESCs) were established in the USA and the UK. This topic has received a recent revival with numerous ambitious programmes announced to deliver large collections of human induced pluripotency cell (hiPSC) lines. This chapter will provide a brief overview charting the development of stem cell banks, their value, and their likely role in the future.

Patent eligibility of stem cells in Europe: where do we stand after 8 years of case law?

PubMed

Storz, Ulrich; Faltus, Timo

2017-01-01

Since 2006, some of the highest ranking European Courts have issued decisions related to the patent eligibility of human embryonic stem cells. The question of patent eligibility of human embryonic stem cells remains, however, still erratic, at least in some aspects. This article will give a short comprehensive overview of the case history, and discuss questions still unsolved.

Differentiation of female Oct4-GFP embryonic stem cells into germ lineage cells.

PubMed

Ma, Xin; Li, Peng; Sun, Xiang; Sun, Yifeng; Hu, Rong; Yuan, Ping

2018-04-01

Due to high infertility ratio nowadays, it is essential to explore efficient ways of enhancing mammalian reproductivity, in particular female reproductivity. Using female Oct4-GFP embryonic stem cells, we mimic the in vivo development procedure to induce ES cells into epiblast cell-like cells (EpiLCs) and then primordial germ cell-like cells (PGCLCs). GFP positive PGCLCs that showed typical PGC markers and epigenetic modification were efficiently obtained. Further transplantation of the GFP positive PGCLC and native ovary cell mixture into ovary of infertile mice revealed that both MVH and GFP positive cells could be developed in ovary, but no later developmental stage germ cells were observed. This study suggested that Oct4-GFP ES cells may be only suitable for tracing early germ cell development. © 2018 International Federation for Cell Biology.

Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.

PubMed Central

Field, S J; Johnson, R S; Mortensen, R M; Papaioannou, V E; Spiegelman, B M; Greenberg, M E

1992-01-01

The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes. Images PMID:1329091

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.

PubMed

Kashuba, Corinna M; Benson, James D; Critser, John K

2014-04-01

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

PubMed

Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

2018-03-03

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

PubMed Central

Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

2014-01-01

Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

Implications of Parkinson's disease pathophysiology for the development of cell replacement strategies and drug discovery in neurodegenerative diseases.

PubMed

Pan-Montojo, Francisco; Funk, Richard H W

2012-11-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder traditionally characterized by the loss of dopaminergic neurons in the substantia nigra (SN) at the midbrain. The potential use of adult or embryonic stem cells, induced pluriputent stem (iPS) cells and endogenous neurogenesis in cell replacement strategies has lead to numerous studies and clinical trials in this direction. It is now possible to differentiate stem cells into dopaminergic neurons in vitro and clinical trials have shown an improvement in PD-related symptoms after intra-striatal embryonic transplants and acceptable cell survival rates on the mid term. However, clinical improvement is transitory and associated with a strong placebo effect. Interestingly, recent pathological studies in PD patients who received embryonic stem cells show that in PD patients, grafted neurons show PD-related pathology. In this manuscript we review the latest findings regarding PD pathophysiology and give an outlook on the implications of these findings in how cell replacement strategies for PD treatment should be tested. These include changes in the type of animal models used, the preparation/conditioning of the cells before intracerebral injection, specially regarding backbone chronic diseases in iPS cells and determining the optimal proliferation, survival, differentiation and migration capacity of the grafted cells.

Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

PubMed Central

Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

2015-01-01

Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

The New Federalism: State Policies Regarding Embryonic Stem Cell Research.

PubMed

Acosta, Nefi D; Golub, Sidney H

2016-09-01

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.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice.

PubMed

Alonso, Maria I; Lamus, Francisco; Carnicero, Estela; Moro, Jose A; de la Mano, Anibal; Fernández, Jose M F; Desmond, Mary E; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice

PubMed Central

Alonso, Maria I.; Lamus, Francisco; Carnicero, Estela; Moro, Jose A.; de la Mano, Anibal; Fernández, Jose M. F.; Desmond, Mary E.; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies. PMID:29311854

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Non-Neuronal Release of Gamma-Aminobutyric Acid by Embryonic Pluripotent Stem Cells

PubMed Central

Teng, Lin; Tang, Ya-Bin; Sun, Fan; An, Shi-Min; Zhang, Chun; Yang, Xin-Jie; Lv, Hao-Yu; Lu, Qin; Cui, Yong-Yao; Hu, Jin-Jia

2013-01-01

γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter. PMID:23799822

Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

PubMed Central

Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene

2015-01-01

Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells.

PubMed

Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios

2016-12-30

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

PubMed Central

Wood, S A; Pascoe, W S; Schmidt, C; Kemler, R; Evans, M J; Allen, N D

1993-01-01

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice. Images Fig. 1 Fig. 2 PMID:8506303

Human embryonic stem cell research: implications from an ethical and legal standpoint.

PubMed

Trepagnier, D M

2000-12-01

The purpose of this paper is to discuss the ethical and legal implications of one of the newest and most controversial medical breakthroughs. Stem cell research has been performed on mice for many years, but human embryonic stem cells are believed by scientists to be the basis for possible treatments and/or cures to many diseases affecting millions of people around the world. In order to perform research on human embryonic stem cells, numerous ethical issues must be addressed. Guidelines and protocols can be established in order to allow scientists to pursue new medical advances while maintaining the highest ethical standards in the use of human embryos. An alternative to using embryos is adult stem cells which have recently proven to be more versatile than previously believed. Opposing views will always be encountered when facing new science technologies. Where should the ethical line be drawn?

Towards a global human embryonic stem cell bank.

PubMed

Lott, Jason P; Savulescu, Julian

2007-08-01

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.

The Architectural Organization of Human Stem Cell Cycle Regulatory Machinery

PubMed Central

Stein, Gary S.; Stein, Janet L.; Wijnen, Andre van J; Lian, Jane B.; Montecino, Martin; Medina, Ricardo; Kapinas, Kristie; Ghule, Prachi; Grandy, Rodrigo; Zaidi, Sayyed K.; Becker, Klaus A.

2013-01-01

Two striking features of human embryonic stem cells that support biological activity are an abbreviated cell cycle and reduced complexity to nuclear organization. The potential implications for rapid proliferation of human embryonic stem cells within the context of sustaining pluripotency, suppressing phenotypic gene expression and linkage to simplicity in the architectural compartmentalization of regulatory machinery in nuclear microenvironments is explored. Characterization of the molecular and architectural commitment steps that license human embryonic stem cells to initiate histone gene expression is providing understanding of the principal regulatory mechanisms that control the G1/S phase transition in primitive pluripotent cells. From both fundamental regulatory and clinical perspectives, further understanding of the pluripotent cell cycle in relation to compartmentalization of regulatory machinery in nuclear microenvironments is relevant to applications of stem cells for regenerative medicine and new dimensions to therapy where traditional drug discovery strategies have been minimally effective. PMID:22394165

Twenty years of embryonic stem cell research in farm animals

USDA-ARS?s Scientific Manuscript database

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...

Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells

PubMed Central

Niu, N; Mercado-Uribe, I; Liu, J

2017-01-01

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse. PMID:28436947

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

PubMed Central

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.

2016-01-01

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

[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?].

PubMed

Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian

2009-12-01

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).

An Overview of Direct Somatic Reprogramming: The Ins and Outs of iPSCs

PubMed Central

Menon, Siddharth; Shailendra, Siny; Renda, Andrea; Longaker, Michael; Quarto, Natalina

2016-01-01

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application. PMID:26805822

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

PubMed

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

2017-04-06

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.

[Research with human embryo stem cells. Foundations and judicial limits].

PubMed

Eser, Albin; Koch, Hans-Georg

2004-01-01

Research with human embryos, and particularly, the use for scientific purposes of human embryonic stem cells has given raise to different sort of problems at the international level. One of the most strict regulation in this field, is this lecture Professors Albin Eser and Hans-Georg Koch analyse the german legal framework in relation with the use of embryos and human embryonic stem cells for scientific purposes.

Inhibition of Embryonic Genes to Control Colorectal Cancer Metastasis

DTIC Science & Technology

2014-09-01

14. ABSTRACT Embryonic core transcription factors (TFs), primarily the retrogene NanogP8, are the master regulators of cancer stem cells (CSC) in...core transcription factors (TFs), primarily the retrogene NanogP8, are the master regulators of cancer stem cells (CSC) in human colorectal carcinoma...maintaining the stemness of colorectal carcinoma (CRC) as well as the identification of two different pathways by which NANOG and NANOGP8 control pluripotency

Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

PubMed Central

Chan, Wen-Hsiung

2007-01-01

The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow

PubMed Central

Zhu, Jie; Wang, Hong; Yang, Shuo; Guo, Liqiao; Li, Zhen; Wang, Wei; Wang, Suhan; Huang, Wenting; Wang, Liping; Yang, Tan; Ma, Qiang; Bi, Yongyi

2013-01-01

Benzene is an occupational toxicant and an environmental pollutant that potentially causes hematotoxicity and leukemia in exposed populations. Epidemiological studies suggest an association between an increased incidence of childhood leukemia and benzene exposure during the early stages of pregnancy. However, experimental evidence supporting the association is lacking at the present time. It is believed that benzene and its metabolites target hematopoietic stem cells (HSCs) to cause toxicity and cancer in the hematopoietic system. In the current study, we compared the effects of hydroquinone (HQ), a major metabolite of benzene in humans and animals, on mouse embryonic yolk sac hematopoietic stem cells (YS-HSCs) and adult bone marrow hematopoietic stem cells (BM-HSCs). YS-HSCs and BM-HSCs were isolated and enriched, and were exposed to HQ at increasing concentrations. HQ reduced the proliferation and the differentiation and colony formation, but increased the apoptosis of both YS-HSCs and BM-HSCs. However, the cytotoxic and apoptotic effects of HQ were more apparent and reduction of colony formation by HQ was more severe in YS-HSCs than in BM-HSCs. Differences in gene expression profiles were observed in HQ-treated YS-HSCs and BM-HSCs. Cyp4f18 was induced by HQ both in YS-HSCs and BM-HSCs, whereas DNA-PKcs was induced in BM-HSCs only. The results revealed differential effects of benzene metabolites on embryonic and adult HSCs. The study established an experimental system for comparison of the hematopoietic toxicity and leukemogenicity of benzene and metabolites during mouse embryonic development and adulthood. PMID:23940708

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

The embryonic development of the cnidarian Hydractinia echinata.

PubMed

Kraus, Yulia; Flici, Hakima; Hensel, Katrin; Plickert, Günter; Leitz, Thomas; Frank, Uri

2014-01-01

With the rapid increase of the quantity of molecular data, many animals joined the ranks of the so-called 'emerging models' of Evo-Devo. One of the necessary steps in converting an emerging model into an established one is gaining comprehensive knowledge of its normal embryonic development. The marine colonial hydrozoan Hydractinia echinata - an excellent model for research on stem cells, metamorphosis, and allorecognition - has been studied for decades. Yet knowledge of its embryonic development remains fragmentary and incomplete. Here we provide a detailed account of H. echinata embryonic development using in vivo observations, histology, immunohistochemistry, and electron microscopy. Furthermore, we propose a model describing the cellular basis of the morphogenetic movements occurring during development and also reveal a functional link between canonical Wnt signaling and regional differences in the morphology of the embryo. Hydractinia embryogenesis is an example of the diversity and plasticity of hydrozoan development where multiple routes lead to the same result - the formation of a normal planula larva. © 2014 Wiley Periodicals, Inc.

Neural Stem Cells Derived Directly from Adipose Tissue.

PubMed

Petersen, Eric D; Zenchak, Jessica R; Lossia, Olivia V; Hochgeschwender, Ute

2018-05-01

Neural stem cells (NSCs) are characterized as self-renewing cell populations with the ability to differentiate into the multiple tissue types of the central nervous system. These cells can differentiate into mature neurons, astrocytes, and oligodendrocytes. This category of stem cells has been shown to be a promisingly effective treatment for neurodegenerative diseases and neuronal injury. Most treatment studies with NSCs in animal models use embryonic brain-derived NSCs. This approach presents both ethical and feasibility issues for translation to human patients. Adult tissue is a more practical source of stem cells for transplantation therapies in humans. Some adult tissues such as adipose tissue and bone marrow contain a wide variety of stem cell populations, some of which have been shown to be similar to embryonic stem cells, possessing many pluripotent properties. Of these stem cell populations, some are able to respond to neuronal growth factors and can be expanded in vitro, forming neurospheres analogous to cells harvested from embryonic brain tissue. In this study, we describe a method for the collection and culture of cells from adipose tissue that directly, without going through intermediates such as mesenchymal stem cells, results in a population of NSCs that are able to be expanded in vitro and be differentiated into functional neuronal cells. These adipose-derived NSCs display a similar phenotype to those directly derived from embryonic brain. When differentiated into neurons, cells derived from adipose tissue have spontaneous spiking activity with network characteristics similar to that of neuronal cultures.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whittaker, Peter A.

A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparingmore » immunocompatible pluripotent stem cells are indicated.« less

Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

EPA Science Inventory

Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

EPA Science Inventory

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...

Redox Disrupting Potential of ToxCast™Chemicals Ranked by Activity in Mouse Embryonic Stem Cells

EPA Science Inventory

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...

Assessment of a 42 metal salts chemical library in mouse embryonic stem cells

EPA Science Inventory

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...

Factors Expressed by Murine Embryonic Pancreatic Mesenchyme Enhance Generation of Insulin-Producing Cells From hESCs

PubMed Central

Guo, Tingxia; Landsman, Limor; Li, Na; Hebrok, Matthias

2013-01-01

Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro. PMID:23305648

Human Embryonic and Induced Pluripotent Stem Cell Research Trends: Complementation and Diversification of the Field

PubMed Central

Kobold, Sabine; Guhr, Anke; Kurtz, Andreas; Löser, Peter

2015-01-01

Summary Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas. PMID:25866160

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

PubMed Central

Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

2015-01-01

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells.

PubMed

Xu, Huilei; Baroukh, Caroline; Dannenfelser, Ruth; Chen, Edward Y; Tan, Christopher M; Kou, Yan; Kim, Yujin E; Lemischka, Ihor R; Ma'ayan, Avi

2013-01-01

High content studies that profile mouse and human embryonic stem cells (m/hESCs) using various genome-wide technologies such as transcriptomics and proteomics are constantly being published. However, efforts to integrate such data to obtain a global view of the molecular circuitry in m/hESCs are lagging behind. Here, we present an m/hESC-centered database called Embryonic Stem Cell Atlas from Pluripotency Evidence integrating data from many recent diverse high-throughput studies including chromatin immunoprecipitation followed by deep sequencing, genome-wide inhibitory RNA screens, gene expression microarrays or RNA-seq after knockdown (KD) or overexpression of critical factors, immunoprecipitation followed by mass spectrometry proteomics and phosphoproteomics. The database provides web-based interactive search and visualization tools that can be used to build subnetworks and to identify known and novel regulatory interactions across various regulatory layers. The web-interface also includes tools to predict the effects of combinatorial KDs by additive effects controlled by sliders, or through simulation software implemented in MATLAB. Overall, the Embryonic Stem Cell Atlas from Pluripotency Evidence database is a comprehensive resource for the stem cell systems biology community. Database URL: http://www.maayanlab.net/ESCAPE

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

PubMed

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

Applications of human umbilical cord blood cells in central nervous system regeneration.

PubMed

Herranz, Antonio S; Gonzalo-Gobernado, Rafael; Reimers, Diana; Asensio, Maria J; Rodríguez-Serrano, Macarena; Bazán, Eulalia

2010-03-01

In recent decades, there has been considerable amount of information about embryonic stem cells (ES). The dilemma facing scientists interested in the development and use of human stem cells in replacement therapies is the source of these cells, i.e. the human embryo. There are many ethical and moral problems related to the use of these cells. Hematopoietic stem cells from umbilical cord blood have been proposed as an alternative source of embryonic stem cells. After exposure to different agents, these cells are able to express antigens of diverse cellular lineages, including the neural type. The In vitro manipulation of human umbilical cord blood (hUCB) cells has shown their stem capacity and plasticity. These cells are easily accessible, In vitro amplifiable, well tolerated by the host, and with more primitive molecular characteristics that give them great flexibility. Overall, these properties open a promising future for the use of hUCB in regenerative therapies for the Central Nervous System (CNS). This review will focus on the available literature concerning umbilical cord blood cells as a therapeutic tool for the treatment of neurodegenerative diseases.

In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method.

PubMed

Wang, Xiang; Yang, Phillip

2008-07-23

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells.

Characterization of axon formation in the embryonic stem cell-derived motoneuron.

PubMed

Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

2011-01-01

The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

PubMed

Reid, Katherine J; Lang, Kenneth; Froscio, Suzanne; Humpage, Andrew J; Young, Fiona M

2015-11-01

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 μg/L. mES exposed to 0-1 μg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 μg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 μg/mL, the Lowest OEC was 1 μg/mL (p < 0.001, n = 3), and the IC50 was 0.86 μg/mL after 24 h. The ADWG 1 μg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

PubMed

Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

2011-07-01

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

Stem Cell Information: Glossary

MedlinePlus

... germ cells (those that would become sperm and eggs). Embryonic germ cells are thought to have properties ... the male gamete (sperm) and the female gamete (egg). Fetus - In humans, the developing human from approximately ...

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

PubMed Central

Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Heparanase confers a growth advantage to differentiating murine embryonic stem cells, and enhances oligodendrocyte formation.

PubMed

Xiong, Anqi; Kundu, Soumi; Forsberg, Maud; Xiong, Yuyuan; Bergström, Tobias; Paavilainen, Tanja; Kjellén, Lena; Li, Jin-Ping; Forsberg-Nilsson, Karin

2017-10-01

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.

Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

PubMed

Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

2014-05-01

The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

Effect of allo- and xenotransplantation of embryonic nervous tissue and umbilical cord blood-derived stem cells on structural and functional state of cerebral cortex of albino rats in posttraumatic period.

PubMed

Ereniev, S I; Semchenko, V V; Sysheva, E V; Bogdashin, I V; Shapovalova, V V; Khizhnyak, A S; Gasanenko, L N

2005-11-01

Comparative study of the structural and functional state of cerebral cortex of adult albino rats after intracerebral allo- and xenotransplantation of embryonic nervous tissue and intravenous injection of umbilical cord blood-derived stem cells at different terms after diffuse-focal cerebral trauma revealed the best cerebroprotective effect on day 7 of posttraumatic period in animals receiving embryonic nervous tissue.

Fluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Batista, Ana; König, Karsten

2014-02-01

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Neuromuscular junction in a microfluidic device.

PubMed

Park, Hyun Sung; Liu, Su; McDonald, John; Thakor, Nitish; Yang, In Hong

2013-01-01

Malfunctions at the site of neuromuscular junction (NMJ) of post-injuries or diseases are major barriers to recovery of function. The ability to efficiently derive motor neurons (MN) from embryonic stem cells has indicated promise toward the development of new therapies in increasing functional outcomes post injury. Recent advances in micro-technologies have provided advanced culture platforms allowing compartmentalization of sub-cellular components of neurons. In this study, we combined these advances in science and technology to develop a compartmentalized in vitro NMJ model. The developed NMJ system is between mouse embryonic stem cell (mESC)-derived MNs and c2c12 myotubes cultured in a compartmentalized polydimethylsiloxane (PDMS) microfluidic device. While some functional in vitro NMJ systems have been reported, this system would further contribute to research in NMJ-related diseases by providing a system to study the site of action of NMJ aimed at improving promoting better functional recovery.

Controversies in cancer stem cells: targeting embryonic signaling pathways.

PubMed

Takebe, Naoko; Ivy, S Percy

2010-06-15

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. (c) 2010 AACR.

Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

2016-03-22

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

PubMed

Beane, Olivia S; Darling, Eric M

2012-10-01

The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.

Commentary: "re-programming or selecting adult stem cells?".

PubMed

Trosko, James E

2008-01-01

The recent observations that embryonic stemness-associated genes could assist in the "de-differentiation" of adult skin fibroblast cells to "embryonic-like stem cells", using the "somatic cell nuclear transfer" techniques, have been interpreted as indicating a "re-programming" of genes. These reports have demonstrated a "proof of principle" approach to by-pass many, but not all, of the ethical, scientific and medical limitations of the "therapeutic cloning" of embryonic stem cells from embryos. However, while the interpretation that real "re-programming" of all those somatic fibroblastic differentiation genes might be correct, there does exists an alternative hypothesis of these exciting results. Based on the fact that multipotent adult stem cells exist in most, if not all, adult organs, the possibility exists that all these recent "re-programming" results, using the somatic nuclear transfer techniques, actually were the results of transferred rare nuclear material from the adult stem cells residing in the skin of the mouse, monkey and human samples. An examination of the rationale for this challenging hypothesis has been drawn from the hypothesis of the "stem cell theory of cancer", as well as from the field of human adult stem cells research.

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

PubMed

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

2016-02-12

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 that controls 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. Copyright © 2016, American Association for the Advancement of Science.

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis.

PubMed

Lipskind, Shane; Lindsey, Jennifer S; Gerami-Naini, Behzad; Eaton, Jennifer L; O'Connell, Daniel; Kiezun, Adam; Ho, Joshua W K; Ng, Nicholas; Parasar, Parveen; Ng, Michelle; Nickerson, Michael; Demirci, Utkan; Maas, Richard; Anchan, Raymond M

2018-05-01

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 + granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 - cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

Hypertranscription in development, stem cells, and regeneration

PubMed Central

Percharde, Michelle; Bulut-Karslioglu, Aydan; Ramalho-Santos, Miguel

2016-01-01

SUMMARY Cells can globally up-regulate their transcriptome during specific transitions, a phenomenon called hypertranscription. Evidence for hypertranscription dates back over 70 years, but it has gone largely ignored in the genomics era until recently. We discuss data supporting the notion that hypertranscription is a unifying theme in embryonic development, stem cell biology, regeneration and cell competition. We review the history, methods for analysis, underlying mechanisms and biological significance of hypertranscription. PMID:27989554

DNA methylation on N6-adenine in mammalian embryonic stem cells

PubMed Central

Wu, Tao P.; Wang, Tao; Seetin, Matthew G.; Lai, Yongquan; Zhu, Shijia; Lin, Kaixuan; Liu, Yifei; Byrum, Stephanie D.; Mackintosh, Samuel G.; Zhong, Mei; Tackett, Alan; Wang, Guilin; Hon, Lawrence S.; Fang, Gang; Swenberg, James A.; Xiao, Andrew Z.

2016-01-01

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N6-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N6-methyladenine. An increase of N6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N6-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N6-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N6-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. PMID:27027282

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity

PubMed Central

Ratajczak, Mariusz Z.; Zuba-Surma, Ewa K.; Shin, Dong-Myung; Ratajczak, Janina; Kucia, Magda

2011-01-01

Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4+) small stem cells (SCs) from the murine bone marrow (BM) that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs (VSELs). Our data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar SCs were also isolated from human cord blood (CB) and mobilized peripheral blood (mPB). We hypothesize that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells (PSCs) that give rise to tissue committed monopotent SCs and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs. Thus, these cells are potential SCs candidates for regenerative medicine and we envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes. PMID:18601995

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

PubMed

Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

2016-01-01

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

Genome editing: a robust technology for human stem cells.

PubMed

Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

2017-09-01

Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

PubMed

Schraermeyer, U; Thumann, G; Luther, T; Kociok, N; Armhold, S; Kruttwig, K; Andressen, C; Addicks, K; Bartz-Schmidt, K U

2001-01-01

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

EPA Science Inventory

Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

EPA Science Inventory

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

EPA Science Inventory

An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis.

PubMed

Angelova, Alexandra; Tiveron, Marie-Catherine; Cremer, Harold; Beclin, Christophe

2018-01-01

In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production.

Using therapeutic cloning to fight human disease: a conundrum or reality?

PubMed

Hall, Vanessa J; Stojkovic, Petra; Stojkovic, Miodrag

2006-07-01

The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

PubMed

Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

2018-03-09

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

PubMed

Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

2015-05-27

Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating

PubMed Central

Engler, Adam J.; Carag-Krieger, Christine; Johnson, Colin P.; Raab, Matthew; Tang, Hsin-Yao; Speicher, David W.; Sanger, Joseph W.; Sanger, Jean M.; Discher, Dennis E.

2009-01-01

Summary Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A ‘cysteine shotgun’ method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development. PMID:18957515

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification

PubMed Central

Xu, Cheng-Ran; Li, Lin-Chen; Donahue, Greg; Ying, Lei; Zhang, Yu-Wei; Gadue, Paul; Zaret, Kenneth S

2014-01-01

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells. PMID:25107471

Human embryonic stem cells express a unique set of microRNAs.

PubMed

Suh, Mi-Ra; Lee, Yoontae; Kim, Jung Yeon; Kim, Soo-Kyoung; Moon, Sung-Hwan; Lee, Ji Yeon; Cha, Kwang-Yul; Chung, Hyung Min; Yoon, Hyun Soo; Moon, Shin Yong; Kim, V Narry; Kim, Kye-Seong

2004-06-15

Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.

Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.

PubMed

Riebeling, Christian; Schlechter, Katharina; Buesen, Roland; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-06-01

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

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 ofmore » 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.« less

Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long-term maintenance of beating areas.

PubMed

Hamidi, Sofiane; Letourneur, Didier; Aid-Launais, Rachida; Di Stefano, Antonio; Vainchenker, William; Norol, Françoise; Le Visage, Catherine

2014-04-01

Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (p<0.05), MEF2C (p<0.01), and GATA4 (p<0.01), confirmed by flow cytometry analysis for MEF2C and NKX2.5. The ability of Fucoidan scaffolds to locally concentrate and slowly release TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (p<0.001), although only rare beating areas were observed. We postulated that absence of mechanical stress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that could be further combined with mechanical stress to promote sarcomere formation at terminal stages of differentiation.

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

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.

Mechanical forces direct stem cell behaviour in development and regeneration

PubMed Central

Vining, Kyle H.; Mooney, David J.

2018-01-01

Stem cells and their local microenvironment, or niche, communicate through mechanical, cues to regulate cell fate and cell behaviour, and to guide developmental processes. During embryonic development, mechanical forces are involved in patterning and organogenesis. The physical environment of pluripotent stem cells regulates their differentiation and self-renewal. Mechanical and physical cues are also important in adult tissues, where adult stem cells require physical interactions with the extracellular matrix to maintain their potency. In vitro, synthetic models of the stem cell niche can be used to precisely control and manipulate the biophysical and biochemical properties of the stem cell microenvironment and examine how the mode and magnitude of mechanical cues, such as matrix stiffness or applied forces, direct stem cell differentiation and function. Fundamental insights on the mechanobiology of stem cells also inform the design of artificial niches to support stem cells for regenerative therapies. PMID:29115301

Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

PubMed

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

2011-09-01

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).

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

A natural stem cell therapy? How novel findings and biotechnology clarify the ethics of stem cell research

PubMed Central

Patel, P

2006-01-01

The natural replacement of damaged cells by stem cells occurs actively and often in adult tissues, especially rapidly dividing cells such as blood cells. An exciting case in Boston, however, posits a kind of natural stem cell therapy provided to a mother by her fetus—long after the fetus is born. Because there is a profound lack of medical intervention, this therapy seems natural enough and is unlikely to be morally suspect. Nevertheless, we feel morally uncertain when we consider giving this type of therapy to patients who would not naturally receive it. Much has been written about the ethics of stem cell research and therapy; this paper will focus on how recent advances in biotechnology and biological understandings of development narrow the debate. Here, the author briefly reviews current stem cell research practices, revisits the natural stem cell therapy case for moral evaluation, and ultimately demonstrates the importance of permissible stem cell research and therapy, even absent an agreement about the definition of when embryonic life begins. Although one promising technology, blighted ovum utilisation, uses fertilised but developmentally bankrupt eggs, it is argued that utilisation of unfertilised eggs to derive totipotent stem cells obviates the moral debate over when life begins. There are two existing technologies that fulfil this criterion: somatic cell nuclear transfer and parthenogenic stem cell derivation. Although these technologies are far from therapeutic, concerns over the morality of embryonic stem cell derivation should not hinder their advancement. PMID:16574879

Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

PubMed

Xia, P; Liu, Z; Qin, P

2011-04-01

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc. © 2010 Blackwell Verlag GmbH.

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation.

PubMed

Shinde, Vaibhav; Klima, Stefanie; Sureshkumar, Perumal Srinivasan; Meganathan, Kesavan; Jagtap, Smita; Rempel, Eugen; Rahnenführer, Jörg; Hengstler, Jan Georg; Waldmann, Tanja; Hescheler, Jürgen; Leist, Marcel; Sachinidis, Agapios

2015-06-17

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

Editorial: Our top 10 developments in stem cell biology over the last 30 years.

PubMed

Armstrong, Lyle; Lako, Majlinda; Buckley, Noel; Lappin, Terry R J; Murphy, Martin J; Nolta, Jan A; Pittenger, Mark; Stojkovic, Miodrag

2012-01-01

To celebrate 30 years of peer-reviewed publication of cutting edge stem cell research in Stem Cells, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this, we will present our views of the 10 most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments would have occurred in recent years, but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body [Naturliche Schopfungsgeschichte, 1868; Berlin: Georg Reimer] but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960s did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960s-1970s and the first embryonic stem cell in 1981, such adult or tissue-specific stem cells were the only known class. The first issue of Stem Cells was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently, induced pluripotent stem cells (iPSCs) have been developed, and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only in Stem Cells but also in other journals over the last 5 years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation. Copyright © 2011 AlphaMed Press.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

PubMed Central

Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Stem cell potency and the ability to contribute to chimeric organisms.

PubMed

Polejaeva, Irina; Mitalipov, Shoukhrat

2013-03-01

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

A qualified endorsement of embryonic stem cell research, based on two widely shared beliefs about the brain-diseased patients such research might benefit.

PubMed

Disilvestro, R

2008-07-01

Are there persuasive approaches to embryonic stem cell (ESC) research that appeal, not just to those fellow-citizens in one's own ideological camp, nor just to those undecided citizens in the middle, but to those citizens on the other side of the issue? I believe that there are such arguments and in this short paper I try to develop one of them. In particular, I argue that certain beliefs shared by some proponents and some opponents of ESC research--beliefs about the personal identity and moral status of those who are victims of terrible brain diseases--are beliefs that should lead us to adopt a qualified endorsement of ESC research.

[A case of diprosopus in the cat].

PubMed

Aharon, D C; Wouda, W; van Weelden, E

1986-06-15

A case of diprosopus in a spontaneously delivered live-born kitten is reported. All facial components were completely duplicated. Fusion of the skulls had occurred in the temporal region; a single ear was present at the site of fusion. Additional defects were a cleft lip in one face and cleft palates in both faces. The cerebral hemispheres and arterior portions of the brain stem were completely duplicated, whereas the cerebellum and caudal brain stem were partially duplicated. The pathogenesis and aetiology are discussed. It is believed that disprosopus originates during the (pre)gastrulation stage of embryonic development, either by coalescence of two embryonic fields following a double process of gastrulation or by bifurcation of the axial mesoderm during a single gastrulation.

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

PubMed

Zhou, Ruoji; Xu, An; Wang, Donghui; Zhu, Dandan; Mata, Helen; Huo, Zijun; Tu, Jian; Liu, Mo; Mohamed, Alaa M T; Jewell, Brittany E; Gingold, Julian; Xia, Weiya; Rao, Pulivarthi H; Hung, Mien-Chie; Zhao, Ruiying; Lee, Dung-Fang

2018-03-01

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Asymmetric cell division of stem cells in the lung and other systems

PubMed Central

Berika, Mohamed; Elgayyar, Marwa E.; El-Hashash, Ahmed H. K.

2014-01-01

New insights have been added to identification, behavior and cellular properties of embryonic and tissue-specific stem cells over the last few years. The modes of stem cell division, asymmetric vs. symmetric, are tightly regulated during development and regeneration. The proper choice of a stem cell to divide asymmetrically or symmetrically has great consequences for development and disease because inappropriate asymmetric division disrupts organ morphogenesis, whereas uncontrolled symmetric division induces tumorigenesis. Therefore, understanding the behavior of lung stem cells could identify innovative solutions for restoring normal morphogenesis and/or regeneration of different organs. In this concise review, we describe recent studies in our laboratory about the mode of division of lung epithelial stem cells. We also compare asymmetric cell division (ACD) in the lung stem cells with other tissues in different organisms. PMID:25364740

Stem Cells in Skeletal Tissue Engineering: Technologies and Models

PubMed Central

Langhans, Mark T.; Yu, Shuting; Tuan, Rocky S.

2017-01-01

This review surveys the use of pluripotent and multipotent stem cells in skeletal tissue engineering. Specific emphasis is focused on evaluating the function and activities of these cells in the context of development in vivo, and how technologies and methods of stem cell-based tissue engineering for stem cells must draw inspiration from developmental biology. Information on the embryonic origin and in vivo differentiation of skeletal tissues is first reviewed, to shed light on the persistence and activities of adult stem cells that remain in skeletal tissues after embryogenesis. Next, the development and differentiation of pluripotent stem cells is discussed, and some of their advantages and disadvantages in the context of tissue engineering is presented. The final section highlights current use of multipotent adult mesenchymal stem cells, reviewing their origin, differentiation capacity, and potential applications to tissue engineering. PMID:26423296

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

USDA-ARS?s Scientific Manuscript database

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

3 CFR 8446 - Proclamation 8446 of October 30, 2009. National Alzheimer's Disease Awareness Month, 2009

Code of Federal Regulations, 2010 CFR

2010-01-01

..., we must leave no avenue unexplored. Embryonic stem cells may hold the key for us to better understand... Executive Order lifting the ban on Federal funding for embryonic stem cell research, with proper guidelines... are now able to start treatments earlier, slowing the loss of brain cells and the progression of...

Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

EPA Science Inventory

The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives.

PubMed

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

2011-12-22

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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of an adherent mouse embryonic stem cell in vitro assay to predict developmental toxicity of ToxCast chemicals.

EPA Science Inventory

The potential for most environmental chemicals to produce developmental toxicity is unknown. Mouse embryonic stem cell (mESC) assays are an alternative in vitro model to assess chemicals. The chemical space evaluated using mESC and compared to in vivo is limited. We used an adher...

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity.

PubMed

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-04-10

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features

PubMed Central

Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

2016-01-01

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP. PMID:27385131

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

PubMed

Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

2016-07-07

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

Embryonic Stem Cells: Isolation, Characterization and Culture

NASA Astrophysics Data System (ADS)

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.

Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion

PubMed Central

Kidder, Benjamin L.; Oseth, Leann; Miller, Shanna; Hirsch, Betsy; Verfaillie, Catherine

2008-01-01

Abstract Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras. PMID:18338954

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

PubMed

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Therapeutic cloning and cellular reprogramming.

PubMed

Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

2012-01-01

Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use.

Nickel(ii) inhibits the oxidation of DNA 5-methylcytosine in mammalian somatic cells and embryonic stem cells.

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2018-03-01

Nickel is found widely in the environment. It is an essential microelement but also toxic. However, nickel displays only weak genotoxicity and mutagenicity. Exploration of the epigenetic toxicity of nickel is extremely interesting. Iron(ii)- and 2-oxoglutarate-dependent Tet dioxygenases are a class of epigenetic enzymes that catalyze the oxidation of DNA 5-methylcytosine (5mC). Thus, they are critical for DNA demethylation and, importantly, are involved with nuclear reprogramming, embryonic development, and regulation of gene expression. Here, we demonstrated that nickel(ii) dramatically inhibits Tet proteins-mediated oxidation of DNA 5mC in cells ranging from somatic cell lines to embryonic stem cells, as manifested by the consistent observation of a significant decrease in 5-hydroxymethylcytosine, a critical intermediate resulting from the oxidation of 5mC. The inhibitory effects of nickel(ii) were concentration- and time-dependent. Using HEK293T cells overexpressing Tet proteins and ascorbic acid-stimulated Tet-proficient ES cells, we observed that nickel(ii) significantly reduced DNA demethylation at the global level. Interestingly, we also showed that nickel(ii) might affect the naïve or ground state of pluripotent embryonic stem cells. Here we show, for the first time, that nickel(ii) represses the oxidation of DNA 5mC and potentially alters the Tet proteins-regulated DNA methylation landscape in human cells. These findings provide new insights into the epigenetic toxicology of nickel.

Current status of stem cells in cardiac repair.

PubMed

Henning, Robert J

2018-03-01

One out of every two men and one out of every three women greater than the age of 40 will experience an acute myocardial infarction (AMI) at some time during their lifetime. As more patients survive their AMIs, the incidence of congestive heart failure (CHF) is increasing. 6 million people in the USA have ischemic cardiomyopathies and CHF. The search for new and innovative treatments for patients with AMI and CHF has led to investigations and use of human embryonic stem cells, cardiac stem/progenitor cells, bone marrow-derived mononuclear cells and mesenchymal stem cells for treatment of these heart conditions. This paper reviews current investigations with human embryonic, cardiac, bone marrow and mesenchymal stem cells, and also stem cell paracrine factors and exosomes.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems.

PubMed

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-11-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non-adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems

PubMed Central

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-01-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non‐adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development. PMID:23486530

Adult Olfactory Bulb Interneuron Phenotypes Identified by Targeting Embryonic and Postnatal Neural Progenitors

PubMed Central

Figueres-Oñate, Maria; López-Mascaraque, Laura

2016-01-01

Neurons are generated during embryonic development and in adulthood, although adult neurogenesis is restricted to two main brain regions, the hippocampus and olfactory bulb. The subventricular zone (SVZ) of the lateral ventricles generates neural stem/progenitor cells that continually provide the olfactory bulb (OB) with new granule or periglomerular neurons, cells that arrive from the SVZ via the rostral migratory stream. The continued neurogenesis and the adequate integration of these newly generated interneurons is essential to maintain homeostasis in the olfactory bulb, where the differentiation of these cells into specific neural cell types is strongly influenced by temporal cues. Therefore, identifying the critical features that control the generation of adult OB interneurons at either pre- or post-natal stages is important to understand the dynamic contribution of neural stem cells. Here, we used in utero and neonatal SVZ electroporation along with a transposase-mediated stable integration plasmid, in order to track interneurons and glial lineages in the OB. These plasmids are valuable tools to study the development of OB interneurons from embryonic and post-natal SVZ progenitors. Accordingly, we examined the location and identity of the adult progeny of embryonic and post-natally transfected progenitors by examining neurochemical markers in the adult OB. These data reveal the different cell types in the olfactory bulb that are generated in function of age and different electroporation conditions. PMID:27242400

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

PubMed

Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Erythro-Myeloid Progenitors: “definitive” hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells

PubMed Central

Frame, Jenna M.; McGrath, Kathleen E.; Palis, James

2013-01-01

Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes. PMID:24095199

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

PubMed

Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

2011-04-01

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Adipose-derived mesenchymal stem cells and regenerative medicine.

PubMed

Konno, Masamitsu; Hamabe, Atsushi; Hasegawa, Shinichiro; Ogawa, Hisataka; Fukusumi, Takahito; Nishikawa, Shimpei; Ohta, Katsuya; Kano, Yoshihiro; Ozaki, Miyuki; Noguchi, Yuko; Sakai, Daisuke; Kudoh, Toshihiro; Kawamoto, Koichi; Eguchi, Hidetoshi; Satoh, Taroh; Tanemura, Masahiro; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

2013-04-01

Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer.

PubMed

Bora-Singhal, Namrata; Perumal, Deepak; Nguyen, Jonathan; Chellappan, Srikumar

2015-07-01

Non-small cell lung cancer (NSCLC) patients have very low survival rates because the current therapeutic strategies are not fully effective. Although EGFR tyrosine kinase inhibitors are effective for NSCLC patients harboring EGFR mutations, patients invariably develop resistance to these agents. Alterations in multiple signaling cascades have been associated with the development of resistance to EGFR inhibitors. Sonic Hedgehog and associated Gli transcription factors play a major role in embryonic development and have recently been found to be reactivated in NSCLC, and elevated Gli1 levels correlate with poor prognosis. The Hedgehog pathway has been implicated in the functions of cancer stem cells, although the underlying molecular mechanisms are not clear. In this context, we demonstrate that Gli1 is a strong regulator of embryonic stem cell transcription factor Sox2. Depletion of Gli1 or inhibition of the Hedgehog signaling significantly abrogated the self-renewal of stem-like side-population cells from NSCLCs as well as vascular mimicry of such cells. Gli1 was found to transcriptionally regulate Sox2 through its promoter region, and Gli1 could be detected on the Sox2 promoter. Inhibition of Hedgehog signaling appeared to work cooperatively with EGFR inhibitors in markedly reducing the viability of NSCLC cells as well as the self-renewal of stem-like cells. Thus, our study demonstrates a cooperative functioning of the EGFR signaling and Hedgehog pathways in governing the stem-like functions of NSCLC cancer stem cells and presents a novel therapeutic strategy to combat NSCLC harboring EGFR mutations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A novel perfused rotary bioreactor for cardiomyogenesis of embryonic stem cells.

PubMed

Teo, Ailing; Mantalaris, Athanasios; Song, Kedong; Lim, Mayasari

2014-05-01

Developments in bioprocessing technology play an important role for overcoming challenges in cardiac tissue engineering. To this end, our laboratory has developed a novel rotary perfused bioreactor for supporting three-dimensional cardiac tissue engineering. The dynamic culture environments provided by our novel perfused rotary bioreactor and/or the high-aspect rotating vessel produced constructs with higher viability and significantly higher cell numbers (up to 4 × 10(5) cells/bead) than static tissue culture flasks. Furthermore, cells in the perfused rotary bioreactor showed earlier gene expressions of cardiac troponin-T, α- and β-myosin heavy chains with higher percentages of cardiac troponin-I-positive cells and better uniformity of sacromeric α-actinin expression. A dynamic and perfused environment, as provided by this bioreactor, provides a superior culture performance in cardiac differentiation for embryonic stem cells particularly for larger 3D constructs.

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

PubMed

Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

2010-10-01

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

The effect of matrix composition of 3D constructs on embryonic stem cell differentiation.

PubMed

Battista, Sabrina; Guarnieri, Daniela; Borselli, Cristina; Zeppetelli, Stefania; Borzacchiello, Assunta; Mayol, Laura; Gerbasio, Diego; Keene, Douglas R; Ambrosio, Luigi; Netti, Paolo A

2005-11-01

The use of embryonic stem (ES) cells as unlimited cell source in tissue engineering has ignited the hope of regenerating any kind of tissue in vitro. However, the role of the material in control and guidance of their development and commitment into complex and viable three-dimensional (3D) tissues is still poorly understood. In this work, we investigate the role of material composition and structure on promoting ES cells growth and differentiation, by culturing mouse ES cell-derived embryoid bodies (EBs) in various semi-interpenetrating polymer networks (SIPNs), made of collagen, fibronectin (FN) and laminin (LM). We show that both composition and strength of the supportive matrix play an important role in EBs development. High collagen concentrations inhibit EBs cavitation and hence the following EBs differentiation, by inhibiting apoptosis. The presence of FN in 3D collagen constructs strongly stimulates endothelial cell differentiation and vascularization. Conversely, LM increases the ability of ES cells to differentiate into beating cardiomyocytes. Our data suggest that matrix composition has an important role in EBs development and that it is possible to influence stem cell differentiation toward preferential pattern, by modulating the physical and biochemical properties of the scaffold.

A new avenue to the synthesis of GAG-mimicking polymers highly promoting neural differentiation of embryonic stem cells.

PubMed

Wang, Mengmeng; Lyu, Zhonglin; Chen, Gaojian; Wang, Hongwei; Yuan, Yuqi; Ding, Kaiguo; Yu, Qian; Yuan, Lin; Chen, Hong

2015-10-28

A new strategy for the fabrication of glycosaminoglycan (GAG) analogs was proposed by copolymerizing the sulfonated unit and the glyco unit, 'splitted' from the sulfated saccharide building blocks of GAGs. The synthetic polymers can promote cell proliferation and neural differentiation of embryonic stem cells with the effects even better than those of heparin.

75 FR 11185 - Submission for OMB Review; Comment Request; Reinstatement of OMB No. 0925-0601/exp. 02/28/2010...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-10

...; Comment Request; Reinstatement of OMB No. 0925-0601/exp. 02/28/2010, Request for Human Embryonic Stem Cell... period seeking public comment for an information collection entitled ``Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research''. In the second paragraph of the notice...

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

USDA-ARS?s Scientific Manuscript database

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...

Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis

PubMed Central

Angelova, Alexandra; Tiveron, Marie-Catherine; Cremer, Harold; Beclin, Christophe

2018-01-01

In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production. PMID:29511358

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

PubMed

Starborg, Tobias; Kadler, Karl E

2015-03-01

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development. © 2015 Wiley Periodicals, Inc.

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

PubMed

Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

2012-03-01

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

Integration and long distance axonal regeneration in the central nervous system from transplanted primitive neural stem cells.

PubMed

Zhao, Jiagang; Sun, Woong; Cho, Hyo Min; Ouyang, Hong; Li, Wenlin; Lin, Ying; Do, Jiun; Zhang, Liangfang; Ding, Sheng; Liu, Yizhi; Lu, Paul; Zhang, Kang

2013-01-04

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.

Angiomodulin is required for cardiogenesis of embryonic stem cells and is maintained by a feedback loop network of p63 and Activin-A.

PubMed

Wolchinsky, Zohar; Shivtiel, Shoham; Kouwenhoven, Evelyn Nathalie; Putin, Daria; Sprecher, Eli; Zhou, Huiqing; Rouleau, Matthieu; Aberdam, Daniel

2014-01-01

The transcription factor p63, member of the p53 gene family, encodes for two main isoforms, TAp63 and ΔNp63 with distinct functions on epithelial homeostasis and cancer. Recently, we discovered that TAp63 is essential for in vitro cardiogenesis and heart development in vivo. TAp63 is expressed by embryonic endoderm and acts on cardiac progenitors by a cell-non-autonomous manner. In the present study, we search for cardiogenic secreted factors that could be regulated by TAp63 and, by ChIP-seq analysis, identified Angiomodulin (AGM), also named IGFBP7 or IGFBP-rP1. We demonstrate that AGM is necessary for cardiac commitment of embryonic stem cells (ESCs) and its regulation depends on TAp63 isoform. TAp63 directly activates both AGM and Activin-A during ESC cardiogenesis while these secreted factors modulate TAp63 gene expression by a feedback loop mechanism. The molecular circuitry controlled by TAp63 on AGM/Activin-A signaling pathway and thus on cardiogenesis emphasizes the importance of p63 during early cardiac development. © 2013.

Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in-vitro study.

PubMed

Tielens, S; Declercq, H; Gorski, T; Lippens, E; Schacht, E; Cornelissen, M

2007-03-01

Mouse embryonic stem cells were cultured on commercially available biodegradable macroporous microcarriers. A culture period of 1-2 weeks was needed to colonize the microcarriers. Embryonic stem cells retained their pluripotency for up to 14 days when cultured in medium supplemented with leukemia inhibitory factor. Replacing this medium by differentiation medium for 2 weeks initiated osteogenic differentiation. Encapsulation of the cell-loaded microcarriers in photopolymerizable polymers (methacrylate-endcapped poly-D,L-lactide-co-caprolactone), triacetin/hydroxyethylmethacrylate (HEMA) as solvent and with/without gelatin as porogen, resulted in a homogeneous distribution of the microcarriers in the polymer. As observed by transmission electron microscopy, viability of the cells was optimal when gelatin was omitted and when using triacetin instead of HEMA.

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

PubMed

Ishiwata, Isamu; Tamagawa, Tomoharu; Tokieda, Yuko; Iguchi, Megumi; Sato, Kahei; Ishikawa, Hiroshi

2003-03-01

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.

PubMed

Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

2016-01-01

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

'New embryos' - new challenges for the ethics of stem cell research.

PubMed

Holm, Søren

2008-01-01

Among the many ethical issues raised by human embryonic stem cell research (in the following all references to 'stem cells' should be read as references to human embryonic stem cells), two have gained specific prominence: (1) whether stem cell research is ethically problematic because it entails the destruction of human embryos and (2) what kind of control embryo donors should have over the stem cell lines derived from their embryos. In the present paper, I will analyse how these two issues are engaged by various attempts to derive stem cells from anomalous embryos (e.g. embryos in cleavage arrest, embryos not implanted following pre-implantation genetic diagnosis or embryos created by altered nuclear transfer) or in ways that are claimed to be non-destructive for the embryo (e.g. blastocyst or blastomere biopsy). Copyright 2008 S. Karger AG, Basel.

Embryonic Stem Cells Promoting Macrophage Survival and Function are Crucial for Teratoma Development

PubMed Central

Chen, Tianxiang; Wang, Xi; Guo, Lei; Wu, Mingmei; Duan, Zhaoxia; Lv, Jing; Tai, Wenjiao; Renganathan, Hemamalini; Didier, Ruth; Li, Jinhua; Sun, Dongming; Chen, Xiaoming; He, Xijing; Fan, Jianqing; Young, Wise; Ren, Yi

2014-01-01

Stem cell therapies have had tremendous potential application for many diseases in recent years. However, the tumorigenic properties of stem cells restrict their potential clinical application; therefore, strategies for reducing the tumorigenic potential of stem cells must be established prior to transplantation. We have demonstrated that syngeneic transplantation of embryonic stem cells (ESCs) provokes an inflammatory response that involves the rapid recruitment of bone marrow-derived macrophages (BMDMs). ESCs are able to prevent mature macrophages from macrophage colony-stimulating factor (M-CSF) withdrawal-induced apoptosis, and thus prolong macrophage lifespan significantly by blocking various apoptotic pathways in an M-CSF-independent manner. ESCs express and secrete IL-34, which may be responsible for ESC-promoted macrophage survival. This anti-apoptotic effect of ESCs involves activation of extracellular signal-regulated kinase (ERK)1/2 and PI3K/Akt pathways and thus, inhibition of ERK1/2 and PI3K/AKT activation decreases ESC-induced macrophage survival. Functionally, ESC-treated macrophages also showed a higher level of phagocytic activity. ESCs further serve to polarize BMDMs into M2-like macrophages that exhibit most tumor-associated macrophage phenotypic and functional features. ESC-educated macrophages produce high levels of arginase-1, Tie-2, and TNF-α, which participate in angiogenesis and contribute to teratoma progression. Our study suggests that induction of M2-like macrophage activation is an important mechanism for teratoma development. Strategies targeting macrophages to inhibit teratoma development would increase the safety of ESC-based therapies, inasmuch as the depletion of macrophages completely inhibits ESC-induced angiogenesis and teratoma development. PMID:25071759

Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line.

PubMed

Hunt, C P J; Pouton, C W; Haynes, J M

2017-06-01

In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32 WT/eGFP-AMP-LacZ ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

PubMed

Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

2009-06-01

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

Protein kinase A inhibitor, H89, significantly enhances survival rate of dissociated human embryonic stem cells following cryopreservation.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-10-01

Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow-freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESCs following cryopreservation. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89. H89 significantly improved survival level of dissociated hESCs after cryopreservation through ROCK inhibition. H89-treated cells still maintained their pluripotency and differentiation capacity. This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future. © 2016 John Wiley & Sons Ltd.

Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

PubMed

Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

2016-08-01

Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. © The Author(s) 2016.

Large Scale Production of Stem Cells and Their Derivatives

NASA Astrophysics Data System (ADS)

Zweigerdt, Robert

Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Yong; Wang Honglan; Mazzone, Theodore

2006-08-01

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,more » 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.« less

Induction of pluripotent stem cells transplantation therapy for ischemic stroke.

PubMed

Jiang, Mei; Lv, Lei; Ji, Haifeng; Yang, Xuelian; Zhu, Wei; Cai, Liying; Gu, Xiaju; Chai, Changfeng; Huang, Shu; Sun, Jian; Dong, Qiang

2011-08-01

Stroke can cause permanent neurological damage, complications, and even death. However, there is no treatment exists to restore its lost function. Human embryonic stems transplantation therapy was a novel and potential therapeutic approach for stroke. However, as we have seen, the ethical controversy pertains to embryonic stem cell research. Human induced pluripotent stem cells (iPSCs) are the latest generation of stem cells that may be a solution to the controversy of using embryonic cells. In our study, we generated iPSCs from adult human fibroblasts by introduction of four defined transcription factors (Oct4, Sox2, Nanog, and Lin-28). And then, we investigated the efficacy of iPSCs transplantation therapy for stroke on the animal models of middle cerebral artery occlusion. Surprisingly, we found that transplanted iPSCs migrated to injured brain areas, and differentiated into neuron-like cells successfully. After 4-16 days iPSCs grafting, sensorimotor function of rats has been improved significantly. In one word, we may prove that iPSCs therapy in stroke to be an effective form of treatment.

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Sevoflurane represses the self-renewal ability by regulating miR-7a,7b/Klf4 signalling pathway in mouse embryonic stem cells.

PubMed

Wang, Qimin; Li, Guifeng; Li, Baolin; Chen, Qiu; Lv, Dongdong; Liu, Jiaying; Ma, Jieyu; Sun, Nai; Yang, Longqiu; Fei, Xuejie; Song, Qiong

2016-10-01

Sevoflurane is a frequently-used clinical inhalational anaesthetic and can cause toxicity to embryos during foetal development. Embryonic stem cells (ESCs) are derived from the inner cell mass of blastospheres and can be used as a useful model of early development. Here, we found that sevoflurane significantly influenced self-renewal ability of mESCs on stemness maintenance and cell proliferation. The cell cycle was arrested via G1 phase delay. We further found that sevoflurane upregulated expression of miR-7a,7b to repress self-renewal. Next we performed rescue experiments and found that after adding miR-7a,7b inhibitor into mESCs treated with sevoflurane, its influence on self-renewal could be blocked. Further we identified stemness factor Klf4 as the direct target of miR-7a,7b. Overexpression of Klf4 restored self-renewal ability repressed by miR-7a,7b or sevoflurane. In this work, we determined that sevoflurane repressed self-renewal ability by regulating the miR-7a,7b/Klf4 signalling pathway in mESCs. Our study demonstrated molecular mechanism underlying the side effects of sevoflurane during early development, laying the foundation for studies on safe usage of inhalational anaesthetic during non-obstetric surgery. © 2016 John Wiley & Sons Ltd.

75 FR 81283 - Eunice Kennedy Shriver National Institute of Child Health & Human Development; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-27

... Support for Human Embryonic Stem Cell Research Update. Place: National Institutes of Health, Building 31...; 93.865, Research for Mothers and Children; 93.929, Center for Medical Rehabilitation Research; 93.209...

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

PubMed

Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

2011-07-14

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

PubMed Central

Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

2009-01-01

BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells.

PubMed

Xu, Zhuojin; Robitaille, Aaron M; Berndt, Jason D; Davidson, Kathryn C; Fischer, Karin A; Mathieu, Julie; Potter, Jennifer C; Ruohola-Baker, Hannele; Moon, Randall T

2016-10-18

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Survival of priceless cells: active and passive protection of embryonic stem cells against immune destruction.

PubMed

Utermöhlen, Olaf; Krönke, Martin

2007-06-15

This review focuses on our current knowledge of the mechanisms employed by embryonic stem (ES) cells to avoid destruction by cell-mediated immune responses. Recently, ES cells have been found to shield themselves against cytotoxic effector cells by expressing CD95L and serine protease inhibitor SPI-6 mediating apoptosis of the cytotoxic cells and inactivation of granzyme B, respectively. These findings are discussed in view of their implications for using ES cell-derived transplants in regenerative medicine as well as for our understanding of early embryonic stages during invasion and implantation.

Maintaining embryonic stem cell pluripotency with Wnt signaling.

PubMed

Sokol, Sergei Y

2011-10-01

Wnt signaling pathways control lineage specification in vertebrate embryos and regulate pluripotency in embryonic stem (ES) cells, but how the balance between progenitor self-renewal and differentiation is achieved during axis specification and tissue patterning remains highly controversial. The context- and stage-specific effects of the different Wnt pathways produce complex and sometimes opposite outcomes that help to generate embryonic cell diversity. Although the results of recent studies of the Wnt/β-catenin pathway in ES cells appear to be surprising and controversial, they converge on the same conserved mechanism that leads to the inactivation of TCF3-mediated repression.

Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era

PubMed Central

Huang, Hongyun; Chen, Lin; Sanberg, Paul

2010-01-01

Recent advances in cell biology, neural injury and repair, and the progress towards development of neurorestorative interventions are the basis for increased optimism. Based on the complexity of the processes of demyelination and remyelination, degeneration and regeneration, damage and repair, functional loss and recovery, it would be expected that effective therapeutic approaches will require a combination of strategies encompassing neuroplasticity, immunomodulation, neuroprotection, neurorepair, neuroreplacement, and neuromodulation. Cell-based restorative treatment has become a new trend, and increasing data worldwide have strongly proven that it has a pivotal therapeutic value in CNS disease. Moreover, functional neurorestoration has been achieved to a certain extent in the CNS clinically. Up to now, the cells successfully used in preclinical experiments and/or clinical trial/treatment include fetal/embryonic brain and spinal cord tissue, stem cells (embryonic stem cells, neural stem/progenitor cells, hematopoietic stem cells, adipose-derived adult stem/precursor cells, skin-derived precursor, induced pluripotent stem cells), glial cells (Schwann cells, oligodendrocyte, olfactory ensheathing cells, astrocytes, microglia, tanycytes), neuronal cells (various phenotypic neurons and Purkinje cells), mesenchymal stromal cells originating from bone marrow, umbilical cord, and umbilical cord blood, epithelial cells derived from the layer of retina and amnion, menstrual blood-derived stem cells, Sertoli cells, and active macrophages, etc. Proof-of-concept indicates that we have now entered a new era in neurorestoratology. PMID:21359168

Stem cell research and policy in India: current scenario and future perspective.

PubMed

Sharma, Alka

2009-01-01

Stem cell research is an exciting area of biomedical research, with potential to advance cell biology, and other new modalities of treatment for many untreatable diseases. The potential resides in the ability of these cells to develop into many different cell types in the body. In India, efforts are being made on several fronts to promote this area in an integrated way. The main features of the strategy are: explore the full potential of adult and embryonic stem cells (ESCs) through basic and translational research; generate patient specific human ESC lines; enhance creation of animal models for pre-clinical studies; virtual network of Centres; creation institutions; generation of well trained manpower; build partnership with large companies in path-breaking areas; promote closer interactions amongst basic scientists, clinical researchers and the industry. Newer initiatives include: establishment of a dedicated institute for stem cell science and regenerative medicine with its translational units; GMP and clean room facilities in medical schools; creation of a system for multi-centric clinical studies using autologous adult stem cells; national and international training courses for providing training to the students and the young scientists in the both embryonic and adult stem cells; and formulation of guidelines to conduct stem cell research in a responsible and ethically sensitive manner in the country. The core capacity must be nurtured and built to create the required critical mass to have impact.

Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells.

PubMed

Chen, Hongwei; Mao, Yu; Wang, Shufen; Li, Bin; Wang, Jinhuan; Li, Jian; Ma, Yuanye

2015-01-01

Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A 2 B 5 -positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo . These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock

PubMed Central

Deschamps, Jacqueline; Duboule, Denis

2017-01-01

Collinear regulation of Hox genes in space and time has been an outstanding question ever since the initial work of Ed Lewis in 1978. Here we discuss recent advances in our understanding of this phenomenon in relation to novel concepts associated with large-scale regulation and chromatin structure during the development of both axial and limb patterns. We further discuss how this sequential transcriptional activation marks embryonic stem cell-like axial progenitors in mammals and, consequently, how a temporal genetic system is further translated into spatial coordinates via the fate of these progenitors. In this context, we argue the benefit and necessity of implementing this unique mechanism as well as the difficulty in evolving an alternative strategy to deliver this critical positional information. PMID:28860158

Revocation of European patent for neural progenitors highlights patent challenges for inventions relating to human embryonic stem cells.

PubMed

Rigby, Barbara

2013-11-01

Cells derived from human embryonic stem cells have great therapeutic potential. Patents are key to allowing companies that develop methods of generating such cells to recuperate their investment. However, in Europe, inventions relating to the use of human embryos for commercial purposes are excluded from patentability on moral grounds. The scope of this morality exclusion was recently tested before Germany's highest court and before the European Patent Office (EPO), with diverging results. The decision by the EPO's Opposition Division to revoke EP1040185 relating to neural precursors and methods for their generation has received a mixed reception. The decision has very recently been appealed, and the outcome of this Appeal should provide more definitive guidance on the scope of the morality exclusion.

The Emergence of Blood and Blood Vessels in the Embryo and Its Relevance to Postnatal Biology and Disease

NASA Astrophysics Data System (ADS)

Sills, Tiffany M.; Hirschi, Karen K.

Blood and blood vessels develop in parallel within mammalian systems, and this temporal and spatial association has led to the confirmation of an endothelial origin of hematopoiesis. The extraembryonic yolk sac and aorto-gonado-mesonephros (AGM) region both contain a specialized population of endothelial cells ("hemogenic endothelium") that function to produce hematopoietic stem and progenitor cells, which then differentiate to provide the full complement of blood cells within the developing embryo and furthermore in the adult system. Therefore, this population has great therapeutic potential in the fields of regenerative medicine and tissue engineering. This chapter reviews the development of the vascular and hematopoietic systems, characterization and function of the hemogenic endothelium within embryonic and embryonic stem cell (ES cell) models, and speculate on the presence of such a population within the adult system. In order to harness this endothelial subtype for clinical application, we must understand both the normal functions of these cells and the potential for misregulation in disease states.

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

PubMed Central

Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

2017-01-01

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

PubMed

Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

High School Students Debate the Use of Embryonic Stem Cells: The Influence of Context on Decision-Making

ERIC Educational Resources Information Center

Molinatti, Gregoire; Girault, Yves; Hammond, Constance

2010-01-01

The present study analyzes decision-making and argumentation by high school students in a debate situation on a socioscientific issue, the use of embryonic stem cells in research and therapy. We tested the influence on the debates of two different contexts. Adolescent students at the high school level in the same grade (mean age 16.4 years) from…

Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

EPA Science Inventory

Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

Stem cells: Balancing resistance and sensitivity to DNA damage

PubMed Central

Liu, Julia C.; Lerou, Paul H.; Lahav, Galit

2015-01-01

Embryonic stem cells are known to be very sensitive to DNA damage and undergo rapid apoptosis even after low damage doses. In contrast, adult stem cells show variable sensitivity to damage. Here we describe the multiple pathways that have been proposed to affect the sensitivity of stem cells to damage, including proximity to the apoptotic threshold (mitochondrial priming) and the p53 signaling pathway, through activation of transcription or direct interaction with pro apoptotic proteins in the cytoplasm. We also discuss which cellular factors might connect mitochondrial priming with pluripotency and the potential therapeutic advances that can be achieved by better understanding the molecular mechanisms leading to sensitivity or resistance of embryonic or adult stem cells from different tissues. PMID:24721782

"Nutrient-sensing" and self-renewal: O-GlcNAc in a new role.

PubMed

Sharma, Nikita S; Saluja, Ashok K; Banerjee, Sulagna

2018-06-01

Whether embryonic, hematopoietic or cancer stem cells, this metabolic reprogramming is dependent on the nutrient-status and bioenergetic pathways that is influenced by the micro-environmental niches like hypoxia. Thus, the microenvironment plays a vital role in determining the stem cell fate by inducing metabolic reprogramming. Under the influence of the microenvironment, like hypoxia, the stem cells have increased glucose and glutamine uptake which result in activation of hexosamine biosynthesis pathway (HBP) and increased O-GlcNAc Transferase (OGT). The current review is focused on understanding how HBP, a nutrient-sensing pathway (that leads to increased OGT activity) is instrumental in regulating self-renewal not only in embryonic and hematopoietic stem cells (ESC/HSC) but also in cancer stem cells.

Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease.

PubMed

Marlicz, Wojciech; Zuba-Surma, Ewa; Kucia, Magda; Blogowski, Wojciech; Starzynska, Teresa; Ratajczak, Mariusz Z

2012-09-01

Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue/organ injury. We sought to determine whether these cells are mobilized into PB in patients with Crohn's disease (CD). Twenty-five patients with active CD, 20 patients in clinical remission, and 25 age-matched controls were recruited and PB samples harvested. The circulating CD133+/Lin-/CD45+ and CD34+/Lin-/CD45+ cells enriched for HSPCs, CD105+/STRO-1+/CD45- cells enriched for MSCs, CD34+/KDR+/CD31+/CD45-cells enriched for EPCs, and small CXCR4+CD34+CD133+ subsets of Lin-CD45- cells that correspond to the population of VSELs were counted by fluorescence-activated cell sorting (FACS) and evaluated by direct immunofluorescence staining for pluripotency embryonic markers and by reverse-transcription polymerase chain reaction (RT-PCR) for expression of messenger (m)RNAs for a panel of genes expressed in intestine epithelial stem cells. The serum concentration of factors involved in stem cell trafficking, such as stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were measured by enzyme-linked immunosorbent assay (ELISA). Our data indicate that cells expressing markers for MSCs, EPCs, and small Oct-4+Nanog+SSEA-4+CXCR4+lin-CD45- VSELs are mobilized into PB in CD. The mobilized cells also expressed at the mRNA level genes playing a role in development and regeneration of gastrointestinal epithelium. All these changes were accompanied by increased serum concentrations of VEGF and HGF. CD triggers the mobilization of MSCs, EPCs, and VSELs, while the significance and precise role of these mobilized cells in repair of damaged intestine requires further study. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

PubMed Central

Sun, Ning; Lee, Andrew; Wu, Joseph C.

2013-01-01

Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

Private sector development of stem cell technology and therapeutic cloning.

PubMed

Lysaght, Michael J; Hazlehurst, Anne L

2003-06-01

Based on data collected in June 2002, more than 30 biotechnology startup firms in 11 countries are pursuing commercial development of stem cell technology and therapeutic cloning. These firms employ 950-1000 scientists and support staff and spend just under $200 million on research and development each year. The field has the look and feel of a high-tech cottage industry, with about half the startups employing fewer than 15 FTEs (full time equivalents). Funding is mostly from venture capitalists and private investors. Participants are geographically dispersed, with about 40% of the activity outside the United States. Focus is equally split between embryonic and adult stem cells. Taken as a whole, both the structure and scope of the private sector in stem cell research seem appropriate to the promise and development time frames of this important new technology.

Alpha-fetoprotein, stem cells and cancer: how study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer.

PubMed

Sell, Stewart

2008-01-01

Identification of the cells in the liver that produce alpha-fetoprotein during development, in response to liver injury and during the early stages of chemical hepatocarcinogenesis led to the conclusion that maturation arrest of liver-determined tissue stem cells was the cellular process that gives rise to hepatocellular carcinomas. When the cellular changes in these processes were compared to that of the formation of teratocarcinomas, the hypothesis arose that all cancers arise from maturation arrest of tissue-determined stem cells. This was essentially a reinterpretation of the embryonal rest theory of cancer whereby tissue stem cells take the role of embryonal rests. A corollary of the stem cell theory of the origin of cancer is that cancers contain the same functional cell populations as normal tissues: stem cells, transit-amplifying cells and mature cells. Cancer stem cells retain the essential feature of normal stem cells: the ability to self-renew. Growth of cancers is due to continued proliferation of cancer transit-amplifying cells that do not differentiate to mature cells (maturation arrest). On the other hand, cancer stem cells generally divide very rarely and contribute little to tumor growth. However, the presence of cancer stem cells in tumors is believed to be responsible for the properties of immortalization, transplantability and resistance to therapy characteristic of cancers. Current therapies for cancer (chemotherapy, radiotherapy, antiangiogenesis and differentiation therapy) are directed against the cancer transit-amplifying cells. When these therapies are discontinued, the cancer reforms from the cancer stem cells. Therapy directed toward interruption of the cell signaling pathways that maintain cancer stem cells could lead to new modalities to the prevention of regrowth of the cancer. Copyright 2008 S. Karger AG, Basel.

ALPHA-FETOPROTEIN (AFP), STEM CELLS, AND CANCER: HOW STUDY OF THE PRODUCTION OF AFP DURING CHEMICAL HEPATOCARCINOGENESIS LED TO REAFFIRMATION OF THE STEM CELL THEORY OF CANCER

PubMed Central

Sell, Stewart

2008-01-01

Identification of the cells in the liver that produce alpha-fetoprotein (AFP) during development, in response to liver injury, and during the early stages of chemical hepatocarcinogenesis led to the conclusion that maturation arrest of liver-determined tissue stem cells was the cellular process that gives rise to hepatocellular carcinomas (HCC). When the cellular changes in these processes were compared that of the formation of teratocarcinomas, the hypothesis arose that all cancers arise from maturation arrest of tissue determined stem cells. This was essentially a reinterpretation of the embryonal rest theory of cancer whereby tissue stem cells take the role of embryonal rests. A corollary of the stem cell theory of the origin of cancer is that cancers contain the same functional cell populations as do normal tissues: stem cells, transit-amplifying cells, and mature cells. Cancer stem cells retain the essential feature of normal stem cells: the ability to self-renew. Growth of cancers is due to continued proliferation of cancer transit-amplifying cells that do not differentiate to mature cells (maturation arrest). On the other hand, cancer stem cells generally divide very rarely and contribute little to tumor growth. However, the presence of cancer stem cells in tumors is believed to be responsible for the properties of immortalization, transplantability and resistance to therapy characteristic of cancers. Current therapies for cancer (chemotherapy, radiotherapy, anti-angiogenesis and differentiation therapy) are directed against the cancer transit amplifying cells. When these therapies are discontinued, the cancer re-forms from the cancer stem cells. Therapy directed toward interruption of the cell-signaling pathways that maintain cancer stem cells could lead to new modalities to the prevention of re-growth of the cancer. PMID:18612221

New perspectives in human stem cell therapeutic research.

PubMed

Trounson, Alan

2009-06-11

Human stem cells are in evaluation in clinical stem cell trials, primarily as autologous bone marrow studies, autologous and allogenic mesenchymal stem cell trials, and some allogenic neural stem cell transplantation projects. Safety and efficacy are being addressed for a number of disease state applications. There is considerable data supporting safety of bone marrow and mesenchymal stem cell transplants but the efficacy data are variable and of mixed benefit. Mechanisms of action of many of these cells are unknown and this raises the concern of unpredictable results in the future. Nevertheless there is considerable optimism that immune suppression and anti-inflammatory properties of mesenchymal stem cells will be of benefit for many conditions such as graft versus host disease, solid organ transplants and pulmonary fibrosis. Where bone marrow and mesenchymal stem cells are being studied for heart disease, stroke and other neurodegenerative disorders, again progress is mixed and mostly without significant benefit. However, correction of multiple sclerosis, at least in the short term is encouraging. Clinical trials on the use of embryonic stem cell derivatives for spinal injury and macular degeneration are beginning and a raft of other clinical trials can be expected soon, for example, the use of neural stem cells for killing inoperable glioma and embryonic stem cells for regenerating beta islet cells for diabetes. The change in attitude to embryonic stem cell research with the incoming Obama administration heralds a new co-operative environment for study and evaluation of stem cell therapies. The Californian stem cell initiative (California Institute for Regenerative Medicine) has engendered global collaboration for this new medicine that will now also be supported by the US Federal Government. The active participation of governments, academia, biotechnology, pharmaceutical companies, and private investment is a powerful consortium for advances in health.

Perspectives on stem cell therapy for cardiac regeneration. Advances and challenges.

PubMed

Choi, Sung Hyun; Jung, Seok Yun; Kwon, Sang-Mo; Baek, Sang Hong

2012-01-01

Ischemic heart disease (IHD) accelerates cardiomyocyte loss, but the developing stem cell research could be useful for regenerating a variety of tissue cells, including cardiomyocytes. Diverse sources of stem cells for IHD have been reported, including embryonic stem cells, induced pluripotent stem cells, skeletal myoblasts, bone marrow-derived stem cells, mesenchymal stem cells, and cardiac stem cells. However, stem cells have unique advantages and disadvantages for cardiac tissue regeneration, which are important considerations in determining the specific cells for improving cell survival and long-term engraftment after transplantation. Additionally, the dosage and administration method of stem cells need to be standardized to increase stability and efficacy for clinical applications. Accordingly, this review presents a summary of the stem cell therapies that have been studied for cardiac regeneration thus far, and discusses the direction of future cardiac regeneration research for stem cells.

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation.

PubMed

Barazzuol, Lara; Jeggo, Penny A

2016-08-01

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

PubMed

Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

2006-07-01

Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

The Role of H1 Linker Histone Subtypes in Preserving the Fidelity of Elaboration of Mesendodermal and Neuroectodermal Lineages during Embryonic Development

PubMed Central

Nguyen, Giang D.; Gokhan, Solen; Molero, Aldrin E.; Yang, Seung-Min; Kim, Byung-Ju; Skoultchi, Arthur I.; Mehler, Mark F.

2014-01-01

H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers. PMID:24802750

TRA-1–60+, SSEA-4+, POU5F1+, SOX2+, NANOG+ Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Testes

PubMed Central

Malecki, Marek; Tombokan, Xenia; Anderson, Mark; Malecki, Raf; Beauchaine, Michael

2013-01-01

Introduction Cancer of the testes is currently the most frequent neoplasm and a leading cause of morbidity in men 15–35 years of age. Its incidence is increasing. Embryonal carcinoma is its most malignant form, which either may be resistant or may develop resistance to therapies, which results in relapses. Cancer stem cells are hypothesized to be drivers of these phenomena. Specific aim The specific aim of this work was identification and isolation of spectra of single, living cancer stem cells, which were acquired directly from the patients’ biopsies, followed by testing of their pluripotency. Patients. Methods Biopsies were obtained from the patients with the clinical and histological diagnoses of the primary, pure embryonal carcinomas of the testes. The magnetic and fluorescent antibodies were genetically engineered. The SSEA-4 and TRA-1–60 cell surface display was analyzed by multiphoton fluorescence spectroscopy (MPFS), flow cytometry (FCM), immunoblotting (IB), nuclear magnetic resonance spectroscopy (NMRS), energy dispersive x-ray spectroscopy (EDXS), and total reflection x-ray spectroscopy (TRXFS). The single, living cells were isolated by magnetic or fluorescent sorting followed by their clonal expansion. The OCT4A, SOX2, and NANOG genes’ transcripts were analyzed by qRTPCR and the products by IB and MPFS. Results The clones of cells, with the strong surface display of TRA-1–60 and SSEA-4, were identified and isolated directly from the biopsies acquired from the patients diagnosed with the pure embryonal carcinomas of the testes. These cells demonstrated high levels of transcription and translation of the pluripotency genes: OCT4A, SOX2, and NANOG. They formed embryoid bodies, which differentiated into ectoderm, mesoderm, and endoderm. Conclusion In the pure embryonal carcinomas of the testes, acquired directly from the patients, we identified, isolated with high viability and selectivity, and profiled the clones of the pluripotent stem cells. These results may help in explaining therapy-resistance and relapses of these neoplasms, as well as, in designing targeted, personalized therapy. PMID:23772337

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vivo differentiation of induced pluripotent stem cells into neural stem cells by chimera formation.

PubMed

Choi, Hyun Woo; Hong, Yean Ju; Kim, Jong Soo; Song, Hyuk; Cho, Ssang Gu; Bae, Hojae; Kim, Changsung; Byun, Sung June; Do, Jeong Tae

2017-01-01

Like embryonic stem cells, induced pluripotent stem cells (iPSCs) can differentiate into all three germ layers in an in vitro system. Here, we developed a new technology for obtaining neural stem cells (NSCs) from iPSCs through chimera formation, in an in vivo environment. iPSCs contributed to the neural lineage in the chimera, which could be efficiently purified and directly cultured as NSCs in vitro. The iPSC-derived, in vivo-differentiated NSCs expressed NSC markers, and their gene-expression pattern more closely resembled that of fetal brain-derived NSCs than in vitro-differentiated NSCs. This system could be applied for differentiating pluripotent stem cells into specialized cell types whose differentiation protocols are not well established.

"Harvesting" and Use of Human (Embryonic) Stem Cells: An Islamic Evaluation.

PubMed

Bouzenita, Anke I

2017-03-01

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.

NFκB signaling regulates embryonic and adult neurogenesis

PubMed Central

ZHANG, Yonggang; HU, Wenhui

2013-01-01

Both embryonic and adult neurogenesis involves the self-renewal/proliferation, survival, migration and lineage differentiation of neural stem/progenitor cells. Such dynamic process is tightly regulated by intrinsic and extrinsic factors and complex signaling pathways. Misregulated neurogenesis contributes much to a large range of neurodevelopmental defects and neurodegenerative diseases. The signaling of NFκB regulates many genes important in inflammation, immunity, cell survival and neural plasticity. During neurogenesis, NFκB signaling mediates the effect of numerous niche factors such as cytokines, chemokines, growth factors, extracellular matrix molecules, but also crosstalks with other signaling pathways such as Notch, Shh, Wnt/β-catenin. This review summarizes current progress on the NFκB signaling in all aspects of neurogenesis, focusing on the novel role of NFκB signaling in initiating early neural differentiation of neural stem cells and embryonic stem cells. PMID:24324484

Overexpression of COUP-TF1 in murine embryonic stem cells reduces retinoic acid-associated growth arrest and increases extraembryonic endoderm gene expression.

PubMed

Zhuang, Yong; Gudas, Lorraine J

2008-09-01

Vitamin A (retinol [Rol]) and its metabolites are essential for embryonic development. The Rol metabolite all-trans retinoic acid (RA) is a biologically active form of Rol. The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription-factors (COUP-TF) proteins have been implicated in the regulation of several important biological processes, such as embryonic development and neuronal cell differentiation. Because there is evidence that COUP-TFs function in the retinoid signaling network during development and differentiation, we generated murine embryonic stem (ES) cell lines which stably and constitutively overexpress COUP-TF1 (NR2F1) and we analyzed RA-induced differentiation. COUP-TF1 overexpression resulted in reduced RA-associated growth arrest. A 2.4+/-0.17-fold higher Nanog mRNA level was seen in COUP-TF1 overexpressing lines, as compared with wild-type (WT) ES cells, after a 72 hr RA treatment. We also showed that COUP-TF1 overexpression enhanced RA-induced extraembryonic endoderm gene expression. Specifically, COUP-TF1 overexpression increased mRNA levels of GATA6 by 3.3+/-0.3-fold, GATA4 by 3.6+/-0.1-fold, laminin B1 (LAMB1) by 3.4+/-0.1-fold, LAMC1 by 3.4+/-0.2-fold, Dab2 by 2.4+0.1-fold, and SOX17 by 2.5-fold at 72 hr after RA treatment plus LIF, as compared with the increases seen in WT ES cells. However, RA-induced neurogenesis was unaffected by COUP-TF1 overexpression, as shown by the equivalent levels of expression of NeuroD1, nestin, GAP43 and other neuronal markers. Our results revealed for the first time that COUP-TF1 is an important signaling molecule during vitamin A (Rol)-mediated very early stage of embryonic development.

Normal embryonic and germ cell development in mice lacking alpha 1,3-fucosyltransferase IX (Fut9) which show disappearance of stage-specific embryonic antigen 1.

PubMed

Kudo, Takashi; Kaneko, Mika; Iwasaki, Hiroko; Togayachi, Akira; Nishihara, Shoko; Abe, Kuniya; Narimatsu, Hisashi

2004-05-01

Stage-specific embryonic antigen 1 (SSEA-1), an antigenic epitope defined as a Lewis x carbohydrate structure, is expressed during the 8-cell to blastocyst stages in mouse embryos and in primordial germ cells, undifferentiated embryonic stem cells, and embryonic carcinoma cells. For many years, SSEA-1 has been implicated in the development of mouse embryos as a functional carbohydrate epitope in cell-to-cell interaction during morula compaction. In a previous study, alpha 1,3-fucosyltransferase IX (Fut9) exhibited very strong activity for the synthesis of Lewis x compared to other alpha 1,3-fucosyltransferases in an in vitro substrate specificity assay. Fut4 and Fut9 transcripts were expressed in mouse embryos. The Fut9 transcript was detected in embryonic-day-13.5 gonads containing primordial germ cells, but the Fut4 transcript was not. In order to identify the role of SSEA-1 and determine the key enzyme for SSEA-1 synthesis in vivo, we have generated Fut9-deficient (Fut9(-/-)) mice. Fut9(-/-) mice develop normally, with no gross phenotypic abnormalities, and are fertile. Immunohistochemical analysis revealed an absence of SSEA-1 expression in early embryos and primordial germ cells of Fut9(-/-) mice. Therefore, we conclude that expression of the SSEA-1 epitope in the developing mouse embryo is not essential for embryogenesis in vivo.

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

NASA Astrophysics Data System (ADS)

Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

2013-04-01

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Steady advance of stem cell therapies: report from the 2011 World Stem Cell Summit, Pasadena, California, October 3-5.

PubMed

Swan, Melanie

2011-12-01

Stem cell research and related therapies (including regenerative medicine and cellular therapies) could have a significant near-term impact on worldwide public health and aging. One reason is the industry's strong linkage between policy, science, industry, and patient advocacy, as was clear in the attendance and programming at the 7(th) annual World Stem Cell Summit held in Pasadena, California, October 3-5, 2011. A special conference session sponsored by the SENS Foundation discussed how stem cell therapies are being used to extend healthy life span. Stem cells are useful not only in cell-replacement therapies, but also in disease modeling, drug discovery, and drug toxicity screening. Stem cell therapies are currently being applied to over 50 diseases, including heart, lung, neurodegenerative, and eye disease, cancer, and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Dozens of companies are developing therapeutic solutions that are in different stages of clinical use and clinical trials. Some high-profile therapies include Dendreon's Provenge for prostate cancer, Geron's first-ever embryonic stem cell trials for spinal cord injury, Fibrocell's laViv cellular therapy for wrinkles, and well-established commercial skin substitutes (Organogenesis' Apligraf and Advanced BioHealing's Dermagraft). Stem cell policy issues under consideration include medical tourism, standards for large-scale stem cell manufacturing, and lingering ethical debates over the use of embryonic stem cells. Contemporary stem cell science advances include a focus on techniques for the direct reprogramming of cells from one lineage to another without returning to pluripotency as an intermediary step, improved means of generating and characterizing induced pluripotent cells, and progress in approaches to neurodegenerative disease.

Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

PubMed

Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

2015-05-15

Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead.

PubMed

Bhartiya, Deepa; Shaikh, Ambreen; Anand, Sandhya; Patel, Hiren; Kapoor, Sona; Sriraman, Kalpana; Parte, Seema; Unni, Sreepoorna

2016-12-01

Both pluripotent very small embryonic-like stem cells (VSELs) and induced pluripotent stem (iPS) cells were reported in 2006. In 2012, a Nobel Prize was awarded for iPS technology whereas even today the very existence of VSELs is not well accepted. The underlying reason is that VSELs exist in low numbers, remain dormant under homeostatic conditions, are very small in size and do not pellet down at 250-280g. The VSELs maintain life-long tissue homeostasis, serve as a backup pool for adult stem cells and are mobilized under stress conditions. An imbalance in VSELs function (uncontrolled proliferation) may result in cancer. The electronic database 'Medline/Pubmed' was systematically searched with the subject heading term 'very small embryonic-like stem cells'. The most primitive stem cells that undergo asymmetric cell divisions to self-renew and give rise to progenitors still remain elusive in the hematopoietic system and testes, while the presence of stem cells in ovary is still being debated. We propose to review the available literature on VSELs, the methods of their isolation and characterization, their ontogeny, how they compare with embryonic stem (ES) cells, primordial germ cells (PGCs) and iPS cells, and their role in maintaining tissue homeostasis. The review includes a look ahead on how VSELs will result in paradigm shifts in basic reproductive biology. Adult tissue-specific stem cells including hematopoietic, spermatogonial, ovarian and mesenchymal stem cells have good proliferation potential and are indeed committed progenitors (with cytoplasmic OCT-4), which arise by asymmetric cell divisions of pluripotent VSELs (with nuclear OCT-4). VSELs are the most primitive stem cells and postulated to be an overlapping population with the PGCs. Rather than migrating only to the gonads, PGCs migrate and survive in various adult body organs throughout life as VSELs. VSELs express both pluripotent and PGC-specific markers and are epigenetically and developmentally more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Use of pluripotent stem cells for reproductive medicine: are we there yet?

PubMed

Duggal, Galbha; Heindryckx, Björn; Deroo, Tom; De Sutter, Petra

2014-01-01

In recent years, pluripotent stem cells have demonstrated to be exciting tools to understand embryonic development, cell lineage specification, tissue generation and repair, and various other biological processes. In addition, the identification and isolation of germ line stem cells has given more insight into germ cell biology at the molecular level and into the underlying causes of infertility which was not possible earlier. The recent derivation of in vitro derived sperm and oocytes from pluripotent stem cells in the mouse model represents a major breakthrough in the field and substantiates the critical relevance of stem cells as a potential alternative resource for treating infertility. Although the past years have yielded compelling information in understanding germ cell development via in vitro stem cell assays, extended investigative research is necessary in order to derive fully functional 'artificial gametes' in a safe way for future therapeutic applications.

Regulatory RNA Key Player in p53-Mediated Apoptosis in Embryonic Stem Cells | Center for Cancer Research

Cancer.gov

Embryonic stem cells (ESCs) must maintain the integrity of their genomes or risk passing potentially deleterious mutations on to numerous tissues. Thus, ESCs have a unique genome surveillance system and easily undergo apoptosis or differentiation when DNA damage is detected. The protein p53 is known to promote differentiation in mouse ESCs (mESCs), but its role in DNA

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.

PubMed

Souquet, Benoit; Freed, Ellen; Berto, Alessandro; Andric, Vedrana; Audugé, Nicolas; Reina-San-Martin, Bernardo; Lacy, Elizabeth; Doye, Valérie

2018-05-22

Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

PubMed

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

PubMed Central

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-01-01

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors. DOI: http://dx.doi.org/10.7554/eLife.26287.001 PMID:28394252

Chick stem cells: Current progress and future prospects

PubMed Central

Intarapat, Sittipon; Stern, Claudio D.

2013-01-01

Chick embryonic stem cells (cESCs) can be derived from cells obtained from stage X embryos (blastoderm stage); these have the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of stem cells that are able to contribute to the germ line can be established from chick primordial germ cells (cPGCs) and embryonic germ cells (cEGCs). This review provides information on avian stem cells, emphasizing different sources of cells and current methods for derivation and culture of pluripotent cells from chick embryos. We also review technologies for isolation and derivation of chicken germ cells and the production of transgenic birds. PMID:24103496

Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

USDA-ARS?s Scientific Manuscript database

Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

3 CFR - Guidelines for Human Stem Cell Research

Code of Federal Regulations, 2010 CFR

2010-01-01

... 3 The President 1 2010-01-01 2010-01-01 false Guidelines for Human Stem Cell Research Presidential Documents Other Presidential Documents Memorandum of July 30, 2009 Guidelines for Human Stem Cell Research..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent...

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

PubMed

Acharya, Aviseka; Brungs, Sonja; Henry, Margit; Rotshteyn, Tamara; Singh Yaduvanshi, Nirmala; Wegener, Lucia; Jentzsch, Simon; Hescheler, Jürgen; Hemmersbach, Ruth; Boeuf, Helene; Sachinidis, Agapios

2018-06-15

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-β signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

Stem cell function during plant vascular development

PubMed Central

Miyashima, Shunsuke; Sebastian, Jose; Lee, Ji-Young; Helariutta, Yka

2013-01-01

The plant vascular system, composed of xylem and phloem, evolved to connect plant organs and transport various molecules between them. During the post-embryonic growth, these conductive tissues constitutively form from cells that are derived from a lateral meristem, commonly called procambium and cambium. Procambium/cambium contains pluripotent stem cells and provides a microenvironment that maintains the stem cell population. Because vascular plants continue to form new tissues and organs throughout their life cycle, the formation and maintenance of stem cells are crucial for plant growth and development. In this decade, there has been considerable progress in understanding the molecular control of the organization and maintenance of stem cells in vascular plants. Noticeable advance has been made in elucidating the role of transcription factors and major plant hormones in stem cell maintenance and vascular tissue differentiation. These studies suggest the shared regulatory mechanisms among various types of plant stem cell pools. In this review, we focus on two aspects of stem cell function in the vascular cambium, cell proliferation and cell differentiation. PMID:23169537

Stem Cells and Society: An Undergraduate Course Exploring the Intersections among Science, Religion, and Law

PubMed Central

Friedrichsen, Patricia

2009-01-01

The intersection of science and our society has led to legal and ethical issues in which we all play a part. To support development of scientific literacy, college science courses need to engage students in difficult dialogues around ethical issues. We describe a new course, Stem Cells and Society, in which students explore the basic biology of stem cell research and the controversy surrounding it. As part of the course, we highlight the nature of science, looking at the methods and norms within the scientific community. To gain a perspective on the current stem cell controversy, we examine the public debates in the 1970s surrounding in vitro fertilization, the stem cell initiative in Missouri, and the personal and religious viewpoints that have emerged relative to the stem cell debate. In the Stem Cells and Society course, students are challenged to develop and clarify their own personal positions concerning embryonic stem cell research. These positions are grounded in science, religion or personal philosophy, and law. PMID:19255139

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis.

PubMed

Saito, Kanako; Dubreuil, Veronique; Arai, Yoko; Wilsch-Bräuninger, Michaela; Schwudke, Dominik; Saher, Gesine; Miyata, Takaki; Breier, Georg; Thiele, Christoph; Shevchenko, Andrej; Nave, Klaus-Armin; Huttner, Wieland B

2009-05-19

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5-E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1-independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis.

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis

PubMed Central

Saito, Kanako; Dubreuil, Veronique; Arai, Yoko; Wilsch-Bräuninger, Michaela; Schwudke, Dominik; Saher, Gesine; Miyata, Takaki; Breier, Georg; Thiele, Christoph; Shevchenko, Andrej; Nave, Klaus-Armin; Huttner, Wieland B.

2009-01-01

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5–E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1–independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis. PMID:19416849

Concise Review: Kidney Generation with Human Pluripotent Stem Cells.

PubMed

Morizane, Ryuji; Miyoshi, Tomoya; Bonventre, Joseph V

2017-11-01

Chronic kidney disease (CKD) is a worldwide health care problem, resulting in increased cardiovascular mortality and often leading to end-stage kidney disease, where patients require kidney replacement therapies such as hemodialysis or kidney transplantation. Loss of functional nephrons contributes to the progression of CKD, which can be attenuated but not reversed due to inability to generate new nephrons in human adult kidneys. Human pluripotent stem cells (hPSCs), by virtue of their unlimited self-renewal and ability to differentiate into cells of all three embryonic germ layers, are attractive sources for kidney regenerative therapies. Recent advances in stem cell biology have identified key signals necessary to maintain stemness of human nephron progenitor cells (NPCs) in vitro, and led to establishment of protocols to generate NPCs and nephron epithelial cells from human fetal kidneys and hPSCs. Effective production of large amounts of human NPCs and kidney organoids will facilitate elucidation of developmental and pathobiological pathways, kidney disease modeling and drug screening as well as kidney regenerative therapies. We summarize the recent studies to induce NPCs and kidney cells from hPSCs, studies of NPC expansion from mouse and human embryonic kidneys, and discuss possible approaches in vivo to regenerate kidneys with cell therapies and the development of bioengineered kidneys. Stem Cells 2017;35:2209-2217. © 2017 AlphaMed Press.

Concise Review: One Stone for Multiple Birds: Generating Universally Compatible Human Embryonic Stem Cells.

PubMed

Zheng, Dejin; Wang, Xiaofang; Xu, Ren-He

2016-09-01

With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including β-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016;34:2269-2275. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Induction of pluripotent stem cells from fibroblast cultures.

PubMed

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Isolation and characterisation of cancer stem cells from canine osteosarcoma.

PubMed

Wilson, H; Huelsmeyer, M; Chun, R; Young, K M; Friedrichs, K; Argyle, D J

2008-01-01

There is increasing evidence that cancer is a stem cell disease. This study sought to isolate and characterise cancer stem cells from canine osteosarcoma. One human and three canine cell lines were cultured in non-adherent culture conditions using serum-starved, semi-solid media. Primitive sarcosphere colonies from all cell lines were identified under these conditions and were characterised using molecular and cytochemical techniques for embryonic stem cell markers. Expression of the embryonic stem cell-associated genes Nanog, Oct4 and STAT3 indicated a primitive phenotype. Sarcospheres could be reproduced consistently when passaged multiple times and produced adherent cell cultures when returned to normal growth conditions. Similarities between human and canine osteosarcoma cell lines add credence to the potential of the dog as a model for human disease.

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay: Book Chapter

EPA Science Inventory

There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adher...

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay-Book Chapter*

EPA Science Inventory

There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adhere...

Embryonic stem cells and the next generation of developmental toxicity testing.

PubMed

Kugler, Josephine; Huhse, Bettina; Tralau, Tewes; Luch, Andreas

2017-08-01

The advent of stem cell technology has seen the establishment of embryonic stem cells (ESCs) as molecular model systems and screening tools. Although ESCs are nowadays widely used in research, regulatory implementation for developmental toxicity testing is pending. Areas Covered: This review evaluates the performance of current ESC, including human (h)ESC testing systems, trying to elucidate their potential for developmental toxicity testing. It shall discuss defining parameters and mechanisms, their relevance and contemplate what can realistically be expected. Crucially this includes the question of how to ascertain the quality of currently employed cell lines and tests based thereon. Finally, the use of hESCs will raise ethical concerns which should be addressed early on. Expert Opinion: While the suitability of (h)ESCs as tools for research and development goes undisputed, any routine use for developmental toxicity testing currently still seems premature. The reasons for this comprise inherent biological deficiencies as well as cell line quality and system validation. Overcoming these issues will require collaboration of scientists, test developers and regulators. Also, validation needs to be made worthwhile for academia. Finally we have to continuously rethink existing strategies, making room for improved testing and innovative approaches.

Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.

PubMed

Lacham-Kaplan, Orly; Chy, Hun; Trounson, Alan

2006-02-01

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Patient-specific embryonic stem cells derived from human SCNT blastocysts.

PubMed

Hwang, Woo Suk; Roh, Sung Il; Lee, Byeong Chun; Kang, Sung Keun; Kwon, Dae Kee; Kim, Sue; Kim, Sun Jong; Park, Sun Woo; Kwon, Hee Sun; Lee, Chang Kyu; Lee, Jung Bok; Kim, Jin Mee; Ahn, Curie; Paek, Sun Ha; Chang, Sang Sik; Koo, Jung Jin; Yoon, Hyun Soo; Hwang, Jung Hye; Hwang, Youn Young; Park, Ye Soo; Oh, Sun Kyung; Kim, Hee Sun; Park, Jong Hyuk; Moon, Shin Yong; Schatten, Gerald

2005-06-17

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.

A role for thrombopoietin in hemangioblast development.

PubMed

Perlingeiro, Rita C R; Kyba, Michael; Bodie, Susan; Daley, George Q

2003-01-01

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast, an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor, c-Mpl, regulate primitive hematopoietic populations, including bone marrow hematopoietic stem cells, we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC), TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions, TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies, as well as endothelial cells, confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation, suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation.

Adult Human Nasal Mesenchymal-Like Stem Cells Restore Cochlear Spiral Ganglion Neurons After Experimental Lesion

PubMed Central

Bas, Esperanza; Van De Water, Thomas R.; Lumbreras, Vicente; Rajguru, Suhrud; Goss, Garrett; Hare, Joshua M.

2014-01-01

A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes deafness, affecting millions of people. Currently, there is no effective therapy to repair the inner ear sensory structures in humans. Cochlear implantation can restore input, but only if auditory neurons remain intact. Efforts to develop stem cell-based treatments for deafness have demonstrated progress, most notably utilizing embryonic-derived cells. In an effort to bypass limitations of embryonic or induced pluripotent stem cells that may impede the translation to clinical applications, we sought to utilize an alternative cell source. Here, we show that adult human mesenchymal-like stem cells (MSCs) obtained from nasal tissue can repair spiral ganglion loss in experimentally lesioned cochlear cultures from neonatal rats. Stem cells engraft into gentamicin-lesioned organotypic cultures and orchestrate the restoration of the spiral ganglion neuronal population, involving both direct neuronal differentiation and secondary effects on endogenous cells. As a physiologic assay, nasal MSC-derived cells engrafted into lesioned spiral ganglia demonstrate responses to infrared laser stimulus that are consistent with those typical of excitable cells. The addition of a pharmacologic activator of the canonical Wnt/β-catenin pathway concurrent with stem cell treatment promoted robust neuronal differentiation. The availability of an effective adult autologous cell source for inner ear tissue repair should contribute to efforts to translate cell-based strategies to the clinic. PMID:24172073

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

PubMed

Stadlmann, Johannes; Taubenschmid, Jasmin; Wenzel, Daniel; Gattinger, Anna; Dürnberger, Gerhard; Dusberger, Frederico; Elling, Ulrich; Mach, Lukas; Mechtler, Karl; Penninger, Josef M

2017-09-28

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Translating stem cell therapies: the role of companion animals in regenerative medicine

PubMed Central

Volk, Susan W.; Theoret, Christine

2013-01-01

Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell-based therapies involving hematopoietic (HSC) and mesenchymal stem cells (MSC) as well as other adult regenerative cells and recently embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the “One Health Initiative”, the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored. PMID:23627495

Data on the potential impact of food supplements on the growth of mouse embryonic stem cells.

PubMed

Correia, Marcelo; Sousa, Maria I; Rodrigues, Ana S; Perestrelo, Tânia; Pereira, Sandro L; Ribeiro, Marcelo F; Ramalho-Santos, João

2016-06-01

The use of new compounds as dietary supplements is increasing, but little is known in terms of possible consequences of their use. Pluripotent stem cells are a promising research tool for citotoxicological research for evaluation of proliferation, cell death, pluripotency and differentiation. Using the mouse embryonic stem cell (mESC) model, we present data on three different compounds that have been proposed as new potential supplements for co-adjuvant disease treatments: kaempferol, berberine and Tauroursodeoxycholic acid (TUDCA). Cell number and viability were monitored following treatment with increased concentrations of each drug in pluripotent culture conditions.

PTBP1 Is Required for Embryonic Development before Gastrulation

PubMed Central

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A. Francis; Solimena, Michele

2011-01-01

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures. PMID:21423341

PTBP1 is required for embryonic development before gastrulation.

PubMed

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

PKCλ/ι signaling-a common node for normal cellular development and breast oncogenesis.

PubMed

Paul, Arindam; Paul, Soumen

2015-01-01

We recently demonstrated that PKCλ/ι signaling is an important contributor to breast cancer development. Strikingly, PKCλ/ι signaling is also important to balance self-renewal versus differentiation in pluripotent stem cells and is essential for embryonic development. This commentary highlights some key functions of PKCλ/ι signaling that are integral to both normal development and cancer progression.

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

NASA Astrophysics Data System (ADS)

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

Effect of the timing of the first cleavage on the developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells.

PubMed

Kobayashi, T; Kato, Y; Tsunoda, Y

2004-09-01

The present study examined whether the timing of the first cleavage has an effect on the in vitro and in vivo developmental potential of nuclear-transferred mouse oocytes receiving embryonic stem cells. First, the timing of the first cleavage and the developmental potential of nuclear-transferred oocytes were examined every hour from 12 to 24 h after the start of culture and compared with in vitro-fertilized oocytes. The developmental potential of in vitro-fertilized oocytes decreased gradually according to the time required for cleavage (84% (32/38) for 15 h to 50% (1/2) for 20 h), but intermediate-cleaved (15-16 h) nuclear-transferred oocytes had a higher potential to develop into blastocysts (55% (17/31) to 67% (45/67) versus 0-43% (6/14)]. Second the nuclear-transferred oocytes were divided into three groups according to the timing of the first cleavage; each group was cultured to blastocysts in vitro, and then transferred to recipients. The potential of intermediate-cleaved oocytes (15-16 h) to develop into blastocysts was significantly higher than fast-cleaved (before 15 h) and slow-cleaved (after 16 h) oocytes (65, 46, and 37%). The proportion of fetuses on Day 10.5 of pregnancy was highest in the intermediate-cleaved group (4 versus 2 and 1%, respectively) and a full-term fetus was obtained from this group. The present study demonstrated that the timing of the first cleavage could be used to determine the potential of nuclear-transferred oocytes with embryonic stem cells to develop to the blastocyst stage in vitro, but not to determine post-implantation viability after transfer to recipients.

Quantitative proteome analysis of pluripotent cells by iTRAQ mass tagging reveals post-transcriptional regulation of proteins required for ES cell self-renewal.

PubMed

O'Brien, Robert N; Shen, Zhouxin; Tachikawa, Kiyoshi; Lee, Pei Angel; Briggs, Steven P

2010-10-01

Embryonic stem cells and embryonal carcinoma cells share two key characteristics: pluripotency (the ability to differentiate into endoderm, ectoderm, and mesoderm) and self-renewal (the ability to grow without change in an untransformed, euploid state). Much has been done to identify and characterize transcription factors that are necessary or sufficient to maintain these characteristics. Oct-4 and Nanog are necessary to maintain pluripotency; they are down-regulated at the mRNA level by differentiation. There may be additional regulatory genes whose mRNA levels are unchanged but whose proteins are destabilized during differentiation. We generated proteome-wide, quantitative profiles of ES and embryonal carcinoma cells during differentiation, replicating a microarray-based study by Aiba et al. (Aiba, K., Sharov, A. A., Carter, M. G., Foroni, C., Vescovi, A. L., and Ko, M. S. (2006) Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells. Stem Cells 24, 889-895) who triggered differentiation by treatment with 1 μM all-trans-retinoic acid. We identified several proteins whose levels decreased during differentiation in both cell types but whose mRNA levels were unchanged. We confirmed several of these cases by RT-PCR and Western blot. Racgap1 (also known as mgcRacgap) was particularly interesting because it is required for viability of preimplantation embryos and hematopoietic stem cells, and it is also required for differentiation. To confirm our observation that RACGAP-1 declines during retinoic acid-mediated differentiation, we used multiple reaction monitoring, a targeted mass spectrometry-based quantitation method, and determined that RACGAP-1 levels decline by half during retinoic acid-mediated differentiation. We knocked down Racgap-1 mRNA levels using a panel of five shRNAs. This resulted in a loss of self-renewal that correlated with the level of knockdown. We conclude that RACGAP-1 is post-transcriptionally regulated during blastocyst development to enable differentiation by inhibiting ES cell self-renewal.

Enhancement of cardiomyogenesis in stem cells by low intensity pulsed ultrasound

NASA Astrophysics Data System (ADS)

Teo, Ailing; Morshedi, Amir; Wang, Jen-Chieh; Lim, Mayasari; Zhou, Yufeng

2017-03-01

Low intensity pulsed ultrasound (LIPUS) has been shown to enhance bone and cartilage regeneration from stem cells. Gene expression of angiotensin II type 1 (AT1) receptor can be increased in LIPUS-treated osteoblasts. The AT1 receptor is a known mechanoreceptor in cardiomyocytes. It suggests that LIPUS may enhance cardiomyogenesis via mechanotransduction by increasing AT1 expression. Murine embryonic stem cells (ESCs) were treated daily by 10-min 1MHz LIPUS at spatial-average temporal-peak acoustic intensities of 30 mW/cm2 and 300 mW/cm2 in both continuous and pulsed wave (20% duty cycle) for 10 days. Polymerase chain reaction (PCR), immunocytochemistry, and beating rate were used to evaluate the cardiac viability quantitatively. After the treatment of LIPUS, beating rate of contractile areas and cardiac gene expression, such as α- and β-myosin heavy chain, were improved. Furthermore, no deleterious effects to the development of cardiac proteins were observed. All results suggest that LIPUS stimulation has the capacity of enhancing cardiomyogenesis from embryonic stem cells. With the benefit and the ease in incorporating LIPUS into various culture platforms, LIPUS has the potential to produce cardiomyocytes for clinical use in the future.

4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila

NASA Astrophysics Data System (ADS)

Yadav, Jhillu S.; Lavanya, Madugula P.; Das, Pragna P.; Bag, Indira; Krishnan, Anita; Jagannadh, Bulusu; Mohapatra, Debendra K.; Pal Bhadra, Manika; Bhadra, Utpal

2010-04-01

p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

PubMed Central

Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P.B.; Panicker, Mitradas M.

2014-01-01

Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies. PMID:25068130

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Saitou, Mitinori

2013-08-01

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Waigel, Sabine J; Enkhmandakh, Badam; Bayarsaihan, Dashzeveg

2012-04-01

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation. © 2011 Wiley Periodicals, Inc.

Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

PubMed

Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

2017-03-01

Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34 + CD43 + hematopoietic progenitor cells (HPCs) and CD34 + CD45 + HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro . In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

Stem cells, embryos, and the environment: a context for both science and ethics.

PubMed

Towns, C R; Jones, D G

2004-08-01

Debate on the potential and uses of human stem cells tends to be conducted by two constituencies-ethicists and scientists. On many occasions there is little communication between the two, with the result that ethical debate is not informed as well as it might be by scientific insights. The aim of this paper is to highlight those scientific insights that may be of relevance for ethical debate. Environmental factors play a significant role in identifying stem cells and their various subtypes. Research related to the role of the microenvironment has led to emphasis upon "plasticity", which denotes the ability of one type of stem cell to undergo a transition to cells from other lineages. This could increase the value given to adult stem cells, in comparison with embryonic stem cell research. Any such conclusion should be treated with caution, however, since optimism of this order is not borne out by current research. The role of the environment is also important in distinguishing between the terms totipotency and pluripotency. We argue that blastocysts (early embryos) and embryonic stem cells are only totipotent if they can develop within an appropriate environment. In the absence of this, they are merely pluripotent. Hence, blastocysts in the laboratory are potentially totipotent, in contrast to their counterparts within the human body which are actually totipotent. This may have implications for ethical debate, suggesting as it does that arguments based on potential for life may be of limited relevance.

Arid3a is essential to execution of the first cell fate decision via direct embryonic and extraembryonic transcriptional regulation

PubMed Central

Rhee, Catherine; Lee, Bum-Kyu; Beck, Samuel; Anjum, Azeen; Cook, Kendra R.; Popowski, Melissa

2014-01-01

Despite their origin from the inner cell mass, embryonic stem (ES) cells undergo differentiation to the trophectoderm (TE) lineage by repression of the ES cell master regulator Oct4 or activation of the TE master regulator Caudal-type homeobox 2 (Cdx2). In contrast to the in-depth studies of ES cell self-renewal and pluripotency, few TE-specific regulators have been identified, thereby limiting our understanding of mechanisms underlying the first cell fate decision. Here we show that up-regulation and nuclear entry of AT-rich interactive domain 3a (Arid3a) drives TE-like transcriptional programs in ES cells, maintains trophoblast stem (TS) cell self-renewal, and promotes further trophoblastic differentiation both upstream and independent of Cdx2. Accordingly, Arid3a−/− mouse post-implantation placental development is severely impaired, resulting in early embryonic death. We provide evidence that Arid3a directly activates TE-specific and trophoblast lineage-specific genes while directly repressing pluripotency genes via differential regulation of epigenetic acetylation or deacetylation. Our results identify Arid3a as a critical regulator of TE and placental development through execution of the commitment and differentiation phases of the first cell fate decision. PMID:25319825

Contribution of Caudal Müllerian Duct Mesenchyme to Prostate Development

PubMed Central

Brechka, Hannah; McAuley, Erin M.; Lamperis, Sophia M.; Paner, Gladell P.

2016-01-01

A fundamental understanding of prostate development and tissue homeostasis has the high potential to reveal mechanisms for prostate disease initiation and identify novel therapeutic approaches for disease prevention and treatment. Our current understanding of prostate lineage specification stems from the use of developmental model systems that rely upon the embryonic preprostatic urogenital sinus mesenchyme to induce the formation of mature prostate epithelial cells. It is unclear, however, how the urogenital sinus epithelium can derive both adult urethral glands and prostate epithelia. Furthermore, the vast disparity in disease initiation between these two glands highlights key developmental factors that predispose prostate epithelia to hyperplasia and cancer. In this study we demonstrate that the caudal Müllerian duct mesenchyme (CMDM) drives prostate epithelial differentiation and is a key determinant in cell lineage specification between urethral glands and prostate epithelia. Utilizing both human embryonic stem cells and mouse embryonic tissues, we document that the CMDM is capable of inducing the specification of androgen receptor, prostate-specific antigen, NKX3.1, and Hoxb13-positive prostate epithelial cells. These results help to explain key developmental differences between prostate and urethral gland differentiation, and implicate factors secreted by the caudal Müllerian duct as novel targets for prostate disease prevention and treatment. PMID:27595922

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

PubMed

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Bustin, Michael

2017-04-07

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation

PubMed Central

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M.; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe

2017-01-01

Abstract An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. PMID:27923998