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Sample records for murine embryonic stem

  1. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  2. Permissive and restricted virus infection of murine embryonic stem cells.

    PubMed

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey; Kellam, Paul

    2012-10-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  3. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  4. Magnetic field-magnetic nanoparticle culture system used to grow in vitro murine embryonic stem cells.

    PubMed

    de Freitas, Erika Regina Leal; Soares, Paula Roberta Otaviano; de Santos, Rachel Paula; dos Santos, Regiane Lopes; Porfírio, Elaine Paulucio; Báo, Sônia N; Lima, Emília Celma Oliveira; Guillo, Lídia Andreu

    2011-01-01

    The in vitro growth of embryonic stem cells (ESCs) is usually obtained in the presence of murine embryonic fibroblasts (MEF), but new methods for in vitro expansion of ESCs should be developed due to their potential clinical use. This study aims to establish a culture system to expand and maintain ESCs in the absence of MEF by using murine embryonic stem cells (mECS) as a model of embryonic stem cell. Magnetic nanoparticles (MNPs) were used for growing mESCs in the presence of an external magnetic field, creating the magnetic field-magnetic nanoparticle (MF-MNP) culture system. The growth characteristics were evaluated showing a doubling time slightly higher for mESCs cultivated in the presence of the system than in the presence of the MEF. The undifferentiated state was characterized by RT-PCR, immunofluorescence, alkaline phosphatase activity and electron microscopy. Murine embryonic stem cells cultivated in presence of the MF-MNP culture system exhibited Oct-4 and Nanog expression and high alkaline phosphatase activity. Ultrastructural morphology showed that the MF-MNP culture system did not interfere with processes that cause structural changes in the cytoplasm or nucleus. The MF-MNP culture system provides a tool for in vitro expansion of mESCs and could contribute to studies that aim the therapeutic use of embryonic stem cells. PMID:21446404

  5. Characterization of Dicer-deficient murine embryonic stem cells.

    PubMed

    Murchison, Elizabeth P; Partridge, Janet F; Tam, Oliver H; Cheloufi, Sihem; Hannon, Gregory J

    2005-08-23

    Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells. PMID:16099834

  6. Characterization of Dicer-deficient murine embryonic stem cells

    PubMed Central

    Murchison, Elizabeth P.; Partridge, Janet F.; Tam, Oliver H.; Cheloufi, Sihem; Hannon, Gregory J.

    2005-01-01

    Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells. PMID:16099834

  7. Compactin enhances osteogenesis in murine embryonic stem cells.

    PubMed

    Phillips, B W; Belmonte, N; Vernochet, C; Ailhaud, G; Dani, C

    2001-06-01

    Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and beta-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation. PMID:11394905

  8. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  9. DAZL regulates Tet1 translation in murine embryonic stem cells

    PubMed Central

    Welling, Maaike; Chen, Hsu-Hsin; Muñoz, Javier; Musheev, Michael U; Kester, Lennart; Junker, Jan Philipp; Mischerikow, Nikolai; Arbab, Mandana; Kuijk, Ewart; Silberstein, Lev; Kharchenko, Peter V; Geens, Mieke; Niehrs, Christof; van de Velde, Hilde; van Oudenaarden, Alexander; Heck, Albert JR; Geijsen, Niels

    2015-01-01

    Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state. PMID:26077710

  10. Regulation of Glycan Structures in Murine Embryonic Stem Cells

    PubMed Central

    Nairn, Alison V.; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J. Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W.

    2012-01-01

    The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development. PMID:22988249

  11. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  12. Sorting and Expansion of Murine Embryonic Stem Cell Colonies using Micropallet Arrays

    PubMed Central

    Shadpour, Hamed; Sims, Christopher E.; Thresher, Randy J.; Allbritton, Nancy L.

    2008-01-01

    Background Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Methods Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. Results The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Conclusions Qualitative and quantitative analyses of sorting, collection efficiency and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. PMID:19012319

  13. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model

    PubMed Central

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E.

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology. PMID:24723848

  14. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model.

    PubMed

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology. PMID:24723848

  15. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  16. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells.

    PubMed

    Haase, Ingo; Knaup, Renate; Wartenberg, Maria; Sauer, Heinrich; Hescheler, Jürgen; Mahrle, Gustav

    2007-12-01

    Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments. PMID:17716780

  17. Induction of murine embryonic stem cell differentiation by medicinal plant extracts.

    PubMed

    Reynertson, Kurt A; Charlson, Mary E; Gudas, Lorraine J

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. PMID:20955699

  18. Induction of murine embryonic stem cell differentiation by medicinal plant extracts

    PubMed Central

    Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.

    2010-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly-cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from twelve species of ethnomedically utilized plants, we found fractions from three species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. PMID:20955699

  19. Induction of murine embryonic stem cell differentiation by medicinal plant extracts

    SciTech Connect

    Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.

  20. 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...

  1. Differentiation of murine embryonic stem cells in skeletal muscles of mice.

    PubMed

    Tian, Chai; Lu, Yifan; Gilbert, Rénald; Karpati, George

    2008-01-01

    Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such cultures, very rare contracting myotubes were still present. Suspensions of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles (ATM) of different cohorts of mice for the study of their survival and possible myogenic differentiation. The different cohorts of mice included isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections, the injected ATM of nonisogenic mice did not contain cells positive for LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact mdx mice contained very rare examples of muscle fibers positive for dystrophin and/or embryonic myosin heavy chain. In the ATM of the isogenic normal and the immunosuppressed mdx mice, as expected, large teratomas developed containing the usual diverse cell types. In some teratomas of immunosuppressed mdx mice, small pockets of muscle fibers expressed dystrophin and myosin heavy chain. Our studies indicated that in muscles of animals nonisogenic with the used ESCs, only very rare ESCs survived with myogenic differentiation. These studies also indicated that ESCs will not undergo significant, selective, and preferential myogenic differentiation in vitro or in vivo in any of the models studied. It is probable that this strain of murine ESC requires some experimentally induced alteration of its gene expression profile to secure significant myogenicity and suppress tumorogenicity. PMID:18522235

  2. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

    PubMed Central

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Wang, Xiaozhong

    2015-01-01

    The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation. PMID:25992972

  3. Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

    PubMed Central

    Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

    2016-01-01

    TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

  4. Leptin enhances endothelial cell differentiation and angiogenesis in murine embryonic stem cells.

    PubMed

    Kurtovic, Silvia; Ng, Tina T; Gupta, Ankur; Arumugaswami, Vaithilingaraja; Chaiboonma, Kira L; Aminzadeh, Mohammad Amin; Makkar, Raj; Dafoe, Donald C; Talavera-Adame, Dodanim

    2015-01-01

    The metabolic regulation of leptin and its angiogenic effects have been well characterized in adult mammals. However, the role of leptin in the differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs) has not been characterized. We hypothesized that leptin enhances the generation of ECs derived from ESCs and, in this way, promotes angiogenesis in embryonic vessels. To address this hypothesis, we utilized an in vitro model consisting of murine ESCs-derived embryoid bodies (EBs). Vascular density, EC and angiogenesis markers as well as phosphorylation levels of signal transducer and activator of transcription 3 (pSTAT3) were investigated in leptin-treated EBs and in untreated EBs as controls. ESC-derived ECs were isolated by magnetic sorting based on the expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31). Significant upregulation of EC and angiogenic markers as well as higher vessel density were found in leptin-treated EBs compared to controls. CD31 positive enriched cells derived from leptin-treated EBs had improved proliferation and survival rate and showed higher levels of pSTAT3. These results suggested that leptin promotes EC differentiation and angiogenesis in mouse EBs and that janus tyrosine kinase (JAK)/STAT pathway can play a role in this biological process. Leptin-mediated EC differentiation and angiogenesis in ESCs can be a useful application towards regenerative medicine and tissue engineering. PMID:25250519

  5. Reduced serum concentration is permissive for increased in vitro endocrine differentiation from murine embryonic stem cells.

    PubMed

    Vincent, Robert K; Odorico, Jon S

    2009-07-01

    Embryonic stem cells (ESCs) have been shown to be capable of differentiating into pancreatic progenitors and insulin-producing cells in vitro. However, before ESC derivatives can be used in clinical settings, efficient selective differentiation needs to be achieved. Essential to improving ESC differentiation to islet endocrine cells is an understanding of the influences of extrinsic signals and transcription factors on cell specification. Herein, we investigate the influence of serum-supplemented growth conditions on the differentiation of murine ESCs to endocrine lineages in the context of over-expression of two pancreatic transcription factors, Pdx1 and Ngn3. To study the effect of different serum formulations and concentrations on the ability of murine ESCs to differentiate into endocrine cells in vitro, cells were grown into embryoid bodies and then differentiated in various serum replacement (SR), fetal calf serum (FCS) and serum-free conditions. Using immunohistochemistry and quantitative real-time RT-PCR (QPCR), we found that, of the conditions tested, 1% SR differentiation medium resulted in the highest levels of insulin-1 mRNA and significantly increased the total number of insulin-expressing cells. Applying this knowledge to cell lines in which Pdx1 or Ngn3 transgene expression could be induced by exposure to doxycycline we differentiated TetPDX1 and TetNgn3 ESCs under conditions of either 10% FCS or 1% SR medium. In the presence of 10% serum, induced expression of either Pdx1 or Ngn3 in differentiating ESCs resulted in modest increases in hormone transcripts and cell counts. However, changing the serum formulation from 10% FCS to 1% SR significantly enhanced the number of insulin+/C-peptide+ cells in parallel with increased insulin-1 transcript levels in both inducible cell lines. In summary, these data demonstrate that induced expression of key pancreatic transcription factors in combination with low serum/SR concentrations increases endocrine cell

  6. CrxOS maintains the self-renewal capacity of murine embryonic stem cells

    SciTech Connect

    Saito, Ryota; Yamasaki, Tokiwa; Nagai, Yoko; Wu, Jinzhan; Kajiho, Hiroaki; Yokoi, Tadashi; Noda, Eiichiro; Nishina, Sachiko; Niwa, Hitoshi; Azuma, Noriyuki; Katada, Toshiaki; Nishina, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

  7. The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells

    PubMed Central

    Obeidi, Narges; Pourfathollah, Ali Akbar; Soleimani, Masoud; Nikougoftar Zarif, Mahin; Kouhkan, Fatemeh

    2016-01-01

    Objective MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. Materials and Methods In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction. Results Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Conclusion Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines. PMID:27540521

  8. Large-Scale Identification of MicroRNA Targets in Murine Dgcr8-Deficient Embryonic Stem Cell Lines

    PubMed Central

    Davis, Matthew P. A.; Abreu-Goodger, Cei; van Dongen, Stijn; Lu, Dong; Tate, Peri H.; Bartonicek, Nenad; Kutter, Claudia; Liu, Pentao; Skarnes, William C.; Enright, Anton J.; Dunham, Ian

    2012-01-01

    Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3′ UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation. PMID:22912678

  9. Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

    PubMed Central

    Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

    2009-01-01

    Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

  10. Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells

    PubMed Central

    Munier, Mathilde; Meduri, Geri; Viengchareun, Say; Leclerc, Phillipe; Le Menuet, Damien; Lombès, Marc

    2010-01-01

    Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem (ES) cell lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene Green Fluorescent Protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites) and the increase in the expression of neuronal markers (nestin, β-tubulin III, MAP2) as demonstrated by immunocytochemistry and qPCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively and correlated with MR expression. Finally, while progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by siRNA. Concluding, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases. PMID:20207834

  11. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro.

    PubMed

    Srivastava, Anand S; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A; Carrier, Ewa

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of

  12. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

    SciTech Connect

    Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A.; Carrier, Ewa . E-mail: assrivastava@ucsd.edu

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the

  13. Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

    PubMed

    Molcanyi, Marek; Mehrjardi, Narges Zare; Schäfer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jürgen; Bosche, Bert

    2014-01-01

    Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed. PMID:25249934

  14. Differentiation of Murine Embryonic Stem Cells Induces Progesterone Receptor Gene Expression

    PubMed Central

    Sauter, Carley N.; McDermid, Rebecca L.; Weinberg, Amy L.; Greco, Tamara L.; Xu, Xiaojie; Murdoch, Fern E.; Fritsch, Michael K.

    2005-01-01

    The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERα, ERβ), are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen dependent manner by endogenous ERs or by transiently overexpressed ERα. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation PMID:16223481

  15. MEF2C as a Neurogenic and Anti-Apoptotic Transcription Factor in Murine Embryonic Stem Cells

    PubMed Central

    Li, Zhen; McKercher, Scott R.; Cui, Jiankun; Nie, Zhiguo; Soussou, Walid; Roberts, Amanda J.; Sallmen, Tina; Okamoto, Shu-ichi; Lipton, Jeffrey H.; Talantova, Maria; Lipton, Stuart A.

    2008-01-01

    Cell-based therapies require a reliable source of cells that can be easily grown, undergo directed differentiation, and remain viable after transplantation. Here we generated stably transformed murine embryonic stem (ES) cells that express a constitutively active form of myocyte enhancer factor 2C (MEF2CA). MEF2C has been implicated as a calcium-dependent transcription factor that enhances survival and affects synapse formation of neurons as well as differentiation of cardiomyocytes. We now report that expression of MEF2CA, both in vitro and in vivo, under regulation of the nestin enhancer effectively produces ‘neuronal’ progenitor cells that differentiate into a virtually pure population of neurons. Histological, electrophysiological and behavioral analyses demonstrate that MEF2C-directed neuronal progenitor cells transplanted into a mouse model of cerebral ischemia can successfully differentiate into functioning neurons and ameliorate stroke-induced behavioral deficits. PMID:18579729

  16. 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.

  17. Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate.

    PubMed

    Zhang, Ying-Ying; Tang, Lei-Lei; Zheng, Bei; Ge, Ren-Shan; Zhu, Dan-Yan

    2016-05-01

    Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α-actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L-threonine dehydrogenase, X-ray repair cross-complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26178269

  18. In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium

    PubMed Central

    Chung, Jaehoon; Kee, Kehkooi; Barral, Joëlle K.; Dash, Rajesh; Kosuge, Hisanori; Wang, Xi; Weissman, Irving; Robbins, Robert C.; Nishimura, Dwight; Quertermous, Thomas; Reijo-Pera, Renee A.; Yang, Phillip C.

    2012-01-01

    Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, non-specific intracellular accumulation of SPIO limits long-term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO-conjugated monoclonal antibody against these antigens (SPIO-MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO-MAb. The human and mouse ESCs were transduced with the RG (ESC-RGs) and transplanted into the peri-infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO-MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC-derived teratoma formation in the injured murine myocardium. PMID:21604295

  19. Transplantation of Neural Stem Cells Clonally Derived from Embryonic Stem Cells Promotes Recovery After Murine Spinal Cord Injury

    PubMed Central

    Salewski, Ryan P.; Mitchell, Robert A.; Shen, Carl

    2015-01-01

    The pathology of spinal cord injury (SCI) makes it appropriate for cell-based therapies. Treatments using neural stem cells (NSCs) in animal models of SCI have shown positive outcomes, although uncertainty remains regarding the optimal cell source. Pluripotent cell sources such as embryonic stem cells (ESCs) provide a limitless supply of therapeutic cells. NSCs derived using embryoid bodies (EB) from ESCs have shown tumorigenic potential. Clonal neurosphere generation is an alternative method to generate safer and more clinically relevant NSCs without the use of an EB stage for use in cell-based therapies. We generated clonally derived definitive NSCs (dNSCs) from ESC. These cells were transplanted into a mouse thoracic SCI model. Embryonic stem cell-derived definitive neural stem cell (ES-dNSC)-transplanted mice were compared with controls using behavioral measures and histopathological analysis of tissue. In addition, the role of remyelination in injury recovery was investigated using transmission electron microscopy. The SCI group that received ES-dNSC transplantation showed significant improvements in locomotor function compared with controls in open field and gait analysis. The cell treatment group had a significant enhancement of spared neural tissue. Immunohistological assessments showed that dNSCs differentiated primarily to oligodendrocytes. These cells were shown to express myelin basic protein, associate with axons, and support nodal architecture as well as display proper compact, multilayer myelination in electron microscopic analysis. This study provides strong evidence that dNSCs clonally derived from pluripotent cells using the default pathway of neuralization improve motor function after SCI and enhance sparing of neural tissue, while remaining safe and clinically relevant. PMID:25119334

  20. Improved Survival and Hematopoietic Differentiation of Murine Embryonic Stem Cells on Electrospun Polycaprolactone Nanofiber

    PubMed Central

    Dehdilani, Nima; Shamsasenjan, Karim; Movassaghpour, Aliakbar; Akbarzadehlaleh, Parvin; Amoughli Tabrizi, Bahram; Parsa, Hamed; Sabagi, Fatemeh

    2016-01-01

    Objective Three-dimensional (3D) biomimetic nanofiber scaffolds have widespread ap- plications in biomedical tissue engineering. They provide a suitable environment for cel- lular adhesion, survival, proliferation and differentiation, guide new tissue formation and development, and are one of the outstanding goals of tissue engineering. Electrospinning has recently emerged as a leading technique for producing biomimetic scaffolds with mi- cro to nanoscale topography and a high porosity similar to the natural extracellular matrix (ECM). These scaffolds are comprised of synthetic and natural polymers for tissue engi- neering applications. Several kinds of cells such as human embryonic stem cells (hESCs) and mouse ESCs (mESCs) have been cultured and differentiated on nanofiber scaffolds. mESCs can be induced to differentiate into a particular cell lineage when cultured as em- bryoid bodies (EBs) on nano-sized scaffolds. Materials and Methods We cultured mESCs (2500 cells/100 µl) in 96-well plates with knockout Dulbecco’s modified eagle medium (DMEM-KO) and Roswell Park Memorial Institute-1640 (RPMI-1640), both supplemented with 20% ESC grade fetal bovine serum (FBS) and essential factors in the presence of leukemia inhibitory factor (LIF). mESCs were seeded at a density of 2500 cells/100 µl onto electrospun polycaprolactone (PCL) nanofibers in 96-well plates. The control group comprised mESCs grown on tissue cul- ture plates (TCP) at a density of 2500 cells/100 µl. Differentiation of mESCs into mouse hematopoietic stem cells (mHSCs) was performed by stem cell factor (SCF), interleukin-3 (IL-3), IL-6 and Fms-related tyrosine kinase ligand (Flt3-L) cytokines for both the PCL and TCP groups. We performed an experimental study of mESCs differentiation. Results PCL was compared to conventional TCP for survival and differentiation of mESCs to mHSCs. There were significantly more mESCs in the PCL group. Flowcyto- metric analysis revealed differences in hematopoietic

  1. Stress-induced enzyme activation primes murine embryonic stem cells to differentiate toward the first extraembryonic lineage.

    PubMed

    Slater, Jill A; Zhou, Sichang; Puscheck, Elizabeth Ella; Rappolee, Daniel A

    2014-12-15

    Extracellular stresses influence transcription factor (TF) expression and therefore lineage identity in the peri-implantation mouse embryo and its stem cells. This potentially affects pregnancy outcome. To understand the effects of stress signaling during this critical period of pregnancy, we exposed cultured murine embryonic stem cells (mESCs) to hyperosmotic stress. We then measured stress-enzyme-dependent regulation of key pluripotency and lineage TFs. Hyperosmotic stress slowed mESC accumulation due to slowing of the cell cycle over 72 h, after a small apoptotic response within 12 h. Phosphoinositide 3-kinase (PI3K) enzymatic signaling was responsible for stem cell survival under stressed conditions. Stress initially triggered mESC differentiation after 4 h through MEK1, c-Jun N-terminal kinase (JNK), and PI3K enzymatic signaling, which led to proteasomal degradation of Oct4, Nanog, Sox2, and Rex1 TF proteins. Concurrent with this post-transcriptional effect was the decreased accumulation of potency TF mRNA transcripts. After 12-24 h of stress, cells adapted, cell cycle resumed, and Oct4 and Nanog mRNA and protein expression returned to approximately normal levels. The TF protein recovery was mediated by p38MAPK and PI3K signaling, as well as by MEK2 and/or MEK1. However, due to JNK signaling, Rex1 expression did not recover. Probing for downstream lineages revealed that although mESCs did not differentiate morphologically during 24 h of stress, they were primed to differentiate by upregulating markers of the first lineage differentiating from mESCs, extraembryonic endoderm. Thus, although two to three TFs that mark pluripotency recover expression by 24 h of stress, there is nonetheless sustained Rex1 suppression and a priming of mESCs for differentiation to the earliest lineage. PMID:25144240

  2. Stress-Induced Enzyme Activation Primes Murine Embryonic Stem Cells to Differentiate Toward the First Extraembryonic Lineage

    PubMed Central

    Slater, Jill A.; Zhou, Sichang; Puscheck, Elizabeth Ella

    2014-01-01

    Extracellular stresses influence transcription factor (TF) expression and therefore lineage identity in the peri-implantation mouse embryo and its stem cells. This potentially affects pregnancy outcome. To understand the effects of stress signaling during this critical period of pregnancy, we exposed cultured murine embryonic stem cells (mESCs) to hyperosmotic stress. We then measured stress-enzyme-dependent regulation of key pluripotency and lineage TFs. Hyperosmotic stress slowed mESC accumulation due to slowing of the cell cycle over 72 h, after a small apoptotic response within 12 h. Phosphoinositide 3-kinase (PI3K) enzymatic signaling was responsible for stem cell survival under stressed conditions. Stress initially triggered mESC differentiation after 4 h through MEK1, c-Jun N-terminal kinase (JNK), and PI3K enzymatic signaling, which led to proteasomal degradation of Oct4, Nanog, Sox2, and Rex1 TF proteins. Concurrent with this post-transcriptional effect was the decreased accumulation of potency TF mRNA transcripts. After 12–24 h of stress, cells adapted, cell cycle resumed, and Oct4 and Nanog mRNA and protein expression returned to approximately normal levels. The TF protein recovery was mediated by p38MAPK and PI3K signaling, as well as by MEK2 and/or MEK1. However, due to JNK signaling, Rex1 expression did not recover. Probing for downstream lineages revealed that although mESCs did not differentiate morphologically during 24 h of stress, they were primed to differentiate by upregulating markers of the first lineage differentiating from mESCs, extraembryonic endoderm. Thus, although two to three TFs that mark pluripotency recover expression by 24 h of stress, there is nonetheless sustained Rex1 suppression and a priming of mESCs for differentiation to the earliest lineage. PMID:25144240

  3. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  4. Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells.

    PubMed

    He, Xiaoming; Park, Eric Y H; Fowler, Alex; Yarmush, Martin L; Toner, Mehmet

    2008-06-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2M). The cryoprotectants used included 2M 1,2-propanediol (PROH, cell membrane permeable) and 0.5M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  5. Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Microcapillary: A Study Using Murine Embryonic Stem Cells

    PubMed Central

    He, Xiaoming; Park, Eric Y.H.; Fowler, Alex; Yarmush, Martin L.; Toner, Mehmet

    2009-01-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  6. All-Trans Retinoic Acid Directs Urothelial Specification of Murine Embryonic Stem Cells via GATA4/6 Signaling Mechanisms

    PubMed Central

    Mauney, Joshua R.; Ramachandran, Aruna; Yu, Richard N.; Daley, George Q.

    2010-01-01

    The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP). To date, our understanding of the developmental processes responsible for urothelial differentiation has been hampered due to the lack of suitable models. In this study, we describe a novel in vitro cell culture system for derivation of urothelial cells from murine embryonic stem cells (ESCs) following cultivation on collagen matrices in the presence all trans retinoic acid (RA). Upon stimulation with micromolar concentrations of RA, ESCs significantly downregulated the pluripotency factor OCT-4 but markedly upregulated UP1A, UP1B, UP2, UP3A, and UP3B mRNA levels in comparison to naïve ESCs and spontaneously differentiating controls. Pan-UP protein expression was associated with both p63- and cytokeratin 20-positive cells in discrete aggregating populations of ESCs following 9 and 14 days of RA stimulation. Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations. GATA4−/− and GATA6−/− transgenic ESC lines revealed substantial attenuation of RA-mediated UP expression in comparison to wild type controls. In addition, EMSA analysis revealed that RA treatment induced formation of transcriptional complexes containing GATA4/6 on both UP1B and UP2 promoter fragments containing putative GATA factor binding sites. Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6–dependent processes. PMID:20644631

  7. An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres

    SciTech Connect

    Wang, Yisong; Giannone, Richard J; Wu, Jun; Gomez, Marla V; Liu, Yie

    2005-01-01

    Telomerase deficiency leads to a progressive loss of telomeric DNA that eventually triggers cell apoptosis in human primary cells during prolonged growth in culture. Rare survivors can maintain telomere length through either activation of telomerase or recombination-based telomere lengthening, and thus proliferate indefinitely. We have explored the possibility that telomeres may be maintained through telomere sister chromatid exchange (T-SCE) in murine telomere reverse transcriptase-deficient (mTert -/-) splenocytes and ES cells. Because telomerase deficiency leads to gradual loss of telomeric DNA in mTert -/- splenocytes and ES cells and eventually to chromosomes with telomere signal-free ends (SFEs), we examined these cell types for evidence of sister chromatid exchange at telomeres, and observed an increase in T-SCEs only in a subset of mTert -/- splenocytes or ES cells that possessed multiple SFEs. Furthermore, T-SCEs were more often detected in ES cells than in splenocytes that harbored a similar frequency of SFEs. In mTert heterozygous (mTert +/-) ES cells or splenocytes, which are known to exhibit a decrease in average telomere length but no SFEs, no increase in T-SCE was observed. In addition to T-SCE, other genomic rearrangements (i.e., SCE) were also significantly increased in mTert -/- ES cells possessing critically short telomeres, but not in splenocytes. Our results suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened.

  8. 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

  9. Calmidazolium chloride inhibits growth of murine embryonal carcinoma cells, a model of cancer stem-like cells.

    PubMed

    Lee, Jina; Kim, Min Seong; Kim, Min Aeh; Jang, Yeun Kyu

    2016-09-01

    Calmidazolium chloride (CMZ) is widely used as a calmodulin (CaM) antagonist, but is also known to induce apoptosis in certain cancer cell lines. However, in spite of the importance of cancer stem cells (CSCs) in cancer therapy, the effects of CMZ on CSCs are not yet well understood. We investigated the effects of CMZ on the F9 embryonal carcinoma cell (ECC) line as a surrogate model of CSCs. To avoid bias due to culture conditions, F9 ECCs and E14 embryonic stem cells (ESCs) were grown in the same culture medium. Results obtained using a cell-counting kit showed that CMZ significantly inhibited growth in F9 ECCs compared with growth in E14 ESCs. CMZ also induced apoptosis of F9 ECCs, but not of E14 ESCs, which was associated with caspase-3 activation and an increased fraction of the sub-G1 cell population. In addition, our data revealed that the expression of stemness-related genes including c-Myc was selectively down regulated in CMZ-treated F9 ECCs. Our results suggest that CMZ can inhibit the growth of ECCs by inducing apoptosis and down regulating stemness-related genes, without causing any harm to normal stem cells. These findings indicate a potential application of CMZ in the development of anti-CSC therapeutics. PMID:27247146

  10. Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells.

    PubMed

    Yang, Jianchang; Chai, Li; Fowles, Taylor C; Alipio, Zaida; Xu, Dan; Fink, Louis M; Ward, David C; Ma, Yupo

    2008-12-16

    Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls. PMID:19060217

  11. Impurity of Stem Cell Graft by Murine Embryonic Fibroblasts – Implications for Cell-Based Therapy of the Central Nervous System

    PubMed Central

    Molcanyi, Marek; Mehrjardi, Narges Zare; Schäfer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jürgen; Bosche, Bert

    2014-01-01

    Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts – MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed. PMID:25249934

  12. Single cell derived murine embryonic stem cell clones stably express Rex1-specific green fluorescent protein and their differentiation study

    SciTech Connect

    Chen Xiaopan; Wu Rongrong; Feng Shumei; Gu Bin; Dai Licheng; Zhang Ming; Zhao Xiaoli

    2007-10-19

    Embryonic stem cells (ESCs) often display high rates of apoptosis and spontaneous differentiation in routine culture, thus bring the proliferation of these cells highly inefficient. Moreover, little is known about the factors that are indispensable for sustaining self-renewal. To surmount these issues, we established transgenic mES cell lines expressing the enhanced green fluorescent protein (EGFP) under the control of the Rex1 promoter which is a key regulator of pluripotency in ES cells. In addition, we provided a simplified and improved protocol to derive transgenic mESCs from single cell. Finally, we showed that embryoid body (EB) development was faster than adherent differentiation in terms of differentiation ratio by real-time tracking of the EGFP expression. Therefore, these cell lines can be tracked and selected both in vitro and in vivo and should be invaluable for studying the factors that are indispensable for maintaining pluripotency.

  13. Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell-derived cardiomyocytes for cardiac tissue engineering.

    PubMed

    Parrag, Ian C; Zandstra, Peter W; Woodhouse, Kimberly A

    2012-03-01

    Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC-derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC-derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre-seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length-to-diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC-derived cardiac cells. PMID:22006660

  14. A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis.

    PubMed

    Yu, Ruoxing; Miyamura, Norio; Okamoto-Uchida, Yoshimi; Arima, Norie; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi

    2015-01-01

    Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA) formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat.) D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST) that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium. PMID:26682887

  15. A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis

    PubMed Central

    Yu, Ruoxing; Miyamura, Norio; Okamoto-Uchida, Yoshimi; Arima, Norie; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Nishina, Hiroshi

    2015-01-01

    Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA) formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat.) D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST) that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium. PMID:26682887

  16. Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

    PubMed

    Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  17. Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

    PubMed Central

    Pfeiffer, Susanne M.; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A.

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  18. Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia

    PubMed Central

    Hammoud, Aya Abou; Kirstein, Nina; Mournetas, Virginie; Darracq, Anais; Broc, Sabine; Blanchard, Camille; Zeineddine, Dana; Mortada, Mohamad; Boeuf, Helene

    2016-01-01

    Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF–mediated reprogramming are poorly understood. By employing a LIF-dependent ‘plasticity’ test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of Pluri and Master gene expression in comparison to 20% O2. PMID:26731538

  19. Distinct Contributions of JNK and p38 to Chromium Cytotoxicity and Inhibition of Murine Embryonic Stem Cell Differentiation

    PubMed Central

    Chen, Liang; Ovesen, Jerald L.; Puga, Alvaro; Xia, Ying

    2009-01-01

    Background Potassium dichromate [Cr(VI)] is a widespread environmental toxicant responsible for increased risk of several human diseases. Cr(VI) exposure leads to activation of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK)1/2, p38, and extracellular-signal regulated kinase (ERK)1/2. Objectives We evaluated the contribution of MAPKs to Cr(VI) toxicity. Methods Phosphorylation of MAPKs and their downstream effectors was evaluated by Western immunoblotting; reactive oxygen species were measured by DCFDA (5′,6′-chloromethyl-2′-7′-dichlorofluorescin diacetate) labeling and flow cytometry, and glutathione and glutathione disulfide levels were determined by monochrome graphic spectroflurometer. Cytotoxicity was assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation. Embryoid body (EB) differentiation was evaluated by contracting cardiomyocyte formation, and real-time polymerase chain reaction (RT-PCR) was used for cardiomyocyte-specific and stem-cell-specific gene expression. Results Acute treatment of mouse embryonic stem (ES) cells with 50 μM Cr(VI) induced the rapid phosphorylation of JNK, p38, and ERK and their respective downstream transcription factors, c-JUN, activating transcription factor-2, and ELK1. MAPK activation and cytotoxicity induction were partially blocked by pretreatment with the antioxidant N-acetyl cysteine. Ablation of the upstream MAP kinase kinase (MAP2K7) in ES cells prevented JNK activation, whereas ablation of MAP2K4 prevented both JNK and p38 activation. Using specific MAPK inhibitors and MAP2K4- and MAP2K7-deficient ES cells, we showed that JNK reduced acute Cr(VI) cytotoxicity, p38 potentiated it, and ERK had no effect. At low submicromolar concentrations, Cr(VI) caused MAP2K4/7-dependent JNK activation and MAP2K4-dependent p38 activation and strongly inhibited contracting cardiomyocyte development in wild-type ES

  20. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

    PubMed Central

    Wu, H; Liu, X; Jaenisch, R

    1994-01-01

    A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation. Images PMID:8146196

  1. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    SciTech Connect

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V.; Shively, John E.

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  2. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    PubMed

    Fang, Jennifer S; Gritz, Emily C; Marcelo, Kathrina L; Hirschi, Karen K

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  3. Isolation of Murine Embryonic Hemogenic Endothelial Cells

    PubMed Central

    Marcelo, Kathrina L.; Hirschi, Karen K.

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  4. 28. Embryonic and adult stem cell therapy.

    PubMed

    Henningson, Carl T; Stanislaus, Marisha A; Gewirtz, Alan M

    2003-02-01

    Stem cells are characterized by the ability to remain undifferentiated and to self-renew. Embryonic stem cells derived from blastocysts are pluripotent (able to differentiate into many cell types). Adult stem cells, which were traditionally thought to be monopotent multipotent, or tissue restricted, have recently also been shown to have pluripotent properties. Adult bone marrow stem cells have been shown to be capable of differentiating into skeletal muscle, brain microglia and astroglia, and hepatocytes. Stem cell lines derived from both embryonic stem and embryonic germ cells (from the embryonic gonadal ridge) are pluripotent and capable of self-renewal for long periods. Therefore embryonic stem and germ cells have been widely investigated for their potential to cure diseases by repairing or replacing damaged cells and tissues. Studies in animal models have shown that transplantation of fetal, embryonic stem, or embryonic germ cells may be able to treat some chronic diseases. In this review, we highlight recent developments in the use of stem cells as therapeutic agents for three such diseases: Diabetes, Parkinson disease, and congestive heart failure. We also discuss the potential use of stem cells as gene therapy delivery cells and the scientific and ethical issues that arise with the use of human stem cells. PMID:12592319

  5. Lack of Phenotypical and Morphological Evidences of Endothelial to Hematopoietic Transition in the Murine Embryonic Head during Hematopoietic Stem Cell Emergence

    PubMed Central

    Iizuka, Kazuhide; Yokomizo, Tomomasa; Watanabe, Naoki; Tanaka, Yosuke; Osato, Motomi; Takaku, Tomoiku; Komatsu, Norio

    2016-01-01

    During mouse ontogeny, hematopoietic cells arise from specialized endothelial cells, i.e., the hemogenic endothelium, and form clusters in the lumen of arterial vessels. Hemogenic endothelial cells have been observed in several embryonic tissues, such as the dorsal aorta, the placenta and the yolk sac. Recent work suggests that the mouse embryonic head also produces hematopoietic stem cells (HSCs)/progenitors. However, a histological basis for HSC generation in the head has not yet been determined because the hematopoietic clusters and hemogenic endothelium in the head region have not been well characterized. In this study, we used whole-mount immunostaining and 3D confocal reconstruction techniques to analyze both c-Kit+ hematopoietic clusters and Runx1+ hemogenic endothelium in the whole-head vasculature. The number of c-Kit+ hematopoietic cells was 20-fold less in the head arteries than in the dorsal aorta. In addition, apparent nascent hematopoietic cells, which are characterized by a “budding” structure and a Runx1+ hemogenic endothelium, were not observed in the head. These results suggest that head HSCs may not be or are rarely generated from the endothelium in the same manner as aortic HSCs. PMID:27227884

  6. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  7. 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

  8. Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Ikeda, Nobuhito; Nakayama, Yuji; Nakazawa, Natsumi; Yoshida, Akio; Ninomiya, Haruaki; Shirayoshi, Yasuaki

    2016-01-01

    Background The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP+ cells include undifferentiated cells with a capacity to develop into tumors. Methods PrP+ cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT–PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5GFP/+ (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. Results PrP+ cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP+ cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP+ cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP+ cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP+/SSEA1– cells did not proliferate and expressed cardiac cell markers, while PrP+/SSEA1+ did proliferate. Conclusion PrP+ cells isolated from EB included undifferentiated cells in day 21. PrP+/SSEA1– cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes. PMID:27493483

  9. From teratocarcinomas to embryonic stem cells.

    PubMed Central

    Andrews, Peter W

    2002-01-01

    The recent derivation of human embryonic stem (ES) cell lines, together with results suggesting an unexpected degree of plasticity in later, seemingly more restricted, stem cells (so-called adult stem cells), have combined to focus attention on new opportunities for regenerative medicine, as well as for understanding basic aspects of embryonic development and diseases such as cancer. Many of the ideas that are now discussed have a long history and much has been underpinned by the earlier studies of teratocarcinomas, and their embryonal carcinoma (EC) stem cells, which present a malignant surrogate for the normal stem cells of the early embryo. Nevertheless, although the potential of EC and ES cells to differentiate into a wide range of tissues is now well attested, little is understood of the key regulatory mechanisms that control their differentiation. Apart from the intrinsic biological interest in elucidating these mechanisms, a clear understanding of the molecular process involved will be essential if the clinical potential of these cells is to be realized. The recent observations of stem-cell plasticity suggest that perhaps our current concepts about the operation of cell regulatory pathways are inadequate, and that new approaches for analysing complex regulatory networks will be essential. PMID:12028783

  10. Common stemness regulators of embryonic and cancer stem cells

    PubMed Central

    Hadjimichael, Christiana; Chanoumidou, Konstantina; Papadopoulou, Natalia; Arampatzi, Panagiota; Papamatheakis, Joseph; Kretsovali, Androniki

    2015-01-01

    Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies. PMID:26516408

  11. Human embryonic stem cells and lung regeneration

    PubMed Central

    Varanou, A; Page, C P; Minger, S L

    2008-01-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically. PMID:18724383

  12. Embryonic and adult stem cell therapy.

    PubMed

    Brignier, Anne C; Gewirtz, Alan M

    2010-02-01

    There are many types of stem cells. All share the characteristics of being able to self-renew and to give rise to differentiated progeny. Over the last decades, great excitement has been generated by the prospect of being able to exploit these properties for the repair, improvement, and/or replacement of damaged organs. However, many hurdles, both scientific and ethical, remain in the path of using human embryonic stem cells for tissue-engineering purposes. In this report we review current strategies for isolating, enriching, and, most recently, inducing the development of human pluripotent stem cells. In so doing, we discuss the scientific and ethical issues associated with this endeavor. Finally, progress in the use of stem cells as therapies for type 1 diabetes mellitus, congestive heart failure, and various neurologic and immunohematologic disorders, and as vehicles for the delivery of gene therapy, is briefly discussed. PMID:20061008

  13. 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. PMID:20579256

  14. Engineering tissue from human embryonic stem cells

    PubMed Central

    Metallo, CM; Azarin, SM; Ji, L; De Pablo, JJ; Palecek, SP

    2008-01-01

    Abstract Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome. PMID:18194458

  15. GATA factors efficiently direct cardiac fate from embryonic stem cells.

    PubMed

    Turbendian, Harma K; Gordillo, Miriam; Tsai, Su-Yi; Lu, Jia; Kang, Guoxin; Liu, Ting-Chun; Tang, Alice; Liu, Susanna; Fishman, Glenn I; Evans, Todd

    2013-04-01

    The GATA4 transcription factor is implicated in promoting cardiogenesis in combination with other factors, including TBX5, MEF2C and BAF60C. However, when expressed in embryonic stem cells (ESCs), GATA4 was shown to promote endoderm, not cardiac mesoderm. The capacity of related GATA factors to promote cardiogenesis is untested. We found that expression of the highly related gene, Gata5, very efficiently promotes cardiomyocyte fate from murine ESCs. Gata5 directs development of beating sheets of cells that express cardiac troponin T and show a full range of action potential morphologies that are responsive to pharmacological stimulation. We discovered that by removing serum from the culture conditions, GATA4 and GATA6 are each also able to efficiently promote cardiogenesis in ESC derivatives, with some distinctions. Thus, GATA factors can function in ESC derivatives upstream of other cardiac transcription factors to direct the efficient generation of cardiomyocytes. PMID:23487308

  16. Programming embryonic stem cells to neuronal subtypes

    PubMed Central

    Peljto, Mirza; Wichterle, Hynek

    2010-01-01

    Richness of neural circuits and specificity of neuronal connectivity depends on the diversification of nerve cells into functionally and molecularly distinct subtypes. While efficient methods for directed differentiation of embryonic stem cells (ESCs) into multiple principal neuronal classes have been established, only a few studies systematically examined the subtype diversity of in vitro derived nerve cells. Here we review evidence based on molecular and in vivo transplantation studies that ESC-derived spinal motor neurons and cortical layer V pyramidal neurons acquire subtype specific functional properties. We discuss similarities and differences in the role of cell intrinsic transcriptional programs, extrinsic signals and cell-cell interactions during subtype diversification of the two classes of nerve cells. We conclude that the high degree of fidelity with which differentiating ESCs recapitulate normal embryonic development provides a unique opportunity to explore developmental processes underlying specification of mammalian neuronal diversity in a simplified and experimentally accessible system. PMID:20970319

  17. Debating restrictions on embryonic stem cell research.

    PubMed

    McClain, Colleen

    2009-09-01

    This study investigates the emotional and behavioral effects of interpersonal online communication, focusing on the controversy surrounding the loosening of restrictions on human embryonic stem cell research. The issue, central to national and statewide elections in 2008, generated heated debate among candidates and voters and evoked strong emotional sentiments among partisans. Using the theory of affective intelligence, this study proposes a predictive model connecting levels of enthusiasm and anxiety with behavioral and information-seeking outcomes. Cognitive appraisal theory is also employed to provide a role for political emotion in accounting for interactive media effects. To investigate the ways that online deliberation may influence discussions surrounding stem cell research, a between-subjects experimental study was conducted that systematically varied the tone of feedback received (reinforcing or challenging) and type of interaction (synchronous or asynchronous) experienced by users. Results indicate that emotional responses play a significant role in predicting behavioral intentions arising from the user-to-user interactive experience. PMID:20205522

  18. Electrospun Polyacrylonitrile-Based Nanofibers Maintain Embryonic Stem Cell Stemness via TGF-Beta Signaling.

    PubMed

    Liu, Shih-Ping; Lin, Chen-Huan; Lin, Shao-Ji; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Hsu, Chung Y; Shyu, Woei-Cherng

    2016-04-01

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. Culturing ESCs on mouse embryonic fibroblast-derived and cell-based feeder layers to maintain pluripotency is a standard laboratory procedure. However, xenogeneic contamination and the large amount of time required for feeder cell preparation are two challenges that encourage the use of a murine-based feeder layer. A novel biomaterial is required to replace the current cell-based feeder system. Toward this goal, we applied a combination of biocompatible polyacrylonitrile (PAN) and electrospinning technology to establish a non-cell-based feeder layer. According to results from stem cell marker staining, scanning electron microscopy, and embryoid body formation tests, optimal ESC stemness and pluripotency were noted in three electrospun groups (2, 4, and 8 minutes), with the longer electrospinning times producing higher feeder-layer densities. KEGG pathway microarray results identified TGF-beta signaling as one of the major deregulatory pathways on electrospun-based feeder layers. Western blot data indicate significant increases in TGF-beta receptor II, phosphorylated Smad3, and Nanog protein levels in the 4- and 8-minute electrospun-based feeder layer groups compared to the non-feeder layer group. Combined, the data suggest that electrospun-based feeder layers are good candidates for maintaining ESC and iPSC pluripotency in clinical applications. PMID:27301199

  19. Culturing murine embryonic organs: Pros, cons, tips and tricks.

    PubMed

    McClelland, Kathryn S; Bowles, Josephine

    2016-01-01

    There are three established techniques described for ex vivo culture of the early embryonic organs: filter culture, agar block culture and hanging drop culture. Each of these protocols has advantages and disadvantages; here we assess the merits of each approach. Agar block culture has a long history and has been well described. This method results in good embryonic organ morphology. Filter culture has been used to culture a number of different embryonic organs and there are a variety of filter choices available. The key disadvantage of agar-block and filter based culture is that the large amount of media required can make the approach expensive, especially if biologicals such as growth factors are necessary; in addition, using these methods it can be difficult to track particular samples. Hanging drop culture is most commonly used to enable the aggregation of embryonic stem cells into embryoid bodies but it has also been employed for ex vivo organ culture. This method requires only 40μL of media per drop and isolates every organ to a trackable unit. We describe each of these methods and the use of different medias and provide the user with a matrix to help determine the optimal culture method for their needs. Glass-based culture methods required for live imaging are not discussed here. PMID:26988290

  20. Graphene Oxide promotes embryonic stem cell differentiation to haematopoietic lineage

    PubMed Central

    Garcia-Alegria, Eva; Iluit, Maria; Stefanska, Monika; Silva, Claudio; Heeg, Sebastian; Kimber, Susan J.; Kouskoff, Valerie; Lacaud, Georges; Vijayaraghavan, Aravind; Batta, Kiran

    2016-01-01

    Pluripotent stem cells represent a promising source of differentiated tissue-specific stem and multipotent progenitor cells for regenerative medicine and drug testing. The realisation of this potential relies on the establishment of robust and reproducible protocols of differentiation. Several reports have highlighted the importance of biomaterials in assisting directed differentiation. Graphene oxide (GO) is a novel material that has attracted increasing interest in the field of biomedicine. In this study, we demonstrate that GO coated substrates significantly enhance the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. GO does not affect cell proliferation or survival of differentiated cells but rather enhances the transition of haemangioblasts to haemogenic endothelial cells, a key step during haematopoietic specification. Importantly, GO also improves, in addition to murine, human ES cell differentiation to blood cells. Taken together, our study reveals a positive role for GO in haematopoietic differentiation and suggests that further functionalization of GO could represent a valid strategy for the generation of large numbers of functional blood cells. Producing these cells would accelerate haematopoietic drug toxicity testing and treatment of patients with blood disorders or malignancies. PMID:27197878

  1. Graphene Oxide promotes embryonic stem cell differentiation to haematopoietic lineage.

    PubMed

    Garcia-Alegria, Eva; Iluit, Maria; Stefanska, Monika; Silva, Claudio; Heeg, Sebastian; Kimber, Susan J; Kouskoff, Valerie; Lacaud, Georges; Vijayaraghavan, Aravind; Batta, Kiran

    2016-01-01

    Pluripotent stem cells represent a promising source of differentiated tissue-specific stem and multipotent progenitor cells for regenerative medicine and drug testing. The realisation of this potential relies on the establishment of robust and reproducible protocols of differentiation. Several reports have highlighted the importance of biomaterials in assisting directed differentiation. Graphene oxide (GO) is a novel material that has attracted increasing interest in the field of biomedicine. In this study, we demonstrate that GO coated substrates significantly enhance the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. GO does not affect cell proliferation or survival of differentiated cells but rather enhances the transition of haemangioblasts to haemogenic endothelial cells, a key step during haematopoietic specification. Importantly, GO also improves, in addition to murine, human ES cell differentiation to blood cells. Taken together, our study reveals a positive role for GO in haematopoietic differentiation and suggests that further functionalization of GO could represent a valid strategy for the generation of large numbers of functional blood cells. Producing these cells would accelerate haematopoietic drug toxicity testing and treatment of patients with blood disorders or malignancies. PMID:27197878

  2. Interaction of Salmonella Typhimurium with Dendritic Cells Derived from Pluripotent Embryonic Stem Cells

    PubMed Central

    Rossi, Raffaella; Hale, Christine; Goulding, David; Andrews, Robert; Abdellah, Zarah; Fairchild, Paul J.; Dougan, Gordon

    2012-01-01

    Using an in vitro differentiation protocol we isolated cells with the properties of dendritic cells (DCs) from immunologically refractive pluripotent murine embryonic stem cells (ESCs). These ES-derived dendritic cells (ESDCs) expressed cytokines and were able to present antigen to a T cell line. Infection of ESDCs with Salmonella Typhimurium stimulated the expression of immune cell markers and thousands of murine genes, many associated with the immune response. Consequently, this system provides a novel in vitro model, amenable to genetic modification, for monitoring host/pathogen interactions. PMID:23284947

  3. Embryonic Stem Cells/Induced Pluripotent Stem Cells

    PubMed Central

    Feng, Xuezhu; Zhang, Jiuchun; Smuga-Otto, Kimberly; Tian, Shulan; Yu, Junying; Stewart, Ron; Thomson, James A.

    2012-01-01

    Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition. PMID:22213079

  4. Human embryonic stem cells: preclinical perspectives

    PubMed Central

    Deb, Kaushik Dilip; Sarda, Kanchan

    2008-01-01

    Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic. PMID:18230169

  5. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  6. Genetic Manipulation of Human Embryonic Stem Cells.

    PubMed

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells. PMID:25520283

  7. Thermoresponsive hydrogel maintains the mouse embryonic stem cell "naïve" pluripotency phenotype.

    PubMed

    Mangani, Christian; Lilienkampf, Annamaria; Roy, Marcia; de Sousa, Paul A; Bradley, Mark

    2015-10-15

    A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N'-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a "naïve" state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture. PMID:26372076

  8. Probing Embryonic Stem Cell Autocrine and Paracrine Signaling Using Microfluidics

    NASA Astrophysics Data System (ADS)

    Przybyla, Laralynne; Voldman, Joel

    2012-07-01

    Although stem cell fate is traditionally manipulated by exogenously altering the cells' extracellular signaling environment, the endogenous autocrine and paracrine signals produced by the cells also contribute to their two essential processes: self-renewal and differentiation. Autocrine and/or paracrine signals are fundamental to both embryonic stem cell self-renewal and early embryonic development, but the nature and contributions of these signals are often difficult to fully define using conventional methods. Microfluidic techniques have been used to explore the effects of cell-secreted signals by controlling cell organization or by providing precise control over the spatial and temporal cellular microenvironment. Here we review how such techniques have begun to be adapted for use with embryonic stem cells, and we illustrate how many remaining questions in embryonic stem cell biology could be addressed using microfluidic technologies.

  9. Nanog: a new recruit to the embryonic stem cell orchestra.

    PubMed

    Cavaleri, Fatima; Schöler, Hans R

    2003-05-30

    In this issue of Cell, and add a new transcriptional operating system to the known Oct4 and Stat3 systems required for early embryonal stem cell potency and self-renewal. Nanog, a homeobox transcription factor, plays a crucial role in the second embryonic cell fate specification following formation of the blastocyst. PMID:12787492

  10. Nucleosome Organization in Human Embryonic Stem Cells

    PubMed Central

    Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

    2015-01-01

    The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a “ground state” of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this “ground state” by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of

  11. Human Embryonic Stem Cells and Cardiac Repair

    PubMed Central

    Zhu, Wei-Zhong; Hauch, Kip; Xu, Chunhui; Laflamme, Michael A.

    2008-01-01

    The muscle lost after a myocardial infarction is replaced with non-contractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. hESC-derived cardiomyocytes (hESC-CMs) exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain to the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-CMs and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges. PMID:18657407

  12. Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

    PubMed

    Treff, Nathan R; Vincent, Robert K; Budde, Melisa L; Browning, Victoria L; Magliocca, Joseph F; Kapur, Vivek; Odorico, Jon S

    2006-11-01

    Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression. PMID:16809427

  13. Screening of nanoparticle embryotoxicity using embryonic stem cells.

    PubMed

    Campagnolo, Luisa; Fenoglio, Ivana; Massimiani, Micol; Magrini, Andrea; Pietroiusti, Antonio

    2013-01-01

    Due to the increasing use of engineered nanoparticles in many consumer products, rapid and economic tests for evaluating possible adverse effects on human health are urgently needed. In the present chapter the use of mouse embryonic stem cells as a valuable tool to in vitro screen nanoparticle toxicity on embryonic tissues is described. This in vitro method is a modification of the embryonic stem cell test, which has been widely used to screen soluble chemical compounds for their embryotoxic potential. The test offers an alternative to animal experimentation, reducing experimental costs and ethical issues. PMID:23592031

  14. Human Naive Embryonic Stem Cells: How Full Is the Glass?

    PubMed

    Wang, Yixuan; Gao, Shaorong

    2016-03-01

    Human naive embryonic stem cells in the ground state of pluripotency provide a new opportunity to study human developmental biology and potential clinical applications. Two studies now report related work in human naive stem cell derivation and DNA methylation analysis, with one reporting some differences from oocyte and blastocyst profiles. PMID:26942847

  15. Embryonic stem cell identity grounded in the embryo

    PubMed Central

    Plusa, Berenika; Hadjantonakis, Anna-Katerina

    2016-01-01

    Pluripotent embryonic stem cells (ESCs) can be derived from blastocyst-stage mouse embryos. However, the exact in vivo counterpart of ESCs has remained elusive. A combination of expression profiling and stem cell derivation identifies epiblast cells from late-stage blastocysts as the source, and functional equivalent, of ESCs. PMID:24875737

  16. Study of Gap Junctions in Human Embryonic Stem Cells.

    PubMed

    Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency. PMID:24859928

  17. 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. PMID:11660628

  18. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  19. Human Embryonic Stem Cells Form Functional Thyroid Follicles

    PubMed Central

    Latif, Rauf; Davies, Terry F.

    2015-01-01

    Objective: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Methods: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Results: Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. Conclusion: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be

  20. Nuclear lamins are not required for lamina-associated domain organization in mouse embryonic stem cells

    PubMed Central

    Amendola, Mario; van Steensel, Bas

    2015-01-01

    In mammals, the nuclear lamina interacts with hundreds of large genomic regions, termed lamina-associated domains (LADs) that are generally in a transcriptionally repressed state. Lamins form the major structural component of the lamina and have been reported to bind DNA and chromatin. Here, we systematically evaluate whether lamins are necessary for the LAD organization in murine embryonic stem cells. Surprisingly, removal of essentially all lamins does not have any detectable effect on the genome-wide interaction pattern of chromatin with emerin, a marker of the inner nuclear membrane. This suggests that other components of the lamina mediate these interactions. PMID:25784758

  1. Epigenetics and chromatin plasticity in embryonic stem cells

    PubMed Central

    Přikrylová, Terézia; Pacherník, Jiří; Kozubek, Stanislav; Bártová, Eva

    2013-01-01

    The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson’s disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells. PMID:23951389

  2. Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.

    PubMed

    Kubaczka, Caroline; Senner, Claire E; Cierlitza, Monika; Araúzo-Bravo, Marcos J; Kuckenberg, Peter; Peitz, Michael; Hemberger, Myriam; Schorle, Hubert

    2015-11-01

    Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation. PMID:26412560

  3. RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.

    PubMed

    Yang, Junning; Wu, Chuanshen; Stefanescu, Ioana; Jakobsson, Lars; Chervoneva, Inna; Horowitz, Arie

    2016-01-01

    Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid-induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx(+/+) and Syx(-/-) embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx(-/-) embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx(+/+) embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx(-/-) embryoid bodies were thinner than those in Syx(+/+) cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx(-/-) embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion. PMID:27460990

  4. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  6. Twenty years of embryonic stem cell research in farm animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Engineering of the Embryonic and Adult Stem Cell Niches

    PubMed Central

    Hosseinkhani, Mohsen; Shirazi, Reza; Rajaei, Farzad; Mahmoudi, Masoud; Mohammadi, Navid; Abbasi, Mahnaz

    2013-01-01

    Context Stem cells have the potential to generate a renewable source of cells for regenerative medicine due to their ability to self-renew and differentiate to various functional cell types of the adult organism. The extracellular microenvironment plays a pivotal role in controlling stem cell fate responses. Therefore, identification of appropriate environmental stimuli that supports cellular proliferation and lineage-specific differentiation is critical for the clinical application of the stem cell therapies. Evidence Acquisition Traditional methods for stem cells culture offer limited manipulation and control of the extracellular microenvironment. Micro engineering approaches are emerging as powerful tools to control stem cell-microenvironment interactions and for performing high-throughput stem cell experiments. Results In this review, we provided an overview of the application of technologies such as surface micropatterning, microfluidics, and engineered biomaterials for directing stem cell behavior and determining the molecular cues that regulate cell fate decisions. Conclusions Stem cells have enormous potential for therapeutic and pharmaceutical applications, because they can give rise to various cell types. Despite their therapeutic potential, many challenges, including the lack of control of the stem cell microenvironment remain. Thus, a greater understanding of stem cell biology that can be used to expand and differentiate embryonic and adult stem cells in a directed manner offers great potential for tissue repair and regenerative medicine. PMID:23682319

  8. Derivation of Genea002 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  9. 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. PMID:21903672

  10. The molecular nature of very small embryonic-like stem cells in adult tissues.

    PubMed

    Kim, YongHwan; Jeong, Jaeho; Kang, Hyunsook; Lim, Jisun; Heo, Jinbeom; Ratajczak, Janina; Ratajczak, Mariusz Z; Shin, Dong-Myung

    2014-11-01

    Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4(+) SSEA-1(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (∼3-6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells. PMID:25473442

  11. The Molecular Nature of Very Small Embryonic-Like Stem Cells in Adult Tissues

    PubMed Central

    Kim, YongHwan; Jeong, Jaeho; Kang, Hyunsook; Lim, Jisun; Heo, Jinbeom; Ratajczak, Janina; Ratajczak, Mariusz Z.; Shin, Dong-Myung

    2014-01-01

    Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4+ SSEA-1+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (∼3–6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells. PMID:25473442

  12. In vitro myelin formation using embryonic stem cells

    PubMed Central

    Kerman, Bilal E.; Kim, Hyung Joon; Padmanabhan, Krishnan; Mei, Arianna; Georges, Shereen; Joens, Matthew S.; Fitzpatrick, James A. J.; Jappelli, Roberto; Chandross, Karen J.; August, Paul; Gage, Fred H.

    2015-01-01

    Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation. PMID:26015546

  13. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  14. Medical perspectives of adults and embryonic stem cells.

    PubMed

    Cavazzana-Calvo, Marina; André-Schmutz, Isabelle; Lagresle, Chantal; Fischer, Alain

    2002-10-01

    In the last 30 years, allogeneic bone marrow transplantation has become the treatment of choice for many hematologic malignancies or inherited disorders and a number of changes have been registered in terms of long-term survival rate of transplanted patients as well as of available sources of hematopoietic stem cell (HSC). In parallel to the publication of better results in HSC transplantation, several recent discoveries have opened a scientific and ethical debate on the therapeutical potential of stem cells isolated from adult or embryonic tissues. One of the major discoveries in this field is the capacity of bone marrow-derived stem cells to treat a genetic liver disease in a mouse model, thus justifying the concept of transdifferentiation of adult stem cell and raising hopes on its possible therapeutical applications. We have tried here to summarise the advances in this field and to discuss the limits of these biological data. PMID:12494504

  15. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    PubMed

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well. PMID:24482125

  16. 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. PMID:27587447

  17. DIRECTED DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO BLADDER TISSUE

    PubMed Central

    Oottamasathien, Siam; Wang, YongQing; Williams, Karin; Franco, Omar E.; Wills, Marcia L.; Thomas, John C.; Saba, Katrina; Sharif-Afshar, Ali-Reza; Makari, John H.; Bhowmick, Neil A; DeMarco, Romano T.; Hipkens, Susan; Magnuson, Mark; Brock, John W.; Hayward, Simon W.; Pope, John C.; Matusik, Robert J.

    2007-01-01

    Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies. PMID:17289017

  18. Three-dimensional optical coherence tomography of the embryonic murine cardiovascular system

    NASA Astrophysics Data System (ADS)

    Luo, Wei; Marks, Daniel L.; Ralston, Tyler S.; Boppart, Stephen A.

    2006-03-01

    Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology that has potential as a novel investigational tool in developmental biology and functional genomics. In this study, murine embryos and embryonic hearts are visualized with an OCT system capable of 2-µm axial and 15-µm lateral resolution and with real-time acquisition rates. We present, to our knowledge, the first sets of high-resolution 2- and 3-D OCT images that reveal the internal structures of the mammalian (murine) embryo (E10.5) and embryonic (E14.5 and E17.5) cardiovascular system. Strong correlations are observed between OCT images and corresponding hematoxylin- and eosin-stained histological sections. Real-time in vivo embryonic (E10.5) heart activity is captured by spectral-domain optical coherence tomography, processed, and displayed at a continuous rate of five frames per second. With the ability to obtain not only high-resolution anatomical data but also functional information during cardiovascular development, the OCT technology has the potential to visualize and quantify changes in murine development and in congenital and induced heart disease, as well as enable a wide range of basic in vitro and in vivo research studies in functional genomics.

  19. Modulation of rabbit corneal epithelial cells fate using embryonic stem cell extract

    PubMed Central

    Zhan, Weijiao; Liu, Zhiping; Liu, Ying; Ke, Qicheng; Ding, Yuanyuan; Lu, Xiaoyan

    2010-01-01

    Purpose To develop a new culture system to cultivate differentiated autologous cells in vitro for cell therapy and tissue engineering. Methods After incubation in murine embryonic stem cell (ESC) extract for 1 h, streptolysin-O (SLO) permeabilized cells were resealed with CaCl2 and continually cultured for weeks. The morphological study was analyzed by light microscopy. Isolated colonies were selected and expanded to establish cell lines. Octamer-4 (Oct-4), stage-specific embryonic antigen-1 (SSEA-1), transformation-related protein 63 (p63), ATP-binding cassette subfamily G, member 2 (ABCG2), and cytokeratin3 (K3) were detected by indirect immunofluorescent staining. Oct-4, K3, and p63 were also detected by RT–PCR analysis. To examine the stemness characteristics of the induced cells, both alkaline phosphatase (AKP) staining and tumorigenicity detection were performed, respectively. Results Reprogramming was induced in corneal epithelial cells. The reprogrammed cells showed characteristics similar to ESCs in the early weeks, including colony formation, positive AKP staining, and multi-potential differentiation in vivo. Oct-4 and SSEA1 protein expression was upregulated. However, these changes were not persistent or stable. With the passage of time, the colonies became flat. The ESC markers were downregulated, while epithelial cell related proteins gradually increased. Conclusions Less terminal differentiated rabbit corneal epithelial cells could be induced to a more pluripotent state with embryonic stem cell extract (ESC-E). These cells have the potential to return to the beginning of their own lineage and obtain the ability of long-term growth. Our findings indicate that this culture system can generate low-immunogenic autologous cells for use in regenerative medicine. PMID:20664691

  20. Biobanking human embryonic stem cell lines: policy, ethics and efficiency.

    PubMed

    Holm, Søren

    2015-12-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy discussions relating to their establishment or maintenance. It is argued (1) that 'ethical arguments' are often more important in the establishment phase and 'efficiency arguments' more important in the maintenance phase, and (2) that arguments relating to the interests of embryo and gamete donors are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being ignored in the policy making process. PMID:26758612

  1. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

    PubMed

    Abu Dawud, Raed; Schreiber, Kerstin; Schomburg, Dietmar; Adjaye, James

    2012-01-01

    While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2) while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency. PMID:22768158

  2. Human Embryonic Stem Cells and Embryonal Carcinoma Cells Have Overlapping and Distinct Metabolic Signatures

    PubMed Central

    Schomburg, Dietmar; Adjaye, James

    2012-01-01

    While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O2 while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency. PMID:22768158

  3. Automated analysis of contractility in the embryonic stem cell test, a novel approach to assess embryotoxicity.

    PubMed

    Peters, Annelieke K; Wouwer, Gert Van de; Weyn, Barbara; Verheyen, Geert R; Vanparys, Philippe; Gompel, Jacques Van

    2008-12-01

    The embryonic stem cell test (EST) is an ECVAM-validated assay to detect embryotoxicity. The output of the assay is the effect of test compounds on the differentiation of murine-derived embryonic stem cells (D3 cells), recorded by visual analysis of contracting cardiomyocyte-like cells. Incorporation of a system to assess the contractility in an automated manner is proposed, to increase the throughput in the EST independent of observer bias. The automated system is based on image recording of each well, resulting in the area (pixels) and frequency of contractility (Hz). Four test compounds were assessed for their embryotoxic potency in the 96-well version of the EST, with both manual and automated analysis: 6-Aminonicotinamide, Valproic Acid, Boric Acid, and Penicillin G. There was no statistically significant difference in the outcome of both methods in the fraction of contractility (p<0.05), resulting in the same rank-order of Relative Embryotoxic Potency (REP) values: 6-aminonicotinamide (1)>valproic acid (0.007-0.013)>Boric Acid (0.002-0.005)>Penicillin G (0.00001). The automated image recording of contractile cardiomyocyte-like cells in the EST allows for an unbiased high throughput method to assess the embryotoxic potency of test compounds, resulting in an outcome comparable to manual analysis. PMID:18845236

  4. Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

    PubMed Central

    Kim, Peter T. W.; Hoffman, Brad G.; Plesner, Annette; Helgason, Cheryl D.; Verchere, C. Bruce; Chung, Stephen W.; Warnock, Garth L.; Mui, Alice L. F.; Ong, Christopher J.

    2010-01-01

    Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors. PMID:21152387

  5. Hypoxia promotes thyroid differentiation of native murine induced pluripotent stem cells.

    PubMed

    Yang, Yipeng; Lu, Yunshu; Chen, Tong; Zhang, Shenglai; Chu, Bingfeng; Gong, Yurong; Zhao, Weixin; Zhu, Jian; Liu, Yingbin

    2016-01-01

    Hypothyroidism is a very common hormonal deficiency and the stem cell technology which developed in the recent years may offer a therapeutic strategy for treating this disorder. Hypoxia has been demonstrated to play an important role in embryonic formation and development and to modulate stem cell differentiation. However, the influence of oxygen tension on thyroid differentiation has not been studied. In this study, we used murine induced pluripotent stem (iPS) cells for thyroid cell differentiation under normoxic and hypoxic conditions and compared differentiation efficiency in morphology, function, gene and protein expression under both conditions. We found that hypoxia promoted adhesion and outgrowth of embryoid bodies (EBs) derived from murine iPS cells. Expression of endodermal markers (Foxa2 and Gata4) and thyroid transcription factors (Pax8 and Nkx2.1) was increased by hypoxia at both gene and protein levels during early-mid differentiation stages (p<0.05). And so were the thyroid specific markers NIS and TSHR at the end of the experiment (p<0.05). In addition, functional iodide uptake by differentiated cells was also increased after hypoxia. Thyroid differentiation from iPS cells is enhanced under hypoxia and this may involve hypoxia inducible factors (HIFs) and their downstream gene FGF2. Our data offer a foundation for understanding thyroid development and provide a potentially more efficient way to use cell therapy for treating thyroid deficiency. PMID:27389981

  6. Inner Ear Hair Cell-Like Cells from Human Embryonic Stem Cells

    PubMed Central

    Ronaghi, Mohammad; Nasr, Marjan; Ealy, Megan; Durruthy-Durruthy, Robert; Waldhaus, Joerg; Diaz, Giovanni H.; Joubert, Lydia-Marie; Oshima, Kazuo

    2014-01-01

    In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation. PMID:24512547

  7. Inner ear hair cell-like cells from human embryonic stem cells.

    PubMed

    Ronaghi, Mohammad; Nasr, Marjan; Ealy, Megan; Durruthy-Durruthy, Robert; Waldhaus, Joerg; Diaz, Giovanni H; Joubert, Lydia-Marie; Oshima, Kazuo; Heller, Stefan

    2014-06-01

    In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation. PMID:24512547

  8. Nanoscale topography and chemistry affect embryonic stem cell self-renewal and early differentiation.

    PubMed

    Lapointe, Vanessa L S; Fernandes, Ana Tiago; Bell, Nia C; Stellacci, Francesco; Stevens, Molly M

    2013-12-01

    Adherent cells respond to a wide range of substrate cues, including chemistry, topography, hydrophobicity, and surface energy. The cell-substrate interface is therefore an important design parameter in regenerative medicine and tissue engineering applications, where substrate cues are used to influence cell behavior. Thin films comprising 4.5 nm (average diameter) gold nanoparticles coated with a mixture of two alkanethiols can confer hemispherical topography and specific chemistry to bulk substrates. The behavior of murine embryonic stem cells (ESCs) on the thin films can then be compared with their behavior on self-assembled monolayers of the same alkanethiols on vapor-deposited gold, which lack the topographical features. Cells cultured both with and without differentiation inhibitors are characterized by immunofluorescence for Oct4 and qPCR for Fgf5, Foxa2, Nanog, Pou5f1, and Sox2. Nanoscale chemistry and topography are found to influence stem cell differentiation, particularly the early differentiation markers, Fgf5 and Foxa2. Nanoscale topography also affects Oct4 localization, whereas the chemical composition of the substrate does not have an effect. It is demonstrated for the first time that ESCs can sense topographical features established by 4.5 nm particles, and these findings suggest that nanoscale chemistry and topography can act synergistically to influence stem cell differentiation. This study furthers the understanding of the effects of these substrate properties, improving our ability to design materials to control stem cell fate. PMID:23852884

  9. Genome-wide nucleosome positioning during embryonic stem cell development.

    PubMed

    Teif, Vladimir B; Vainshtein, Yevhen; Caudron-Herger, Maïwen; Mallm, Jan-Philipp; Marth, Caroline; Höfer, Thomas; Rippe, Karsten

    2012-11-01

    We determined genome-wide nucleosome occupancies in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell-type- and protein-specific binding preferences of transcription factors to sites with either low (Myc, Klf4 and Zfx) or high (Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome-depleted regions around transcription start and transcription termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to CpG content or histone methylation marks. Throughout the genome, nucleosome occupancy was correlated with certain histone methylation or acetylation modifications. In addition, the average nucleosome repeat length increased during differentiation by 5-7 base pairs, with local variations for specific regions. Our results reveal regulatory mechanisms of cell differentiation that involve nucleosome repositioning. PMID:23085715

  10. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L.; Wolf, Don; Mitalipov, Shoukhrat

    2013-01-01

    SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state. PMID:23683578

  11. Lessons from the embryonic neural stem cell niche for neural lineage differentiation of pluripotent stem cells.

    PubMed

    Solozobova, Valeriya; Wyvekens, Nicolas; Pruszak, Jan

    2012-09-01

    Pluripotent stem cells offer an abundant and malleable source for the generation of differentiated cells for transplantation as well as for in vitro screens. Patterning and differentiation protocols have been developed to generate neural progeny from human embryonic or induced pluripotent stem cells. However, continued refinement is required to enhance efficiency and to prevent the generation of unwanted cell types. We summarize and interpret insights gained from studies of embryonic neuroepithelium. A multitude of factors including soluble molecules, interactions with the extracellular matrix and neighboring cells cooperate to control neural stem cell self-renewal versus differentiation. Applying these findings and concepts to human stem cell systems in vitro may yield more appropriately patterned cell types for biomedical applications. PMID:22628111

  12. The ethical dilemma of embryonic stem cell research.

    PubMed

    Manzar, Nabeel; Manzar, Bushra; Hussain, Nuzhat; Hussain, M Fawwad Ahmed; Raza, Sajjad

    2013-03-01

    To determine the knowledge, attitude, and ethical concerns of medical students and graduates with regard to Embryonic Stem Cell (ESC) research. This questionnaire based descriptive study was conducted at the Civil Hospital Karachi (CHK), Pakistan from February to July 2008. A well structured questionnaire was administered to medical students and graduate doctors, which included their demographic profile as well as questions in line with the study objective. Informed consent was taken and full confidentiality was assured to the participants. Data were entered in a Statistical Package for Social Sciences (SPSS version.12) and analyzed. A total of 204 male and 216 female medical students and doctors were administered questionnaires out of which 105 males (51.4%) and 108 females (50%) were aware of the embryonic stem cell research and its ethical implications. Forty percent males and 47% of females were of the opinion that life begins at conception. Forty-six percent males and 39% females were in favor of stem cell research while only 31% males and 28% females supported the ESC research. Less than 1/3 of students supported using frozen embryos for research purposes while more than 2/3 indicated that they were unlikely to support abortion for stem cell research purposes. The majority of the students were in favor of stem cell research with some reservations regarding ESC research. A sizeable number of students withheld their views, reflecting their poor understanding of medical ethics. The result of the study indicates a need for incorporating bioethics into the medical curriculum. PMID:22038063

  13. Derivation of Genea057 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  14. Derivation of Genea042 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  15. Derivation of Genea052 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  16. Derivation of human embryonic stem cell line Genea023.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  17. Derivation of Genea015 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  18. Derivation of human embryonic stem cell line Genea022.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  19. Derivation of Genea047 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  20. Derivation of Genea043 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  1. Derivation of Genea016 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  2. Human embryonic stem cell differentiation toward regional specific neural precursors.

    PubMed

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  3. Human Embryonic Stem Cell Differentiation Toward Regional Specific Neural Precursors

    PubMed Central

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  4. Embryonic stem cells and the bigger reprogenetic picture.

    PubMed

    Parens, E

    2000-01-01

    In this country, public policy conversations about reproductive and genetic technologies have been conducted separately. This makes it difficult for policy makers and the public to notice the extent to which reproductive and genetic technologies are converging, and thus the extent to which we are approaching the possibility of reprogenetically shaping our children. Embryonic stem cell research offers a superb opportunity to identify that convergence, and thus to begin considering the creation of a public institution that would be devoted to contemplating the bigger picture of reprogenetically shaping children. PMID:10828546

  5. X Inactivation Lessons from Differentiating Mouse Embryonic Stem Cells.

    PubMed

    Pintacuda, Greta; Cerase, Andrea

    2015-10-01

    X chromosome inactivation (XCI) is the dosage compensation mechanism that evolved in female mammals to correct the genetic imbalance of X-linked genes between sexes. X chromosome inactivation occurs in early development when one of the two X chromosomes of females is nearly-completely silenced. Differentiating Embryonic Stem cells (ESC) are regarded as a useful tool to study XCI, since they recapitulate many events occurring during early development. In this review we aim to summarise the advances in the field and to discuss the close connection between cell differentiation and X chromosome inactivation, with a particular focus on mouse ESCs. PMID:26198263

  6. The topographical regulation of embryonic stem cell differentiation.

    PubMed Central

    Murray, Patricia; Edgar, David

    2004-01-01

    The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis. PMID:15306413

  7. Maintenance of chicken embryonic stem cells in vitro.

    PubMed

    Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

    2006-01-01

    In this chapter, we describe the methods we have used to show that chicken leukemia inhibitory factor (LIF) maintains chicken embryonic stem (ES) cells in an undifferentiated state in culture. Recombinant chicken LIF (rchLIF) was expressed as a fusion protein linked to glutathione S-transferase (GST) and purified to greater than 90% purity in two chromatography stages, the first an affinity step using the GST tail, which was cleaved before further purification by gel chromatography. Chicken ES cells were obtained by culturing chicken blastodermal cells isolated from stage X embryos of freshly laid chicken eggs. These cells can be maintained in media containing rchLIF for at least 9 d without any other cytokines or feeder cells. Chicken ES cells were characterized by the expression of alkaline phosphatase activity, stage-specific embryonic antigen (SSEA)-1 and embryonal carcinoma cell monoclonal antibody-1. In addition, the phosphorylation of signal transducers and activators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis. PMID:16845981

  8. Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

    PubMed Central

    Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

    2013-01-01

    Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

  9. Wnt5a Supports Osteogenic Lineage Decisions in Embryonic Stem Cells.

    PubMed

    Keller, Kevin C; Ding, Huawen; Tieu, Rudy; Sparks, Nicole R L; Ehnes, Devon D; Zur Nieden, Nicole I

    2016-07-01

    The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. PMID:26956615

  10. Isolation and characterization of embryonic stem cell-derived cardiac Purkinje cells.

    PubMed

    Maass, Karen; Shekhar, Akshay; Lu, Jia; Kang, Guoxin; See, Fiona; Kim, Eugene E; Delgado, Camila; Shen, Steven; Cohen, Lisa; Fishman, Glenn I

    2015-04-01

    The cardiac Purkinje fiber network is composed of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies, and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PCs) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their proarrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling, and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis, and screening for novel antiarrhythmic therapies. PMID:25524238

  11. Applicability, usability, and limitations of murine embryonic imaging with optical coherence tomography and optical projection tomography

    PubMed Central

    Singh, Manmohan; Raghunathan, Raksha; Piazza, Victor; Davis-Loiacono, Anjul M.; Cable, Alex; Vedakkan, Tegy J.; Janecek, Trevor; Frazier, Michael V.; Nair, Achuth; Wu, Chen; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

    2016-01-01

    We present an analysis of imaging murine embryos at various embryonic developmental stages (embryonic day 9.5, 11.5, and 13.5) by optical coherence tomography (OCT) and optical projection tomography (OPT). We demonstrate that while OCT was capable of rapid high-resolution live 3D imaging, its limited penetration depth prevented visualization of deeper structures, particularly in later stage embryos. In contrast, OPT was able to image the whole embryos, but could not be used in vivo because the embryos must be fixed and cleared. Moreover, the fixation process significantly altered the embryo morphology, which was quantified by the volume of the eye-globes before and after fixation. All of these factors should be weighed when determining which imaging modality one should use to achieve particular goals of a study. PMID:27375945

  12. Derivation and differentiation of haploid human embryonic stem cells.

    PubMed

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-01

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. PMID:26982723

  13. Lack of vimentin impairs endothelial differentiation of embryonic stem cells.

    PubMed

    Boraas, Liana C; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM -/- ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM -/- EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM -/- EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  14. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J.

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. PMID:26890252

  15. Human embryonic stem cells carrying mutations for severe genetic disorders.

    PubMed

    Frumkin, Tsvia; Malcov, Mira; Telias, Michael; Gold, Veronica; Schwartz, Tamar; Azem, Foad; Amit, Ami; Yaron, Yuval; Ben-Yosef, Dalit

    2010-04-01

    Human embryonic stem cells (HESCs) carrying specific mutations potentially provide a valuable tool for studying genetic disorders in humans. One preferable approach for obtaining these cell lines is by deriving them from affected preimplantation genetically diagnosed embryos. These unique cells are especially important for modeling human genetic disorders for which there are no adequate research models. They can be further used to gain new insights into developmentally regulated events that occur during human embryo development and that are responsible for the manifestation of genetically inherited disorders. They also have great value for the exploration of new therapeutic protocols, including gene-therapy-based treatments and disease-oriented drug screening and discovery. Here, we report the establishment of 15 different mutant human embryonic stem cell lines derived from genetically affected embryos, all donated by couples undergoing preimplantation genetic diagnosis in our in vitro fertilization unit. For further information regarding access to HESC lines from our repository, for research purposes, please email dalitb@tasmc.health.gov.il. PMID:20186514

  16. Lack of vimentin impairs endothelial differentiation of embryonic stem cells

    PubMed Central

    Boraas, Liana C.; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM −/− ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM −/− EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM −/− EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  17. Suppression of Chondrogenesis by Id Helix-Loop-Helix Proteins in Murine Embryonic Orofacial Tissue

    PubMed Central

    Mukhopadhyay, Partha; Rezzoug, Francine; Webb, Cynthia L.; Pisano, M. Michele; Greene, Robert M.

    2009-01-01

    Inhibitors of differentiation (Id) proteins are helix-loop-helix (HLH) transcription factors lacking a DNA binding domain. Id proteins modulate cell proliferation, apoptosis, and differentiation in embryonic/fetal tissue. Perturbation of any of these processes in cells of the developing orofacial region results in orofacial anomalies. Chondrogenesis, a process integral to normal orofacial ontogenesis, is known to be modulated, in part, by Id proteins. In the present study, the mRNA and protein expression patterns of Id1, Id2, Id3 and Id4 were examined in developing murine orofacial tissue in vivo, as well as in murine embryonic maxillary mesenchymal cells in vitro. The functional role of Ids during chondrogenesis was also explored in vitro. Results reveal that cells derived from developing murine orofacial tissue: (1) express Id1, Id2, Id3 and Id4 mRNAs and proteins on each of gestational days 12-14, (2) express all four Id proteins in a developmentally regulated manner, (3) undergo chondrogenesis and express genes encoding various chondrogenic marker proteins (e.g. Runx2, Type X collagen, Sox9) when cultured under micromass conditions, and (4) can have their chondrogenic potential regulated via alteration of Id protein function through overexpression of a basic HLH factor. In summary, results from the current report reveal for the first time, the expression of all four Id proteins in cells derived from developing murine orofacial tissue, and demonstrate a functional role for the Ids in regulating the ability of these cells to undergo chondrogenesis. PMID:19349107

  18. The dynamics of murine mammary stem/progenitor cells

    PubMed Central

    DONG, Qiaoxiang; SUN, Lu-Zhe

    2014-01-01

    The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups. PMID:25580105

  19. Murine Embryonic Fibroblast Cell Lines Differentiate into Three Mesenchymal Lineages to Different Extents: New Models to Investigate Differentiation Processes

    PubMed Central

    Dastagir, Khaled; Lazaridis, Andrea; Jahn, Sabrina; Maurer, Viktor; Strauß, Sarah; Dastagir, Nadjib; Radtke, Christine; Kampmann, Andreas; Bucan, Vesna; Vogt, Peter M.

    2014-01-01

    Abstract Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as “feeder cells.” Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation. PMID:25068630

  20. Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

    PubMed

    Dastagir, Khaled; Reimers, Kerstin; Lazaridis, Andrea; Jahn, Sabrina; Maurer, Viktor; Strauß, Sarah; Dastagir, Nadjib; Radtke, Christine; Kampmann, Andreas; Bucan, Vesna; Vogt, Peter M

    2014-08-01

    Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation. PMID:25068630

  1. 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 ...

  2. Development of an invitro technique to use mouse embryonic stem cell in evaluating effects of xenobiotics

    EPA Science Inventory

    Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combin...

  3. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    EPA Science Inventory

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  4. [Expert consensus on induction of human embryonic stem cells into tenocytes].

    PubMed

    Chen, Xiao; Zou, Xiaohui; Yu, Guangyan; Fu, Xin; Cao, Tong; Xiao, Yin; Ouyang, Hongwei

    2016-03-01

    Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs. PMID:27273982

  5. In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor's Production.

    PubMed

    Fauzi, Iliana; Panoskaltsis, Nicki; Mantalaris, Athanasios

    2016-01-01

    Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs. PMID:26160454

  6. Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine.

    PubMed

    Jung, Jin Hyuk; Iwabuchi, Kumiko; Yang, Zhihong; Loeken, Mary R

    2016-01-01

    The hexose transporter, GLUT2 (SLC2A2), which is expressed by mouse embryos, is important for survival before embryonic day 10.5, but its function in embryos is unknown. GLUT2 can transport the amino sugar glucosamine (GlcN), which could increase substrate for the hexosamine biosynthetic pathway (HBSP) that produces UDP-N-acetylglucosamine for O-linked N-acetylglucosamine modification (O-GlcNAcylation) of proteins. To understand this, we employed a novel murine embryonic stem cell (ESC) line that, like mouse embryos, expresses functional GLUT2 transporters. GlcN stimulated ESC proliferation in a GLUT2-dependent fashion but did not regulate pluripotency. Stimulation of proliferation was not due to increased O-GlcNAcylation. Instead, GlcN decreased dependence of the HBSP on fructose-6-PO4 and glutamine. Consequently, glycolytic- and glutamine-derived intermediates that are needed for anabolic metabolism were increased. Thus, maternally obtained GlcN may increase substrates for biomass accumulation by embryos, as exogenous GlcN does for GLUT2-expressing ESC, and may explain the need for GLUT2 expression by embryos. PMID:27311888

  7. Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine

    PubMed Central

    Jung, Jin Hyuk; Iwabuchi, Kumiko; Yang, Zhihong; Loeken, Mary R.

    2016-01-01

    The hexose transporter, GLUT2 (SLC2A2), which is expressed by mouse embryos, is important for survival before embryonic day 10.5, but its function in embryos is unknown. GLUT2 can transport the amino sugar glucosamine (GlcN), which could increase substrate for the hexosamine biosynthetic pathway (HBSP) that produces UDP-N-acetylglucosamine for O-linked N-acetylglucosamine modification (O-GlcNAcylation) of proteins. To understand this, we employed a novel murine embryonic stem cell (ESC) line that, like mouse embryos, expresses functional GLUT2 transporters. GlcN stimulated ESC proliferation in a GLUT2-dependent fashion but did not regulate pluripotency. Stimulation of proliferation was not due to increased O-GlcNAcylation. Instead, GlcN decreased dependence of the HBSP on fructose-6-PO4 and glutamine. Consequently, glycolytic- and glutamine-derived intermediates that are needed for anabolic metabolism were increased. Thus, maternally obtained GlcN may increase substrates for biomass accumulation by embryos, as exogenous GlcN does for GLUT2-expressing ESC, and may explain the need for GLUT2 expression by embryos. PMID:27311888

  8. 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

  9. 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

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

    2014-03-01

    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

  10. Transcriptional dynamics of the embryonic stem cell switch.

    PubMed

    Chickarmane, Vijay; Troein, Carl; Nuber, Ulrike A; Sauro, Herbert M; Peterson, Carsten

    2006-09-15

    Recent ChIP experiments of human and mouse embryonic stem cells have elucidated the architecture of the transcriptional regulatory circuitry responsible for cell determination, which involves the transcription factors OCT4, SOX2, and NANOG. In addition to regulating each other through feedback loops, these genes also regulate downstream target genes involved in the maintenance and differentiation of embryonic stem cells. A search for the OCT4-SOX2-NANOG network motif in other species reveals that it is unique to mammals. With a kinetic modeling approach, we ascribe function to the observed OCT4-SOX2-NANOG network by making plausible assumptions about the interactions between the transcription factors at the gene promoter binding sites and RNA polymerase (RNAP), at each of the three genes as well as at the target genes. We identify a bistable switch in the network, which arises due to several positive feedback loops, and is switched on/off by input environmental signals. The switch stabilizes the expression levels of the three genes, and through their regulatory roles on the downstream target genes, leads to a binary decision: when OCT4, SOX2, and NANOG are expressed and the switch is on, the self-renewal genes are on and the differentiation genes are off. The opposite holds when the switch is off. The model is extremely robust to parameter changes. In addition to providing a self-consistent picture of the transcriptional circuit, the model generates several predictions. Increasing the binding strength of NANOG to OCT4 and SOX2, or increasing its basal transcriptional rate, leads to an irreversible bistable switch: the switch remains on even when the activating signal is removed. Hence, the stem cell can be manipulated to be self-renewing without the requirement of input signals. We also suggest tests that could discriminate between a variety of feedforward regulation architectures of the target genes by OCT4, SOX2, and NANOG. PMID:16978048

  11. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

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

    SciTech Connect

    Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota

    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 of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  13. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

    PubMed Central

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J.; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C. A.

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  14. [Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

    PubMed

    Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang

    2008-07-01

    In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal. PMID:18837407

  15. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  16. Urine excretion strategy for stem cell-generated embryonic kidneys

    PubMed Central

    Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi

    2015-01-01

    There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney. PMID:26392557

  17. Urine excretion strategy for stem cell-generated embryonic kidneys.

    PubMed

    Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi

    2015-10-20

    There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal's ureter to the cloacal-developed bladder, a technique we called the "stepwise peristaltic ureter" (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney. PMID:26392557

  18. Registration of Human Embryonic Stem Cell Lines: Korea, 2010

    PubMed Central

    Lee, Ji-Yoon; Lee, Dae-Yeon; Choi, Young-Sil; Lee, Kyoung-Jae; Kim, Yong-Ou

    2011-01-01

    In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr). PMID:24159464

  19. [Hybrid embryos as a source of embryonic stem cells].

    PubMed

    Beca I, Juan Pablo

    2007-11-01

    The HFEA (Human Fertilisations & Embryology Authority) recently accepted to perform research in hybrid embryos generated by transferring human somatic cell nucleus to cow enucleated oocytes, named cytoplasmatic hybrids. The aim is to obtain a source of embryonic stem cells without the use of human oocytes. The arguments for the approval are to avoid the risk of obtaining human oocytes and that these embryos will not be transferred to a female's womb for its development. Those who oppose the technique argue that it is a manipulation of the beginning of life and a disrespect to the dignity of human life because of the destruction of embryos. Nevertheless, the real nature of this new entity has not been established. Biologically it is an embryo with 99% of human genome and animal's cytoplasm, not generated from human gametes, it is not a new genome and it will be used only to cultivate stem cells. It does not seem possible to define its nature beyond any doubts. If it were considered as a human embryo it should be respected and protected as every human being. Once more, scientific progress opens new ethical and legal questions that we cannot answer in a definitive way. Researchers are exploring new roads to obtain pluripotential stem cells which should favor the development of innovative therapies. The main objection is the unavoidable destruction of human embryos, although in this case its origin and nature are not clear. PMID:18259646

  20. Enriched retinal ganglion cells derived from human embryonic stem cells.

    PubMed

    Gill, Katherine P; Hung, Sandy S C; Sharov, Alexei; Lo, Camden Y; Needham, Karina; Lidgerwood, Grace E; Jackson, Stacey; Crombie, Duncan E; Nayagam, Bryony A; Cook, Anthony L; Hewitt, Alex W; Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  1. 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

    ... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in...

  2. Osteoblastic differentiation of monkey embryonic stem cells in vitro.

    PubMed

    Yamashita, Akihiro; Takada, Tatsuyuki; Narita, Junko; Yamamoto, Gaku; Torii, Ryuzo

    2005-01-01

    Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification. PMID:16390259

  3. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    SciTech Connect

    Li, Wen-Zhong; Ai, Zhi-Ying; Wang, Zhi-Wei; Chen, Lin-Lin; Guo, Ze-Kun; Zhang, Yong

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  4. Derivation of human embryonic stem cell line Genea019.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  5. Histone variants as emerging regulators of embryonic stem cell identity

    PubMed Central

    Turinetto, Valentina; Giachino, Claudia

    2015-01-01

    Dynamic regulation of chromatin structure is an important mechanism for balancing the pluripotency and cell fate decision in embryonic stem cells (ESCs). Indeed ESCs are characterized by unusual chromatin packaging, and a wide variety of chromatin regulators have been implicated in control of pluripotency and differentiation. Genome-wide maps of epigenetic factors have revealed a unique epigenetic signature in pluripotent ESCs and have contributed models to explain their plasticity. In addition to the well known epigenetic regulation through DNA methylation, histone posttranslational modifications, chromatin remodeling, and non-coding RNA, histone variants are emerging as important regulators of ESC identity. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone variants in ESC pluripotency and ESC fate, focusing, in particular, on H1 variants, H2A variants H2A.X, H2A.Z and macroH2A and H3 variant H3.3. PMID:26114724

  6. Random mitotic activities across human embryonic stem cell colonies.

    SciTech Connect

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  7. Cardiac Repair by Embryonic Stem-Derived cells

    PubMed Central

    Rubart, M.

    2008-01-01

    Cell transplantation approaches offer the potential to promote regenerative growth of diseased hearts. It is well established that donor cardiomyocytes stably engraft into recipient hearts when injected directly into the myocardial wall. Moreover, the transplanted donor cardiomyocytes participate in a functional syncytium with the host myocardium. Thus, transplantation of donor cardiomyocytes resulted in at least partial restoration of lost muscle mass. It is also well established that embryonic stem (ES) cells differentiate into cells of ecto-, endo-, and mesodermal lineages when cultured under appropriate conditions in vitro. Robust cardiomyogenic differentiation was frequently observed in spontaneously differentiating ES cultures. Cellular, molecular and physiologic analyses indicated that ES-derived cells were bona fide cardiomyocytes, with in vitro characteristics typical for cells obtained from early stages of cardiac development. Thus, ES-derived cardiomyocytes constitute a viable source of donor cells for cell transplantation therapies. PMID:16370325

  8. Auxetic nuclei in embryonic stem cells exiting pluripotency

    NASA Astrophysics Data System (ADS)

    Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.

    2014-06-01

    Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

  9. Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells

    PubMed Central

    Sun, Yubing; Villa-Diaz, Luis G.; Lam, Raymond H. W.; Chen, Weiqiang; Krebsbach, Paul H.; Fu, Jianping

    2012-01-01

    Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function. PMID:22615930

  10. Characterization of embryonic stem cell model of polycystic ovary syndrome.

    PubMed

    Zhang, Yue; Zhang, Yun-Shan; Xue, Feng-Xia

    2016-05-01

    The aims of this study are to establish an embryonic stem (ES) cell model of polycystic ovary syndrome and to characterize this ES cell line. ES cells were isolated and cultured from 322 wasted fertilized embryos from polycystic ovary syndrome (PCOS) patients in vitro. They were also characterized by development and differential markers. ES cells from PCOS subject present normal development profile with ES-specific markers such as OCT-4 and SSEA-4. These ES cells can also differentiate into three germ layer derivatives and form teratomas in vivo. ES cells from PCOS patients pose development and differentiation potentials as you would expect of cells from non-PCOS patients; therefore, they can be used as a cellular model to study the pathology of PCOS. PMID:27112161

  11. Pioneer Factors in Embryonic Stem Cells and Differentiation

    PubMed Central

    Smale, Stephen T

    2010-01-01

    Most studies of tissue-specific and developmental stage-specific transcription have focused on the DNA motifs, transcription factors, or chromatin events required for the active transcription of a gene in cells in which the gene is expressed, or for its active or heritable silencing in non-expressing cells. However, accumulating evidence suggests that, in multicellular eukaryotes, enhancers or promoters for tissue-specific genes interact with pioneer transcription factors in embryonic stem cells and at other early stages of development, long before the genes are transcribed. These early interactions, which can lead to the presence of unmethylated CpG dinucleotides, histone modification signatures, and/or chromatin remodeling, may carry out different functions at different classes of genes. PMID:20638836

  12. Deliberating or dithering? Ireland and human embryonic stem cell research.

    PubMed

    Gough, Fionnuala

    2013-04-01

    Disagreement about matters of public policy concerned with moral issues is inevitable in pluralist democracies. One approach to the resolution of moral conflicts in society is the concept of deliberative democracy, which emphasises the process or procedure which ultimately allows a political decision to be reached. The Republic of Ireland effectively has no legislative framework regulating human embryonic stem cell research (hESC research). This article proposes that Irish policymakers establish a procedural framework, similar to that used in other European democracies, to allow the development of appropriate regulations pertaining to hESC research in Ireland. In particular the article will consider how a three-tier model of procedural regulation has been used to achieve certainty in the area ofhESC research in the United Kingdom and Germany and how this model might be applied to Ireland. PMID:23772461

  13. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  14. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    PubMed Central

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  15. Histone content increases in differentiating embryonic stem cells.

    PubMed

    Karnavas, Theodoros; Pintonello, Luisa; Agresti, Alessandra; Bianchi, Marco E

    2014-01-01

    Mouse Embryonic Stem Cells (ESCs) are pluripotent mammalian cells derived from the Inner Cell Mass (ICM) of mouse blastocysts, which give rise to all three embryonic germ layers both in vivo and in vitro. Mouse ESCs have a distinct epigenetic landscape and a more decondensed chromatin compared to differentiated cells. Numerous studies have shown that distinct histone modifications in ESCs serve as hallmarks of pluripotency. However, so far it is still unknown whether the total histone content (as opposed to histone modifications) remains the same in cells of different developmental stage and differentiation capacity. In this work we show that total histone content differs between pluripotent and differentiated cells. In vitro spontaneous differentiation from ESCs to Embryoid Bodies (EBs) and directed differentiation toward neuronal and endodermal cells entails an increase in histone content. Primary MEFs also contain more histones than ESCs. We suggest that the difference in histone content is an additional hallmark of pluripotency, in addition to and besides histone modifications. PMID:25221520

  16. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    PubMed

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material. PMID:25673530

  17. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    PubMed Central

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ‘cryopreserve’ their embryos then all the embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material. PMID:25673530

  18. Serum starvation improves transient transfection efficiency in differentiating embryonic stem cells.

    PubMed

    Wallenstein, Eric J; Barminko, Jeffrey; Schloss, Rene S; Yarmush, Martin L

    2010-01-01

    Control of genetic expression is a critical issue in the field of stem cell biology, where determining a cell fate or reprogramming adult somatic cells into pluripotent cells has become a common experimental practice. In turn, for these cells to have therapeutic clinical potential, techniques for controlling gene expression are needed that minimizes or eliminates the risk of oncogenesis and mutagenesis. Possible routes for achieving this outcome could come in the form of a transient nonviral gene delivery system. In this study, we improved the efficiency of transient gene delivery to differentiating murine embryonic stem (ES) cells via serum starvation for 3 days before transfection. The transient expression of a constitutively-controlled plasmid increased from ∼50% (replated control) to ∼83% when transfected after 3 days of serum starvation but decreased to ∼28% when transfected after 3 days in normal high serum-containing media. When probed with a liver-specific reporter, Cyp7A1, expression increased from ∼1.4% (replated control) to ∼3.7% when transfected after 3 days of serum starvation but decreased to ∼0.7% when transfected after 3 days in high serum-containing media. Cy3-tagged oligonucleotides were used to rapidly quantify DNA uptake and predict ultimate transfection efficiency. This study suggests that modifications in media serum levels before transfection can have a profound effect on improving nonviral gene delivery. PMID:20574993

  19. Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

    PubMed Central

    González, Sheyla; Ibáñez, Elena

    2010-01-01

    Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

  20. Brown adipogenesis of mouse embryonic stem cells in alginate microstrands

    NASA Astrophysics Data System (ADS)

    Unser, Andrea Mannarino

    The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.

  1. Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

    SciTech Connect

    Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

    2011-03-01

    The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 {mu}M) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

  2. Functional differentiation of stem cell-derived neurons from different murine backgrounds

    PubMed Central

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E.

    2014-01-01

    Murine stem cell-derived neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioral and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to 2 days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function. PMID:24600351

  3. Alginate Encapsulation Parameters Influence the Differentiation of Microencapsulated Embryonic Stem Cell Aggregates

    PubMed Central

    Wilson, Jenna L.; Najia, Mohamad Ali; Saeed, Rabbia; McDevitt, Todd C.

    2014-01-01

    Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the encapsulation material. Despite initial studies examining alginate microbeads as a platform for stem cell expansion and directed differentiation, the impact of alginate encapsulation parameters on stem cell phenotype has not been thoroughly investigated. Therefore, the objective of this study was to systematically examine the effects of varying alginate compositions on microencapsulated ESC expansion and phenotype. Pre-formed aggregates of murine ESCs were encapsulated in alginate microbeads composed of a high or low ratio of guluronic to mannuronic acid residues (High G and High M, respectively), with and without a poly-l-lysine (PLL) coating, thereby providing four distinct alginate bead compositions for analysis. Encapsulation in all alginate compositions was found to delay differentiation, with encapsulation within High G alginate yielding the least differentiated cell population. The addition of a PLL coating to the High G alginate prevented cell escape from beads for up to 14 days. Furthermore, encapsulation within High M alginate promoted differentiation toward a primitive endoderm phenotype. Taken together, the findings of this study suggest that distinct ESC expansion capacities and differentiation trajectories emerge depending on the alginate composition employed, indicating that encapsulation material physical properties can be used to control stem cell fate. PMID:24166004

  4. Mesenchymal morphogenesis of embryonic stem cells dynamically modulates the biophysical microtissue niche

    PubMed Central

    Kinney, Melissa A.; Saeed, Rabbia; McDevitt, Todd C.

    2014-01-01

    Stem cell fate and function are dynamically modulated by the interdependent relationships between biochemical and biophysical signals constituting the local 3D microenvironment. While approaches to recapitulate the stem cell niche have been explored for directing stem cell differentiation, a quantitative relationship between embryonic stem cell (ESC) morphogenesis and intrinsic biophysical cues within three-dimensional microtissues has not been established. In this study, we demonstrate that mesenchymal embryonic microtissues induced by BMP4 exhibited increased stiffness and viscosity accompanying differentiation, with cytoskeletal tension significantly contributing to multicellular stiffness. Perturbation of the cytoskeleton during ESC differentiation led to modulation of the biomechanical and gene expression profiles, with the resulting cell phenotype and biophysical properties being highly correlated by multivariate analyses. Together, this study elucidates the dynamics of biophysical and biochemical signatures within embryonic microenvironments, with broad implications for monitoring tissue dynamics, modeling pathophysiological and embryonic morphogenesis and directing stem cell patterning and differentiation. PMID:24598818

  5. Transcriptional regulatory mechanisms that govern embryonic stem cell fate.

    PubMed

    Das, Satyabrata; Levasseur, Dana

    2013-01-01

    Embryonic stem cells (ESCs) are defined by their simultaneous capacity for limitless self-renewal and the ability to specify cells borne of all germ layers. The regulation of ESC pluripotency is governed by a set of core transcription factors that regulate transcription by interfacing with nuclear proteins that include the RNA polymerase II core transcriptional machinery, histone modification enzymes, and chromatin remodeling protein complexes. The growing adoption of systems biological approaches used in stem cell biology over last few years has contributed significantly to our understanding of pluripotency. Multilayered approaches coupling transcriptome profiling and proteomics (Nanog-, Oct4-, and Sox2-centered protein interaction networks or "interactomes") with transcription factor chromatin occupancy and epigenetic footprint measurements have enabled a more comprehensive understanding of ESC pluripotency and self-renewal. Together with the genetic and biochemical characterization of promising pluripotency modifying proteins, these systems biological approaches will continue to clarify the molecular underpinnings of the ESC state. This will most certainly contribute to the improvement of current methodologies for the derivation of pluripotent cells from adult tissues. PMID:23756950

  6. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. Cecilia T.; Wolf, Don; Mitalipov, Shoukhrat

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

  7. Trends in the human embryonic stem cell patent field.

    PubMed

    Karlsson, Ulrika; Hyllner, Johan; Runeberg, Kristina

    2007-01-01

    The successful derivation of human embryonic stem (hES) cell lines in late 1990s marks the birth of a new era in biomedical research. In the USA, this landmark invention is protected by granted composition-of-matter patents. In addition to these patents, several others have been granted on further development of hES cell research, such as on differentiated cell types and in vitro and in vivo use aspects. In Europe, there is presently no consensus pertaining to the patentability of hES cells, and all patent applications pending at the European patent office are therefore awaiting a principal decision by the Enlarged Board of Appeal. The authors argue that it will be of importance to the stem cell industry that patents are granted on inventions downstream in the value chain, e.g on specialised cell types derived from hES cells and different drug discovery applications. Patents and patent applications on such inventions for the three germ layers ectoderm/neuro, endoderm/hepato and mesoderm/cardio have been examined. The number of patents increased in the period 2001 to 2006 for all three lineages with ectoderm/neuro as the most patent intensive field. There where 9-13 times more US patent applications filed related to the three lineages compared to in Europe. PMID:19076035

  8. Tracing Synaptic Connectivity onto Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Garcia, Isabella; Huang, Longwen; Ung, Kevin; Arenkiel, Benjamin R.

    2012-01-01

    Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest. PMID:22996827

  9. Light-activated RNA interference in human embryonic stem cells.

    PubMed

    Huang, Xiao; Hu, Qirui; Braun, Gary B; Pallaoro, Alessia; Morales, Demosthenes P; Zasadzinski, Joseph; Clegg, Dennis O; Reich, Norbert O

    2015-09-01

    We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine. PMID:26086448

  10. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    PubMed

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  11. Animal embryonic stem (ES) cells: self-renewal, pluripotency, transgenesis and nuclear transfer.

    PubMed

    Saito, Shigeo; Liu, Bingbing; Yokoyama, Kazunari

    2004-09-01

    Mouse embryonic stem (ES) cells can be maintained indefinitely in the presence of leukemia inhibitory factor (LIF) and they express markers of self-renewal and pluripotency, which include the transcription factor Oct 4, STAT-3, stage-specific embryonic antigen (SSEA)-1, and alkaline phosphatase (AP). Upon removal of LIF, from the culture medium they cease to express markers such as Oct 4, rapidly losing the capacity for self-renewal and differentiating into a variety of cell types. Gene targeting is feasible in murine ES cells because these cells can be maintained in an undifferentiated state long enough to allow selection of properly targeted cell colonies with a high frequency of homologous recombination. Furthermore, blastocysts cloned from cultured murine ES cells develop to term at an efficiency (10-30%) that is three to ten times higher than blastocysts cloned from the nuclei of differentiated somatic cells. It seems likely that ES cells require less extensive reprogramming than do somatic cells, perhaps because in ES cells, many genes that are essential for early development are already active and thus do not require reactivation. Recently, we succeeded in isolating immortalized equine and bovine ES cells with a normal karyotype, that exhibit features similar to those of murine ES cells and express Oct 4, STAT-3, SSEA-1 and AP. We further confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells, as a source of nuclei for nuclear transfer (NT) and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with differentiated somatic cells. Moreover, bovine ES cells that expressed enhanced green fluorescent protein (EGFP) were incorporated into both the inner cell mass (ICM) and the trophectdermal cells of developing blastocysts. These findings should facilitate

  12. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo.

    PubMed Central

    Guild, B C; Finer, M H; Housman, D E; Mulligan, R C

    1988-01-01

    A series of retrovirus vectors were constructed in which cellular promoter elements derived from the chicken beta-actin and human histone H4 genes were introduced within the proviral transcriptional unit of Moloney murine leukemia virus in order to promote expression of inserted sequences. Each of these vectors gave rise to high titer of virus capable of transferring the expected proviral structure to cells. Inclusion of normal 5' splice sequences or a portion of viral gag sequences in these constructions resulted in significant increases in virus titer. Each construction was transcriptionally active in NIH 3T3 cells and in undifferentiated F9 cells. One of the vectors, HSG-neo, which contained the human histone H4 promoter, was shown to be transcriptionally active in hematopoietic cells derived from long-term reconstituted bone marrow transplant recipients engrafted with transduced stem cells. These vectors should be of general use for obtaining efficient gene expression in embryonal and hematopoietic cells. Images PMID:3418785

  13. 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...

  14. Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System

    PubMed Central

    Zandi, Mohammad; Muzaffar, Musharifa; Shah, Syed Mohmad; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Manik, Radheysham; Chauhan, Manmohan Singh

    2015-01-01

    Objective In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. Materials and Methods In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. Results The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. Conclusion We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells. PMID:26199905

  15. Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

    SciTech Connect

    Weston, W.M.; Greene, R.M. )

    1991-01-01

    Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that,in contrast to what has been observed in other systems, ethanol does not affect the production or activity of G{alpha}s in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

  16. 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

  17. Selection of Stable Reference Genes for Quantitative RT-PCR Comparisons of Mouse Embryonic and Extra-Embryonic Stem Cells

    PubMed Central

    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 m

  18. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    SciTech Connect

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  19. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  20. Cross-species stromal signaling programs human embryonic stem cell differentiation.

    PubMed

    Taylor, Renea A; Risbridger, Gail P

    2014-01-01

    The generation of human embryonic stem cell lines in the late 1990s awakened new prospects in the research arena. The opportunity to define and direct the differentiation of the most primitive stem cells in the laboratory, and their potential application to regenerative medicine, invigorated the developmental biology field. Whilst initial studies used in vitro differentiation techniques, the application of in vivo models greatly enhanced the success of directing the differentiation of human embryonic stem cells. Controlling cell fate decisions in embryonic cells requires thorough knowledge of the intrinsic and extrinsic factors that direct differentiation and the complex cell-cell interactions that exist within different tissues. Thus, tissue-specific approaches were applied to generate various differentiated cell types. In this review, cross-species tissue recombination will be discussed as a successful approach to direct embryonic stem cell differentiation. PMID:24530222

  1. 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

    ... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be... within 60 days of the date of this publication. Proposed Collection: Request for Human Embryonic...

  2. Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

    PubMed Central

    Raof, Nurazhani Abdul; Mooney, Bridget M.; Xie, Yubing

    2011-01-01

    Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES) cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed. PMID:22174624

  3. A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

    PubMed

    Renz, Peter F; Beyer, Tobias A

    2016-01-01

    Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA). PMID:25724912

  4. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells

    PubMed Central

    Mantsoki, Anna; Devailly, Guillaume; Joshi, Anagha

    2015-01-01

    In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a ‘poised’ state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. PMID:26582124

  5. Reproducible culture and differentiation of mouse embryonic stem cells using an automated microwell platform☆

    PubMed Central

    Hussain, Waqar; Moens, Nathalie; Veraitch, Farlan S.; Hernandez, Diana; Mason, Chris; Lye, Gary J.

    2013-01-01

    The use of embryonic stem cells (ESCs) and their progeny in high throughput drug discovery and regenerative medicine will require production at scale of well characterized cells at an appropriate level of purity. The adoption of automated bioprocessing techniques offers the possibility to overcome the lack of consistency and high failure rates seen with current manual protocols. To build the case for increased use of automation this work addresses the key question: “can an automated system match the quality of a highly skilled and experienced person working manually?” To answer this we first describe an integrated automation platform designed for the ‘hands-free’ culture and differentiation of ESCs in microwell formats. Next we outline a framework for the systematic investigation and optimization of key bioprocess variables for the rapid establishment of validatable Standard Operating Procedures (SOPs). Finally the experimental comparison between manual and automated bioprocessing is exemplified by expansion of the murine Oct-4-GiP ESC line over eight sequential passages with their subsequent directed differentiation into neural precursors. Our results show that ESCs can be effectively maintained and differentiated in a highly reproducible manner by the automated system described. Statistical analysis of the results for cell growth over single and multiple passages shows up to a 3-fold improvement in the consistency of cell growth kinetics with automated passaging. The quality of the cells produced was evaluated using a panel of biological markers including cell growth rate and viability, nutrient and metabolite profiles, changes in gene expression and immunocytochemistry. Automated processing of the ESCs had no measurable negative effect on either their pluripotency or their ability to differentiate into the three embryonic germ layers. Equally important is that over a 6-month period of culture without antibiotics in the medium, we have not had any cases

  6. Retroviral Transcriptional Regulation and Embryonic Stem Cells: War and Peace

    PubMed Central

    Schlesinger, Sharon

    2014-01-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. PMID:25547290

  7. Nucleosome positioning changes during human embryonic stem cell differentiation.

    PubMed

    Zhang, Wenjuan; Li, Yaping; Kulik, Michael; Tiedemann, Rochelle L; Robertson, Keith D; Dalton, Stephen; Zhao, Shaying

    2016-06-01

    Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content. PMID:27088311

  8. Common marmoset embryonic stem cell can differentiate into cardiomyocytes

    SciTech Connect

    Chen Hao; Hattori, Fumiyuki; Murata, Mitsushige; Li Weizhen; Yuasa, Shinsuke; Onizuka, Takeshi; Shimoji, Kenichiro; Ohno, Yohei; Sasaki, Erika; Kimura, Kensuke; Hakuno, Daihiko

    2008-05-09

    Common marmoset monkeys have recently attracted much attention as a primate research model, and are preferred to rhesus and cynomolgus monkeys due to their small bodies, easy handling and efficient breeding. We recently reported the establishment of common marmoset embryonic stem cell (CMESC) lines that could differentiate into three germ layers. Here, we report that our CMESC can also differentiate into cardiomyocytes and investigated their characteristics. After induction, FOG-2 was expressed, followed by GATA4 and Tbx20, then Nkx2.5 and Tbx5. Spontaneous beating could be detected at days 12-15. Immunofluorescent staining and ultrastructural analyses revealed that they possessed characteristics typical of functional cardiomyocytes. They showed sinus node-like action potentials, and the beating rate was augmented by isoproterenol stimulation. The BrdU incorporation assay revealed that CMESC-derived cardiomyocytes retained a high proliferative potential for up to 24 weeks. We believe that CMESC-derived cardiomyocytes will advance preclinical studies in cardiovascular regenerative medicine.

  9. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    PubMed

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve. PMID:26473610

  10. Generative models: Human embryonic stem cells and multiple modeling relations.

    PubMed

    Fagan, Melinda Bonnie

    2016-04-01

    Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. PMID:27083092

  11. Kinetics of drug selection systems in mouse embryonic stem cells

    PubMed Central

    2013-01-01

    Background Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters. Results We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order. Conclusions The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy. PMID:23919313

  12. Potential applications of keratinocytes derived from human embryonic stem cells.

    PubMed

    Movahednia, Mohammad M; Kidwai, Fahad K; Jokhun, Doorgesh S; Squier, Christopher A; Toh, Wei Seong; Cao, Tong

    2016-01-01

    Although skin grafting is one of the most advanced cell therapy technique, wide application of skin substitutes is hampered by the difficulty in securing sufficient amount of epidermal substitute. Additionally, in understanding the progression of skin aging and disease, and in screening the cosmetic and pharmaceutical products, there is lack of a satisfactory human skin-specific in vitro model. Recently, human embryonic stem cells (hESCs) have been proposed as an unlimited and reliable cell source to obtain almost all cell types present in the human body. This review focuses on the potential off-the-shelf use of hESC-derived keratinocytes for future clinical applications as well as a powerful in vitro skin model to study skin function and integrity, host-pathogen interactions and disease pathogenesis. Furthermore, we discuss the industrial applications of hESC-derived keratinized multi-layer epithelium which provides a human-like test platform for understanding disease pathogenesis, evaluation of new therapeutic modalities and assessment of the safety and efficacy of skin cosmetics and therapeutics. Overall, we conclude that the hESC-derived keratinocytes have great potential for clinical, research and industrial applications. PMID:26663861

  13. Stochastic Cell Fate Progression in Embryonic Stem Cells

    NASA Astrophysics Data System (ADS)

    Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad

    2013-03-01

    Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund

  14. Signaling Control of Differentiation of Embryonic Stem Cells toward Mesendoderm.

    PubMed

    Wang, Lu; Chen, Ye-Guang

    2016-04-10

    Mesendoderm (ME) refers to the primitive streak in mammalian embryos, which has the ability to further differentiate into mesoderm and endoderm. A better understanding on the regulatory networks of ME differentiation of embryonic stem (ES) cells would provide important insights on early embryo patterning and a possible guidance for ES applications in regenerative medicine. Studies on developmental biology and embryology have offered a great deal of knowledge about key signaling pathways involved in primitive streak formation. Recently, various chemically defined recipes have been formulated to induce differentiation of ES cells toward ME in vitro, which greatly facilitate the elucidation of the regulatory mechanisms of different signals involved in ME specification. Among the extrinsic signals, transforming growth factor-β/Activin signaling and Wnt signaling have been shown to be the most critical ones. On another side, intrinsic epigenetic regulation has been indicated to be important in ME determination. In this review, we summarize the current understanding on the extrinsic and intrinsic regulations of ES cells-to-ME differentiation and the crosstalk among them, aiming to get a general overview on ME specification and primitive streak formation. PMID:26119455

  15. Multicolor karyotype analyses of mouse embryonic stem cells.

    PubMed

    Guo, Jianli; Jauch, Anna; Heidi, Holtgreve-Grez; Schoell, Brigitte; Erz, Dorothee; Schrank, Martina; Janssen, Johannes W G

    2005-01-01

    The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources. PMID:16409114

  16. Female Sex Bias in Human Embryonic Stem Cell Lines

    PubMed Central

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat

    2012-01-01

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  17. Female sex bias in human embryonic stem cell lines.

    PubMed

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat; Eiges, Rachel

    2012-02-10

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  18. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

    PubMed Central

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve. PMID:26473610

  19. Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells.

    PubMed

    Thimm, Terra N; Squirrell, Jayne M; Liu, Yuming; Eliceiri, Kevin W; Ogle, Brenda M

    2015-10-01

    Components of the extracellular matrix (ECM) have recently been shown to influence stem cell specification. However, it has been challenging to assess the spatial and temporal dynamics of stem cell-ECM interactions because most methodologies utilized to date require sample destruction or fixation. We examined the efficacy of utilizing the endogenous optical signals of two important ECM proteins, elastin (Eln), through autofluorescence, and type I collagen (ColI), through second harmonic generation (SHG), during mouse embryonic stem cell differentiation. After finding favorable overlap between antibody labeling and the endogenous fluorescent signal of Eln, we used this endogenous signal to map temporal changes in Eln and ColI during murine embryoid body differentiation and found that Eln increases until day 9 and then decreases slightly by day 12, while Col1 steadily increases over the 12-day period. Furthermore, we combined endogenous fluorescence imaging and SHG with antibody labeling of cardiomyocytes to examine the spatial relationship between Eln and ColI accumulation and cardiomyocyte differentiation. Eln was ubiquitously present, with enrichment in regions with cardiomyocyte differentiation, while there was an inverse correlation between ColI and cardiomyocyte differentiation. This work provides an important first step for utilizing endogenous optical signals, which can be visualized in living cells, to understand the relationship between the ECM and cardiomyocyte development and sets the stage for future studies of stem cell-ECM interactions and dynamics relevant to stem cells as well as other cell and tissue types. PMID:25923353

  20. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective

    PubMed Central

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaitre, Jean-Marc; De Vos, John

    2013-01-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address. PMID:23317057

  1. Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression

    PubMed Central

    Savarese, Fabio; Dávila, Amparo; Nechanitzky, Robert; De La Rosa-Velazquez, Inti; Pereira, Carlos F.; Engelke, Rudolf; Takahashi, Keiko; Jenuwein, Thomas; Kohwi-Shigematsu, Terumi; Fisher, Amanda G.; Grosschedl, Rudolf

    2009-01-01

    Satb1 and the closely related Satb2 proteins regulate gene expression and higher-order chromatin structure of multigene clusters in vivo. In examining the role of Satb proteins in murine embryonic stem (ES) cells, we find that Satb1−/− cells display an impaired differentiation potential and augmented expression of the pluripotency determinants Nanog, Klf4, and Tbx3. Metastable states of self-renewal and differentiation competence have been attributed to heterogeneity of ES cells in the expression of Nanog. Satb1−/− cultures have a higher proportion of Nanoghigh cells, and an increased potential to reprogram human B lymphocytes in cell fusion experiments. Moreover, Satb1-deficient ES cells show an increased expression of Satb2, and we find that forced Satb2 expression in wild-type ES cells antagonizes differentiation-associated silencing of Nanog and enhances the induction of NANOG in cell fusions with human B lymphocytes. An antagonistic function of Satb1 and Satb2 is also supported by the almost normal differentiation potential of Satb1−/−Satb2−/− ES cells. Taken together with the finding that both Satb1 and Satb2 bind the Nanog locus in vivo, our data suggest that the balance of Satb1 and Satb2 contributes to the plasticity of Nanog expression and ES cell pluripotency. PMID:19933152

  2. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

  3. [Human embryonic stem cell research in Germany. The scientific reviewing of applications for the import and use of human embryonic stem cells].

    PubMed

    Kiechle, M

    2008-09-01

    From July 2002 to May 2008, 36 applications for the import and use of human embryonic stem cells (hES) were reviewed by the German Central Ethics Committee for Stem Cell Research (ZES). A flood of applications anticipated by opponents to human embryonic stem cell research has not occurred since the enactment of the German Stem Cell Act in 2002. On the contrary, German hES cell research is below international average in terms of project numbers. The current restrictions for using hES cells in Germany might be causative for the opinion that this type of research is not considered to be very promising. This could hold true especially for research aiming at clinical applications. Consequently, potential research goals of premium importance, especially those of potential clinical relevance, could be seriously jeopardized. PMID:18769900

  4. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    PubMed

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-01-01

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544

  5. Induction of Cardiomyogenesis in Human Embryonic Stem Cells by Human Embryonic Stem Cell-Derived Definitive Endoderm

    PubMed Central

    Van Orman, Jordan R.; Si-Tayeb, Karim; Duncan, Stephen A.

    2012-01-01

    We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails. PMID:21627569

  6. A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

    PubMed

    Chandran, Yogeswari; Kang, Nam-Young; Park, Sung-Jin; Alamudi, Samira Husen; Kim, Jun-Young; Sahu, Srikanta; Su, Dongdong; Lee, Jungyeol; Vendrell, Marc; Chang, Young-Tae

    2015-11-01

    Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities. PMID:26115574

  7. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  8. Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

    2016-01-01

    The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623

  9. Evaluation of the embryotoxic potency of compounds in a newly revised high throughput embryonic stem cell test.

    PubMed

    Peters, Annelieke K; Steemans, Margino; Hansen, Erik; Mesens, Natalie; Verheyen, Geert R; Vanparys, Philippe

    2008-10-01

    The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals. PMID:18593728

  10. Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.

    PubMed

    Bonandrini, Barbara; Figliuzzi, Marina; Papadimou, Evangelia; Morigi, Marina; Perico, Norberto; Casiraghi, Federica; Dipl, Chemistry; Sangalli, Fabio; Conti, Sara; Benigni, Ariela; Remuzzi, Andrea; Remuzzi, Giuseppe

    2014-05-01

    For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Micro-computerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and

  11. 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. PMID:26900863

  12. Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein.

    PubMed

    Carlone, Diana L; Lee, Jeong-Heon; Young, Suzanne R L; Dobrota, Erika; Butler, Jill Sergesketter; Ruiz, Joseph; Skalnik, David G

    2005-06-01

    Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP(-)(/)(-) cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP(-)(/)(-) embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP(-)(/)(-) cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP(-)(/)(-) cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP(-)(/)(-) phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development. PMID:15923607

  13. Generation and application of mammalian haploid embryonic stem cells.

    PubMed

    Bai, M; Wu, Y; Li, J

    2016-09-01

    Haploid cells contain one set of chromosomes and are amenable for genetic analyses. In mammals, haploidy exists only in gametes. An intriguing question is whether haploid cells can be derived from gametes. Recently, by application of haploid cell enrichment using fluorescence-activated cell sorting, stable haploid embryonic stem cells (haESCs) have been successfully derived from oocyte-derived parthenogenetic and sperm-derived androgenetic embryos from several species. Whilst both parthenogenetic and androgenetic (AG)-haESCs enable whole-genome genetic screening at the cellular level, such as screening of drug resistance or disease-related genes, AG-haESCs, after intracytoplasmic injection into oocytes, can also be used to produce alive semi-cloned mice. Nevertheless, one major drawback associated with wild-type AG-haESCs is the very low birth rate of healthy semi-cloned mice. Of interest, after inhibiting the expression of two paternally imprinted genes (H19 and Gtl2) in AG-haESCs by removal of their differentially DNA methylated regions, double-knockout AG-haESCs can efficiently and stably support the generation of healthy semi-cloned pups. Importantly, double-knockout AG-haESCs are feasible for multiple genetic manipulations, followed by efficient generation of semi-cloned mice carrying multiple genetic traits; thus they could be used to validate candidate loci that have been identified in genome-wide association studies of multigenic diseases by generation of mouse models carrying multiple alterations. Of note, by combining a CRISPR-Cas9 library and double-knockout AG-haESCs, semi-cloned mice carrying different mutant genes can be efficiently generated in one step, enabling functional mutagenic screening in mice. HaESCs, therefore, provide a powerful tool for genetic analyses in mammals at both the cellular and organismal levels. PMID:27138065

  14. Therapeutic approaches for treating hemophilia A using embryonic stem cells.

    PubMed

    Kasuda, Shogo; Tatsumi, Kohei; Sakurai, Yoshihiko; Shima, Midori; Hatake, Katsuhiko

    2016-06-01

    Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future. PMID:27131224

  15. The Proper Criteria for Identification and Sorting of Very Small Embryonic-Like Stem Cells, and Some Nomenclature Issues

    PubMed Central

    Suszynska, Malwina; Zuba-Surma, Ewa K.; Maj, Magdalena; Mierzejewska, Kasia; Ratajczak, Janina; Kucia, Magda

    2014-01-01

    Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic. PMID:24299281

  16. The proper criteria for identification and sorting of very small embryonic-like stem cells, and some nomenclature issues.

    PubMed

    Suszynska, Malwina; Zuba-Surma, Ewa K; Maj, Magdalena; Mierzejewska, Kasia; Ratajczak, Janina; Kucia, Magda; Ratajczak, Mariusz Z

    2014-04-01

    Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic. PMID:24299281

  17. Effective Mobilization of Very Small Embryonic-Like Stem Cells and Hematopoietic Stem/Progenitor Cells but Not Endothelial Progenitor Cells by Follicle-Stimulating Hormone Therapy

    PubMed Central

    Zbucka-Kretowska, Monika; Eljaszewicz, Andrzej; Lipinska, Danuta; Grubczak, Kamil; Rusak, Malgorzata; Mrugacz, Grzegorz; Dabrowska, Milena; Ratajczak, Mariusz Z.; Moniuszko, Marcin

    2016-01-01

    Recently, murine hematopoietic progenitor stem cells (HSCs) and very small embryonic-like stem cells (VSELs) were demonstrated to express receptors for sex hormones including follicle-stimulating hormone (FSH). This raised the question of whether FSH therapy at clinically applied doses can mobilize stem/progenitor cells in humans. Here we assessed frequencies of VSELs (referred to as Lin−CD235a−CD45−CD133+ cells), HSPCs (referred to as Lin−CD235a−CD45+CD133+ cells), and endothelial progenitor cells (EPCs, identified as CD34+CD144+, CD34+CD133+, and CD34+CD309+CD133+ cells) in fifteen female patients subjected to the FSH therapy. We demonstrated that FSH therapy resulted in statistically significant enhancement in peripheral blood (PB) number of both VSELs and HSPCs. In contrast, the pattern of responses of EPCs delineated by different cell phenotypes was not uniform and we did not observe any significant changes in EPC numbers following hormone therapy. Our data indicate that FSH therapy mobilizes VSELs and HSPCs into peripheral blood that on one hand supports their developmental origin from germ lineage, and on the other hand FSH can become a promising candidate tool for mobilizing HSCs and stem cells with VSEL phenotype in clinical settings. PMID:26635885

  18. Effective Mobilization of Very Small Embryonic-Like Stem Cells and Hematopoietic Stem/Progenitor Cells but Not Endothelial Progenitor Cells by Follicle-Stimulating Hormone Therapy.

    PubMed

    Zbucka-Kretowska, Monika; Eljaszewicz, Andrzej; Lipinska, Danuta; Grubczak, Kamil; Rusak, Malgorzata; Mrugacz, Grzegorz; Dabrowska, Milena; Ratajczak, Mariusz Z; Moniuszko, Marcin

    2016-01-01

    Recently, murine hematopoietic progenitor stem cells (HSCs) and very small embryonic-like stem cells (VSELs) were demonstrated to express receptors for sex hormones including follicle-stimulating hormone (FSH). This raised the question of whether FSH therapy at clinically applied doses can mobilize stem/progenitor cells in humans. Here we assessed frequencies of VSELs (referred to as Lin(-)CD235a(-)CD45(-)CD133(+) cells), HSPCs (referred to as Lin(-)CD235a(-)CD45(+)CD133(+) cells), and endothelial progenitor cells (EPCs, identified as CD34(+)CD144(+), CD34(+)CD133(+), and CD34(+)CD309(+)CD133(+) cells) in fifteen female patients subjected to the FSH therapy. We demonstrated that FSH therapy resulted in statistically significant enhancement in peripheral blood (PB) number of both VSELs and HSPCs. In contrast, the pattern of responses of EPCs delineated by different cell phenotypes was not uniform and we did not observe any significant changes in EPC numbers following hormone therapy. Our data indicate that FSH therapy mobilizes VSELs and HSPCs into peripheral blood that on one hand supports their developmental origin from germ lineage, and on the other hand FSH can become a promising candidate tool for mobilizing HSCs and stem cells with VSEL phenotype in clinical settings. PMID:26635885

  19. Differentiation of embryonic and adult stem cells into insulin producing cells.

    PubMed

    Zulewski, H

    2008-03-01

    Replacement of insulin producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is successful in experienced centers. The wider application of this therapy, however, is limited by the lack of donor organs. Insulin producing cells generated from stem cells represent an attractive alternative. Stem cells with the potential to differentiate into insulin producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns but research with human ESC may help us to decipher important steps in the differentiation process in vitro since almost all information available on pancreas development are based on animal studies. The present review summarizes the current knowledge on the development of insulin producing cells from embryonic and adult stem cells with special emphasis on pancreatic, hepatic and human mesenchymal stem cells. PMID:18427390

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

    SciTech Connect

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  1. An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

    PubMed Central

    Fromm, George; Cadiz-Rivera, Brenda; de Vries, Christina; Getman, Michael; McGrath, Kathleen E.; Kingsley, Paul D.; Fields, Jennifer; Fiering, Steven

    2011-01-01

    In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure. PMID:21321362

  2. Deletion of Serpina1a, a murine α1-antitrypsin ortholog, results in embryonic lethality.

    PubMed

    Wang, Dongmei; Wang, Weimin; Dawkins, Paul; Paterson, Trevor; Kalsheker, Noor; Sallenave, Jean-Michel; Houghton, A McGarry

    2011-06-01

    Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States Approximately 1% to 2% of COPD patients suffer from α(1)-antitrypsin (A1AT) deficiency, the major inheritable predisposition to COPD/emphysema. To further study the role of A1AT deficiency in the pathogenesis of COPD/emphysema, the authors attempted to generate null-mutant mice for Serpina1a, 1 of 2 A1AT orthologs in mice. Here the authors show that targeted deletion of Serpina1a results in embryonic lethality prior to 8.5 days post conception (dpc). The results are surprising given that A1AT-null humans exist and therefore do not require this gene product for normal development. The Serpina1 gene cluster is substantially different between mouse and man. Through gene duplication, mice have 3 to 5 (depending on the strain) highly homologous proteinase inhibiting (Pi) genes, 2 of which inhibit neutrophil elastase. Despite the abundance of Pi genes in mice, Serpina1a serves a critical, nonredundant function during early mouse development. A1AT-deficient mice have been highly sought after to study emphysema, cancer, and liver disease, and as a model to perfect gene replacement therapy. These results highlight important differences between human and murine serpins and point to the difficulty inherent to using gene-targeted mice to study this common human genetic disease. PMID:21574874

  3. Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

    PubMed Central

    Xiong, Jianhua; Todorova, Dilyana; Su, Ning-Yuan; Kim, Jinchul; Lee, Pei-Jen; Shen, Zhouxin; Briggs, Steven P.

    2015-01-01

    Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)–dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs. PMID:25733712

  4. Dopamine Receptors in Human Embryonic Stem Cell Neurodifferentiation

    PubMed Central

    Belinsky, Glenn S.; Sirois, Carissa L.; Rich, Matthew T.; Short, Shaina M.; Moore, Anna R.; Gilbert, Sarah E.

    2013-01-01

    We tested whether dopaminergic drugs can improve the protocol for in vitro differentiation of H9 human embryonic stem cells (hESCs) into dopaminergic neurons. The expression of 5 dopamine (DA) receptor subtypes (mRNA and protein) was analyzed at each protocol stage (1, undifferentiated hESCs; 2, embryoid bodies [EBs]; 3, neuroepithelial rosettes; 4, expanding neuroepithelium; and 5, differentiating neurons) and compared to human fetal brain (gestational week 17–19). D2-like DA receptors (D2, D3, and D4) predominate over the D1-like receptors (D1 and D5) during derivation of neurons from hESCs. D1 was the receptor subtype with the lowest representation in each protocol stage (Stages 1–5). D1/D5-agonist SKF38393 and D2/D3/D4-agonist quinpirole (either alone or combined) evoked Ca2+ responses, indicating functional receptors in hESCs. To identify when receptor activation causes a striking effect on hESC neurodifferentiation, and what ligands and endpoints are most interesting, we varied the timing, duration, and drug in the culture media. Dopaminergic agonists or antagonists were administered either early (Stages 1–3) or late (Stages 4–5). Early DA exposure resulted in more neuroepithelial colonies, more neuronal clusters, and more TH+ clusters. The D1/D5 antagonist SKF83566 had a strong effect on EB morphology and the expression of midbrain markers. Late exposure to DA resulted in a modest increase in TH+ neuron clusters (∼75%). The increase caused by DA did not occur in the presence of dibutyryl cAMP (dbcAMP), suggesting that DA acts through the cAMP pathway. However, a D2-antagonist (L741) decreased TH+ cluster counts. Electrophysiological parameters of the postmitotic neurons were not significantly affected by late DA treatment (Stages 4–5). The mRNA of mature neurons (VGLUT1 and GAD1) and the midbrain markers (GIRK2, LMX1A, and MSX1) were lower in hESCs treated by DA or a D2-antagonist. When hESCs were neurodifferentiated on PA6 stromal cells, DA also

  5. Recent developments in StemBase: a tool to study gene expression in human and murine stem cells

    PubMed Central

    Sandie, Reatha; Palidwor, Gareth A; Huska, Matthew R; Porter, Christopher J; Krzyzanowski, Paul M; Muro, Enrique M; Perez-Iratxeta, Carolina; Andrade-Navarro, Miguel A

    2009-01-01

    Background Currently one of the largest online repositories for human and mouse stem cell gene expression data, StemBase was first designed as a simple web-interface to DNA microarray data generated by the Canadian Stem Cell Network to facilitate the discovery of gene functions relevant to stem cell control and differentiation. Findings Since its creation, StemBase has grown in both size and scope into a system with analysis tools that examine either the whole database at once, or slices of data, based on tissue type, cell type or gene of interest. As of September 1, 2008, StemBase contains gene expression data (microarray and Serial Analysis of Gene Expression) from 210 stem cell samples in 60 different experiments. Conclusion StemBase can be used to study gene expression in human and murine stem cells and is available at . PMID:19284540

  6. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

    PubMed Central

    2015-01-01

    Background Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. Results We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. Conclusions We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature. PMID:26694224

  7. Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells

    PubMed Central

    Dingwall, Steve; Lee, Jung Bok; Guezguez, Borhane; Fiebig, Aline; McNicol, Jamie; Boreham, Douglas; Collins, Tony J.; Bhatia, Mick

    2015-01-01

    Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs. PMID:26082437

  8. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    PubMed

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471. PMID:27275414

  9. Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

    PubMed

    Yin, Nuoya; Yao, Xinglei; Qin, Zhanfen; Wang, Yuan-Liang; Faiola, Francesco

    2015-10-01

    The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells. PMID:26456621

  10. Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro

    SciTech Connect

    Zhang Enming; Li Xiaolong; Zhang Shufang; Chen Liangqiang; Zheng Xiaoxiang . E-mail: zxx@mail.bme.zju.edu.cn

    2005-08-12

    Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation.

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

    PubMed Central

    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. PMID:26091287

  12. 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

  13. CD13 and ROR2 Permit Isolation of Highly Enriched Cardiac Mesoderm from Differentiating Human Embryonic Stem Cells

    PubMed Central

    Skelton, Rhys J.P.; Brady, Bevin; Khoja, Suhail; Sahoo, Debashis; Engel, James; Arasaratnam, Deevina; Saleh, Kholoud K.; Abilez, Oscar J.; Zhao, Peng; Stanley, Edouard G.; Elefanty, Andrew G.; Kwon, Murray; Elliott, David A.; Ardehali, Reza

    2016-01-01

    Summary The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications. PMID:26771355

  14. Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells

    PubMed Central

    Cattoglio, Claudia; Zhang, Elisa T.; Grubisic, Ivan; Chiba, Kunitoshi; Fong, Yick W.; Tjian, Robert

    2015-01-01

    The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex. PMID:25901318

  15. Lineage Conversion of Murine Extraembryonic Trophoblast Stem Cells to Pluripotent Stem Cells▿†

    PubMed Central

    Kuckenberg, Peter; Peitz, Michael; Kubaczka, Caroline; Becker, Astrid; Egert, Angela; Wardelmann, Eva; Zimmer, Andreas; Brüstle, Oliver; Schorle, Hubert

    2011-01-01

    In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals. PMID:21300784

  16. Control of leucocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue.

    PubMed

    Hannig, Madeleine; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2010-01-01

    Embryonic stem (ES) cells spontaneously differentiate capillary-like structures as well as leucocytes such as monocytes/macrophages, neutrophils, natural killer (NK) cells and cytototoxic T lymphocytes. The interplay between vasculogenesis and leucocyte differentiation as well as the population of tumour tissues with ES cell-derived leucocytes and endothelial cells is, however, not sufficiently specified. In the present study, gene expression of the cell surface markers CD68 and CD14 (expressed on monocytes and macrophages), Mac-1 (CD11b) (expressed on granulocytes, monocytes and NK cells) and CD16 (expressed on neutrophils) was investigated in murine CGR8 ES cells in relation to the endothelial cell markers CD31 and vascular endothelial (VE)-cadherin. Expression of leucocyte markers increased from day 7-8 of cell culture on. Furthermore, addition of macrophage colony-stimulating factor to the cell culture medium resulted in a threefold increase in the number of CD68(+) monocytes/macrophages. Treatment of embryoid bodies with lipopolysaccharide (LPS) up-regulated CD14 thus suggesting functionality of the CD14 LPS receptor. Differentiation of vascular structures positive for CD31 and VE-cadherin preceded leucocyte differentiation by 2 days (i.e. from day 5-6 on) suggesting that vasculogenesis may be a determinant of leucocyte differentiation. Consequently the Flk-1 antagonist SU5416 which inhibits vasculogenesis of ES cells significantly blunted leucocyte differentiation. Confrontation culture of embryoid bodies with multicellular breast tumour spheroids initiated significant increase of leucocyte cell numbers and invasion of leucocytes into the tumour tissue. In summary our data demonstrate that during ES cell differentiation vasculogenesis precedes leucocyte differentiation, and point towards the direction that leucocyte cell invasion into tumour tissue may initiate the pro-inflammatory microenvironment necessary for tumour vascularization. PMID:20414974

  17. Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿ †

    PubMed Central

    Pines, Alex; Kelstrup, Christian D.; Vrouwe, Mischa G.; Puigvert, Jordi C.; Typas, Dimitris; Misovic, Branislav; de Groot, Anton; von Stechow, Louise; van de Water, Bob; Danen, Erik H. J.; Vrieling, Harry; Mullenders, Leon H. F.; Olsen, Jesper V.

    2011-01-01

    Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response. PMID:22006019

  18. Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis.

    PubMed

    Feraud, O; Cao, Y; Vittet, D

    2001-12-01

    The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro differentiation of murine embryonic stem (ES) cells has been shown to be a useful tool to investigate factors and genes potentially involved in vasculogenesis (Hirashima et al, 1999; Risau et al, 1988; Vittet et al, 1996; Wang et al, 1992; Wartenberg et al, 1998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after 11 days of differentiation, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic growth factors, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial outgrowths that can give tube-like structures with concomitant differentiation of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk1 (VEGFR2), flt1 (VEGFR1), tie-1, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic factors with a major contribution of vascular endothelial growth factor (VEGF). Known angiostatic agents, such as platelet factor 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic factors. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell differentiation can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system

  19. Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

    PubMed Central

    Alvarez-Manilla, Gerardo; Warren, Nicole L.; Atwood, James; Orlando, Ron; Dalton, Stephen; Pierce, Michael

    2011-01-01

    Numerous studies have recently focused on the identification of specific glycan biomarkers; given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction), that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin bound fractions, (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2 and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage, or were expressed in both cell stages but carried the lectin bound oligosaccharides in only one of them. Therefore, these lectin bound glycopeptides can be considered as stage specific glycobiomarkers. PMID:19545112

  20. Scale-up of human embryonic stem cell culture using a hollow fibre bioreactor.

    PubMed

    Roberts, Iwan; Baila, Stefano; Rice, R Brent; Janssens, Michiel Etienne; Nguyen, Kim; Moens, Nathalie; Ruban, Ludmila; Hernandez, Diana; Coffey, Pete; Mason, Chris

    2012-12-01

    The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested. PMID:22983716

  1. Beta-adrenoceptor subtype dependence of chronotropy in mouse embryonic stem cell-derived cardiomyocytes.

    PubMed

    Ali, N N; Xu, X; Brito-Martins, M; Poole-Wilson, P A; Harding, S E; Fuller, S J

    2004-11-01

    Cardiomyocytes derived from embryonic stem cells (ESCM) have potential both as an experimental model for investigating cardiac physiology and as a source for tissue repair. For both reasons it is important to characterise the responses of these cells, and one of the key modulators of contraction is the beta-adrenergic system. We therefore undertook a detailed study of the response of the spontaneous beating rate of ESCM to beta-adrenoceptor (betaAR) stimulation. Embryoid bodies (EBs) were generated from murine ES line E14Tg2a by the hanging drop method, followed by plating. Spontaneously beating areas were seen starting from 9-14 days after differentiation: the experiments described here were performed on EBs between developmental day 19 and 48. Beating cell layers were seeded with charcoal to allow tracking of movement by a video-edge detection system. Experiments were performed in physiological medium containing 1 mM Ca2+ at 37 degrees C. Isoprenaline (Iso) increased beating rate with an EC50 value of 52 nM. Iso (0.3 microM) increased basal rate from 67 +/- 7 beats per minute (bpm) to 138 +/- 18 bpm, P < 0.001, n = 22. At earlier developmental time points the response to Iso was not maintained through 5 min exposure; this spontaneous desensitisation only being observed before day 36. A repeat application of Iso after a wash period of 20 min produced reproducible effects on beating rate. Subtype dependence of the betaAR response was determined by comparing an initial response with a second in the presence of selective beta1- or beta2AR antagonists. In the presence of the specific beta1AR-blocker CGP 20712A (300 nM) the increase in rate with Iso was reduced from 207 +/- 42% of basal to 128 +/- 13%, P < 0.01. With the beta2AR-blocker ICI 118,551 (50 nM) there was no significant change in Iso response. Exposure to the muscarinic agonist, carbachol (10 microM), inhibited the increase in frequency mediated by isoprenaline, but had mixed stimulatory and inhibitory

  2. 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...

  3. Study of Pluripotency Markers in Zebrafish Embryos and Transient Embryonic Stem Cell Cultures

    PubMed Central

    Robles, Vanesa; Martí, Mercé

    2011-01-01

    Abstract Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and the expression of some of these genes is recorded throughout embryonic development. Some of the commonly accepted pluripotency markers are also tested in embryonic cells, transient embryonic cell cultures, and differentiated cells. Our results support the hypothesis that stage-specific embryonic antigen 1 (SSEA1) is a marker that precedes the expression of pluripotency genes in a zebrafish embryonic cell colony, in the same way that SOX2 precedes nestin expression in those colonies that have already started differentiation toward neurons. We consider this study a step forward in the knowledge of zebrafish pluripotency markers and, therefore, an important tool for the monitoring of zebrafish embryonic cell cultures. PMID:21563922

  4. Oncogenic KRAS activates an embryonic stem cell-like program in human colon cancer initiation

    PubMed Central

    Le Rolle, Anne-France; Chiu, Thang K.; Zeng, Zhaoshi; Shia, Jinru; Weiser, Martin R.; Paty, Philip B.; Chiu, Vi K.

    2016-01-01

    Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer. PMID:26744320

  5. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    PubMed

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  6. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells

    PubMed Central

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-01-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency-associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  7. Equine Induced Pluripotent Stem Cells have a Reduced Tendon Differentiation Capacity Compared to Embryonic Stem Cells

    PubMed Central

    Bavin, Emma P.; Smith, Olivia; Baird, Arabella E. G.; Smith, Lawrence C.; Guest, Deborah J.

    2015-01-01

    Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs) differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs) have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In two-dimensional differentiation assays, the iPSCs expressed tendon-associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in three-dimensional (3D) differentiation assays, the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3D in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application. PMID:26664982

  8. [Differentiation of human amniotic fluid stem cells into cardiomyocytes through embryonic body formation].

    PubMed

    Wang, Han; Chen, Shuai; Cheng, Xiang; Dou, Zhongying; Wang, Huayan

    2008-09-01

    To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies. PMID:19160841

  9. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

    PubMed Central

    Wu, Yi-Lin; Ma, Shi-Rong; Peng, Tao; Teng, Zeng-Hui; Liang, Xiang-Yan; Guo, Guo-Zhen; Zhang, Hai-Feng; Li, Kang-Chu

    2014-01-01

    Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells. PMID:25196518

  10. Effects of pulsed electromagnetic field on differentiation of HUES-17 human embryonic stem cell line.

    PubMed

    Wu, Yi-Lin; Ma, Shi-Rong; Peng, Tao; Teng, Zeng-Hui; Liang, Xiang-Yan; Guo, Guo-Zhen; Zhang, Hai-Feng; Li, Kang-Chu

    2014-01-01

    Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells. PMID:25196518

  11. Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

    PubMed

    Doering, C B; Coursey, C; Spangler, W; Danner, D J

    1998-06-01

    These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system. PMID:9611264

  12. Stage-specific embryonic antigen-4 identifies human dental pulp stem cells.

    PubMed

    Kawanabe, Noriaki; Murata, Satoko; Fukushima, Hiroaki; Ishihara, Yoshihito; Yanagita, Takeshi; Yanagita, Emmy; Ono, Mitsuaki; Kurosaka, Hiroshi; Kamioka, Hiroshi; Itoh, Tomoo; Kuboki, Takuo; Yamashiro, Takashi

    2012-03-10

    Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells. PMID:22266579

  13. 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. PMID:25394653

  14. Challenges of stem cell therapy for spinal cord injury: human embryonic stem cells, endogenous neural stem cells, or induced pluripotent stem cells?

    PubMed

    Ronaghi, Mohammad; Erceg, Slaven; Moreno-Manzano, Victoria; Stojkovic, Miodrag

    2010-01-01

    Spinal cord injury (SCI) causes myelopathy, damage to white matter, and myelinated fiber tracts that carry sensation and motor signals to and from the brain. The gray matter damage causes segmental losses of interneurons and motoneurons and restricts therapeutic options. Recent advances in stem cell biology, neural injury, and repair, and the progress toward development of neuroprotective and regenerative interventions are the basis for increased optimism. This review summarizes the pathophysiological mechanisms following SCI and compares human embryonic, adult neural, and the induced pluripotent stem cell-based therapeutic strategies for SCI. PMID:19904738

  15. 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...

  16. 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...

  17. 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...

  18. Anthropological and ethical reflections on the production and use of embryonic stem cells.

    PubMed

    Faggioni, M P

    2008-02-01

    Stem cells and their potential therapeutic application have generated tremendous public interest, great enthusiasm among researchers and intense commercial interest. There are diverse sources of stem cells. According to their origin and their biological characteristics, they are classified as embryonic stem cells, germline stem cells and tissue stem cells. Until now, the most concrete therapeutic results have come from some adult tissue stem cells, with promising prospects also being offered by umbilical cord stem cells. Regarding embryonic stem cells, there is concern that they would be difficult to control in vivo. Nonetheless, many researchers are still pursuing their potential uses, convinced that they will be useful not only for study, but also for therapy, especially as a result of their high capacity for self-renewal as well as their broad potential for differentiation. This discussion which is eminently scientific in nature, and not lacking in ethical and political repercussions, will not be entered into above all regarding the allocation of available intellectual and economic resources. PMID:18181948

  19. Functional Analysis of Various Promoters in Lentiviral Vectors at Different Stages of In Vitro Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Hong, Sunghoi; Hwang, Dong-Youn; Yoon, Soonsang; Isacson, Ole; Ramezani, Ali; Hawley, Robert G

    2008-01-01

    Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1α (EF1α) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken β-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1α promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1α promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity. PMID:17609656

  20. A rapid lung de-cellularization protocol supports embryonic stem cell differentiation in vitro and following implantation.

    PubMed

    Jensen, Todd; Roszell, Blair; Zang, Fan; Girard, Eric; Matson, Adam; Thrall, Roger; Jaworski, Diane M; Hatton, Cayla; Weiss, Daniel J; Finck, Christine

    2012-08-01

    Pulmonary diseases represent a large portion of neonatal and adult morbidity and mortality. Many of these have no cure, and new therapeutic approaches are desperately needed. De-cellularization of whole organs, which removes cellular elements but leaves intact important extracellular matrix (ECM) proteins and three-dimensional architecture, has recently been investigated for ex vivo generation of lung tissues. As specific cell culture surfaces, including ECM composition, profoundly affect cell differentiation, this approach offers a potential means of using de-cellularized lungs to direct differentiation of embryonic and other types of stem/progenitor cells into lung phenotypes. Several different methods of whole-lung de-cellularization have been reported, but the optimal method that will best support re-cellularization and generation of lung tissues from embryonic stem cells (ESCs) has not been determined. We present a 24-h approach for de-cellularizing mouse lungs utilizing a detergent-based (Triton-X100 and sodium deoxycholate) approach with maintenance of three-dimensional lung architecture and ECM protein composition. Predifferentiated murine ESCs (mESCs), with phenotypic characteristics of type II alveolar epithelial cells, were seeded into the de-cellularized lung scaffolds. Additionally, we evaluated the effect of coating the de-cellularized scaffold with either collagen or Matrigel to determine if this would enhance cell adhesion and affect mechanics of the scaffold. Finally, we subcutaneously implanted scaffolds in vivo after seeding them with mESCs that are predifferentiated to express pro-surfactant protein C (pro-SPC). The in vivo environment supported maintenance of the pro-SPC-expressing phenotype and further resulted in vascularization of the implant. We conclude that a rapid detergent-based de-cellularization approach results in a scaffold that can maintain phenotypic evidence of alveolar epithelial differentiation of ESCs and support

  1. How to cross immunogenetic hurdles to human embryonic stem cell transplantation.

    PubMed

    de Rham, Casimir; Villard, Jean

    2011-11-01

    Implantation of human embryonic stem cells (hES), derived progenitors or mature cells derived from hES has great therapeutic potential for many diseases. If hES would come from genetically unrelated individuals, it would be probably rejected by the immune system of the recipient. Blood groups, MHC and minor antigens are the immunogenetic hurdles that have to be crossed for successful transplantation. Autologous transplantation with adult stem cells would be the best approach but several elements argue against this option. Classical immunosuppression, depleting antibody, induction of tolerance and stem cell banking are alternative methods that could be proposed to limit the risk of rejection. PMID:21461719

  2. The Genetic and Epigenetic Journey of Embryonic Stem Cells into Mature Neural Cells

    PubMed Central

    Olynik, Brendan M.; Rastegar, Mojgan

    2012-01-01

    Epigenetic changes occur throughout life from embryonic development into adulthood. This results in the timely expression of developmentally important genes, determining the morphology and identity of different cell types and tissues within the body. Epigenetics regulate gene expression and cellular morphology through multiple mechanisms without alteration in the underlying DNA sequences. Different epigenetic mechanisms include chromatin condensation, post-translational modification of histone proteins, DNA cytosine marks, and the activity of non-coding RNA molecules. Epigenetics play key roles in development, stem cell differentiation, and have high impact in human disease. In this review, we will discuss our current knowledge about these epigenetic mechanisms, with a focus on histone and DNA marks. We will then talk about the genetics and epigenetics of embryonic stem cell self-renewal and differentiation into neural stem cells, and further into specific neuronal cell types. PMID:22629283

  3. In vitro imaging of embryonic stem cells using multiphoton luminescence of gold nanoparticles

    PubMed Central

    Nagesha, D; Laevsky, GS; Lampton, P; Banyal, R; Warner, C; DiMarzio, C; Sridhar, S

    2007-01-01

    Recent advances in nonlinear optical techniques and materials such as quantum wells, nanowires and noble-metal nanoparticles have led to advances in cellular imaging wherein various nanoparticles have been shown to improve both in vitro and in vivo visualization. In this paper, we demonstrate in vitro imaging using multi-photon photoluminescence of gold nanoparticles from two different cell types – Dictyostelium discoideum and mouse embryonic stem cells. By observing nanoparticles we show that embryonic stem cells maintained their ability to proliferate for several passages while grown in the presence of gold nanoparticles. The advantages of multi-photon luminescence using gold nanoparticles have important implications for use in stem cell proliferation experiments and in vitro experiments to monitor differentiation. PMID:18203448

  4. Neuronal differentiation of embryonic stem cell derived neuronal progenitors can be regulated by stretchable conducting polymers.

    PubMed

    Srivastava, Nishit; Venugopalan, Vijay; Divya, M S; Rasheed, V A; James, Jackson; Narayan, K S

    2013-09-01

    Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950

  5. Neuronal Differentiation of Embryonic Stem Cell Derived Neuronal Progenitors Can Be Regulated by Stretchable Conducting Polymers

    PubMed Central

    Srivastava, Nishit; Venugopalan, Vijay; Divya, M.S.; Rasheed, V.A.

    2013-01-01

    Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950

  6. 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

  7. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells.

    PubMed

    Bai, Yinshan; Feng, Meiying; Liu, Shanshan; Wei, Hengxi; Li, Li; Zhang, Xianwei; Shen, Chao; Zhang, Shouquan; Ma, Ningfang

    2016-08-01

    Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency‑associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs. PMID:27353491

  8. Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs.

    PubMed

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

    2013-05-01

    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

  9. Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation.

    PubMed

    Nie, Li; Gao, Shi-Jun; Zhao, Ya-Nan; Masika, Jacob; Luo, Hong-Yan; Hu, Xin-Wu; Zhang, Liang-Pin; Zeng, Ying; Hescheler, Jürgen; Liang, Hua-Min

    2016-06-01

    Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation. PMID:27376799

  10. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

    PubMed Central

    Quan, Yuan; Yan, Qing; Morales, John E.

    2015-01-01

    Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells

  11. Human embryonic stem cell responses to ionizing radiation exposures: current state of knowledge and future challenges.

    PubMed

    Sokolov, Mykyta V; Neumann, Ronald D

    2012-01-01

    Human embryonic stem cells, which are derived from the inner cell mass of the blastocyst, have become an object of intense study over the last decade. They possess two unique properties that distinguish them from many other cell types: (i) the ability to self-renew indefinitely in culture under permissive conditions, and (ii) the pluripotency, defined as the capability of giving rise to all cell types of embryonic lineage under the guidance of the appropriate developmental cues. The focus of many recent efforts has been on the elucidating the signaling pathways and molecular networks operating in human embryonic stem cells. These cells hold great promise in cell-based regenerative therapies, disease modeling, drug screening and testing, assessing genotoxic and mutagenic risks associated with exposures to a variety of environmental factors, and so forth. Ionizing radiation is ubiquitous in nature, and it is widely used in diagnostic and therapeutic procedures in medicine. In this paper, our goal is to summarize the recent progress in understanding how human embryonic stem cells respond to ionizing radiation exposures, using novel methodologies based on "omics" approaches, and to provide a critical discussion of what remains unknown; thus proposing a roadmap for the future research in this area. PMID:22966236

  12. Very small embryonic/epiblast-like stem cells (VSELs) and their potential role in aging and organ rejuvenation – an update and comparison to other primitive small stem cells isolated from adult tissues

    PubMed Central

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

    2012-01-01

    Very small embryonic-like stem cells (VSELs) are a population of developmentally early stem cells residing in adult tissues. These rare cells, which are slightly smaller than red blood cells, i) become mobilized during stress situations into peripheral blood, ii) are enriched in the Sca1+Lin−CD45− cell fraction in mice and the CD133+ Lin−CD45− cell fraction in humans, iii) express markers of pluripotent stem cells (e.g., Oct4, Nanog, and SSEA), and iv) display a distinct morphology characterized by a high nuclear/cytoplasmic ratio and undifferentiated chromatin. Recent evidence indicates that murine VSELs are kept quiescent in adult tissues and protected from teratoma formation by epigenetic modification of imprinted genes that regulate insulin/insulin like growth factor signaling (IIS). The successful reversal of these epigenetic changes in VSELs that render them quiescent will be crucial for efficient expansion of these cells. The most recent data in vivo from our and other laboratories demonstrated that both murine and human VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells (LT-HSCs), are at the top of the hierarchy in the mesenchymal lineage, and may differentiate into organ-specific cells (e.g., cardiomyocytes). Moreover, as recently demonstrated the number of these cells positively correlates in several murine models with longevity. Finally, while murine BM-derived VSELs have been extensively characterized more work is needed to better characterize these small cells at the molecular level in humans. PMID:22498452

  13. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    SciTech Connect

    Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  14. 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. PMID:17459325

  15. Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation.

    PubMed

    Rose, Laura C; Fitzsimmons, Ross; Lee, Poh; Krawetz, Roman; Rancourt, Derrick E; Uludağ, Hasan

    2013-05-01

    Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder-free monolayers, up to 40 ng/ml of exogenous bFGF did not support self-renewal of mES without LIF during cell expansion. During spontaneous differentiation in high-density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage-specific embryonic antigen-1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca-1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP-2. These results suggest that bFGF could be utilized to expand the cell population in high-density cultures in addition to enriching the BMP-2 responsiveness of mES cells. PMID:22674886

  16. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm.

    PubMed

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Moeller, Jonas Bech; Hansson, Mattias; Dufva, Martin

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation. PMID:26713616

  17. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Chowdhury, Farhan; Na, Sungsoo; Li, Dong; Poh, Yeh-Chuin; Tanaka, Tetsuya S.; Wang, Fei; Wang, Ning

    2010-01-01

    Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.

  18. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    PubMed Central

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Moeller, Jonas Bech

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation. PMID:26713616

  19. Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung.

    PubMed

    Aguilar, Susana; Nye, Emma; Chan, Jerry; Loebinger, Michael; Spencer-Dene, Bradley; Fisk, Nick; Stamp, Gordon; Bonnet, Dominique; Janes, Sam M

    2007-06-01

    Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well-differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies. Disclosure of potential conflicts of interest is found at the end of this article. PMID:17363552

  20. The ventral hippocampus is the embryonic origin for adult neural stem cells in the dentate gyrus

    PubMed Central

    Li, Guangnan; Fang, Li; Fernández, Gloria; Pleasure, Samuel J.

    2013-01-01

    SUMMARY Adult neurogenesis represents a unique form of plasticity in the dentate gyrus requiring the presence of long-lived neural stem cells (LL-NSCs). However, the embryonic origin of these LL-NSCs remains unclear. The prevailing model assumes that the dentate neuroepithelium throughout the longitudinal axis of the hippocampus generates both the LL-NSCs and embryonically produced granule neurons. Here we show that the NSCs initially originate from the ventral hippocampus during late gestation and then relocate into the dorsal hippocampus. The descendants of these cells are the source for the LL-NSCs in the subgranular zone (SGZ). Furthermore, we show that the origin of these cells and their maintenance in the dentate are controlled by distinct sources of Sonic Hedgehog (Shh). The revelation of the complexity of both the embryonic origin of hippocampal LL-NSCs and the sources of Shh has important implications for the functions of LL-NSCs in the adult hippocampus. PMID:23643936

  1. Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system.

    PubMed

    Aflatoonian, Behrouz; Ruban, Ludmila; Shamsuddin, Shamsul; Baker, Duncan; Andrews, Peter; Moore, Harry

    2010-04-01

    The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs. PMID:20224972

  2. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay

    EPA Science Inventory

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...

  3. [Embryonic stem cell test in screening of medicine and other chemicals].

    PubMed

    Zhao, Qing; Xu, Jinsen

    2005-02-01

    The technique of embryonic stem cell test (EST) has been developed and used in vitro to screen new medicines and other chemicals. According to toxicity, such medicines and chemicals can be classified as: non-toxic, weak toxic and/or strong toxic. EST shows merits such as no requirement of the sacrifice of pregnant animals, no side-effects on human or animals for candidate medicines and chemicals, higher sensitivity of embryonic stem cells when compared with the sensitivity of ordinary tissues of adult samples in toxicologic researches, higher accuracy when combined with computing techniques, and possible quantitation based on techniques of molecular biology. Advances in utility of EST technique were reviewed and the prospect of technique was also discussed in this paper. PMID:15762145

  4. Data describing Rax positive optic-vesicle generation from mouse embryonic stem cells in vitro.

    PubMed

    Takata, Nozomu; Eiraku, Mototsugu; Sakakura, Eriko

    2016-09-01

    This article contains data related to the research article entitled "Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture" Sakakura (2016) [1]. Mouse embryonic stem cells (ESC) were used for the generation of optic vesicle-like tissues in vitro. In this article we described data in which a Rax::GFP knock-in ESC line was used to monitor the formation of optic tissues. In addition, we also described the data of regional marker expression of Rax, Sox2 and Pax6 in vivo around the forebrain and the eye tissues for comparative purposes. These data can be valuable to researchers interested in investigating forebrain and eye tissue development. PMID:27358906

  5. Inference of Transcriptional Network for Pluripotency in Mouse Embryonic Stem Cells

    NASA Astrophysics Data System (ADS)

    Aburatani, S.

    2015-01-01

    In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells.

  6. Cell Cycle-Dependent Turnover of 5-Hydroxymethyl Cytosine in Mouse Embryonic Stem Cells

    PubMed Central

    Sharif, Jafar; Endo, Takaho A.; Mishima, Yuichi; Kawakami, Toru; Koseki, Haruhiko; Shirakawa, Masahiro; Suetake, Isao; Tajima, Shoji

    2013-01-01

    Hydroxymethylcytosine in the genome is reported to be an intermediate of demethylation. In the present study, we demonstrated that maintenance methyltransferase Dnmt1 scarcely catalyzed hemi-hydroxymethylated DNA and that the hemi-hydroxymethylated DNA was not selectively recognized by the SRA domain of Uhrf1, indicating that hydroxymethylcytosine is diluted in a replication-dependent manner. A high level of 5-hydroxymethylcytosine in mouse embryonic stem cells was produced from the methylcytosine supplied mainly by de novo-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the HoxA gene cluster showed a high hydroxymethylation level whilst the methylcytosine level was quite low, suggesting that methylated CpG is actively hydroxylated during proliferation. All the results indicate that removal and production of hydroxymethylcytosine are regulated in replication-dependent manners in mouse embryonic stem cells. PMID:24340069

  7. The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

    PubMed

    Sperber, Henrik; Mathieu, Julie; Wang, Yuliang; Ferreccio, Amy; Hesson, Jennifer; Xu, Zhuojin; Fischer, Karin A; Devi, Arikketh; Detraux, Damien; Gu, Haiwei; Battle, Stephanie L; Showalter, Megan; Valensisi, Cristina; Bielas, Jason H; Ericson, Nolan G; Margaretha, Lilyana; Robitaille, Aaron M; Margineantu, Daciana; Fiehn, Oliver; Hockenbery, David; Blau, C Anthony; Raftery, Daniel; Margolin, Adam A; Hawkins, R David; Moon, Randall T; Ware, Carol B; Ruohola-Baker, Hannele

    2015-12-01

    For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development. PMID:26571212

  8. Paracrine signalling events in embryonic stem cell renewal mediated by affinity targeted nanoparticles.

    PubMed

    Corradetti, Bruna; Freile, Paz; Pells, Steve; Bagnaninchi, Pierre; Park, Jason; Fahmy, Tarek M; de Sousa, Paul A

    2012-10-01

    Stem cell growth and differentiation is controlled by intrinsic and extrinsic factors. The latter includes growth factors, which are conventionally supplied in vitro in media exchanged daily. Here, we illustrate the use of affinity targeted biodegradable nanoparticles to mediate paracrine stimulation as an alternative approach to sustain the growth and pluripotency of mouse embryonic stem cells. Leukaemia Inhibitory Factor (LIF) was encapsulated in biodegradable nanoparticles and targeted to the cell surface using an antibody to the oligosaccharide antigen SSEA-1. Sustained release of LIF from nanoparticles composed of a solid Poly(lactide-co-glycolic acid) polyester or a hydrogel-based liposomal system, we term Nanolipogel, replenished once after each cell passage, proved as effective as daily replenishment with soluble LIF for maintenance of pluripotency after 5 passages using 10(4)-fold less LIF. Our study constitutes an alternative paradigm for stem cell culture, providing dynamic microenvironmental control of extrinsic bioactive factors benefiting stem cell manufacturing. PMID:22749449

  9. caBIG® Spotlight - Solving Research Problems: Analyze Mouse Embryonic Stem Cell Transcriptional Profiles —

    Cancer.gov

    Read a case study to learn more about how Dr. Bradley Merrill of the University of Illinois at Chicago and his lab were able to perform their first gene expression array experiment comparing a mutant mouse embryonic stem cell line to a non-mutant control line using GenePattern, an application supported by the Molecular Analysis Tools Knowledge Center which provides bioinformatics tools for gene expression, proteomic and SNP analysis.

  10. Derivation of human embryonic stem cell from spinal muscular atrophy patient.

    PubMed

    Xie, Pingyuan; Zhou, Hao; Zhou, Xiaoying; Zhao, Xiaomeng; Du, Juan; Lu, Guangxiu; Lin, Ge; Ouyang, Qi

    2016-03-01

    We established a human embryonic stem cell (hESC) line chHES-427 from the abnormal embryo carrying homozygous deletion of exon 7 of survival motor neuron gene (SMN). This cell line maintained a normal karyotype 46, XX during long-term culture. Further characteristic analysis suggested that the cells expressed the pluripotency-related markers and had the capacity to differentiate into the derivatives from the three germ layers in vitro. PMID:27345972

  11. Derivation of the human embryonic stem cell line RCe007-A (RC-3).

    PubMed

    De Sousa, P A; Tye, B; Bruce, K; Dand, P; Russell, G; Gardner, J; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe007-A (RC-3) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available. PMID:27346192

  12. Derivation of the human embryonic stem cell line RCe011-A (RC-7).

    PubMed

    De Sousa, P A; Tye, B J; Collins, D M; Bruce, K; Dand, P; Russell, G; Bradburn, H; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available. PMID:27346020

  13. Derivation of the human embryonic stem cell line RCe010-A (RC-6).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Bradburn, H; Gardner, J; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available. PMID:27346019

  14. Derivation of the human embryonic stem cell line RCe008-A (RC-4).

    PubMed

    De Sousa, P A; Tye, B; Bruce, K; Dand, P; Gardner, J; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is available. PMID:27346193

  15. X chromosome inactivation in human parthenogenetic embryonic stem cells following prolonged passaging

    PubMed Central

    QI, QUAN; DING, CHENHUI; HONG, PINGPING; YANG, GANG; XIE, YANXIN; WANG, JING; HUANG, SUNXING; HE, KE; ZHOU, CANQUAN

    2015-01-01

    The present study aimed to investigate the X chrochromosome inactivation (XCI) status in long-term cultured human parthenogenetic embryonic stem cells. One human embryonic stem (hES) cell line and 2 human parthenogenetic embryonic stem (hPES) cell lines were subjected to long-term culture in vitro (>50 passages). Karyotyping, array-based comparative genomic hybridization (aCGH), X-inactive specific transcript (XIST) RNA, immunofluorescence staining and real-time PCR were used to assess the chromosome karyotypes of these cells and the XCI status. X chromosome microdeletion was observed in the hPES-2 cells following culture for 50 passages. As early as 20 passages, XIST RNA expression was detected in the hPES-2 cells and was followed by low X-linked gene expression. The XIST RNA expression level was higher in the differentiated hPES-2 cells. The hPES-2′ cells that were subclones of hPES-2 retained the XCI status, and had low XIST and X-linked gene expression. XIST RNA expression remained at a low level in the differentiated hPES-2′ cells. The human biparental embryonic stem (hBES)-1 and hPES-1 cells did not exhibit XCI, and the differentiated hPES-1 cells had high expression levels of XIST RNA. In conclusion, the chromosome karyotypes of some hPES cell lines revealed instabilities. Similar to the hES cells, the hPES cells exhibited 3 XCI statuses. The unstable XCI status of the hPES-2 line may have been related to chromosome instability. These unstable chromosomes renedered these cells susceptible to environmental conditions and freezing processes, which may be the result of environmental adaptations. PMID:25524499

  16. Agouti C57BL/6N embryonic stem cells for mouse genetic resources

    PubMed Central

    Pettitt, Stephen J.; Liang, Qi; Rairdan, Xin Y.; Moran, Jennifer L.; Prosser, Haydn M.; Beier, David R.; Lloyd, Kent; Bradley, Allan; Skarnes, William C.

    2010-01-01

    We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant Agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background. PMID:19525957

  17. Plasma treatment of biomaterials to direct the differentiation of embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Hanley, Erik

    In this work, we explore how embryonic stem (ES) cell differentiation patterns are affected by surface interactions with plasma-processed materials. We hypothesize that mouse embryonic stem-cell exposure to certain plasma-polymerized tetraglyme surfaces will direct their differentiation into endothelial cells. R1 mouse embryonic stem (ES) cells were plated on surfaces onto which tetraglyme was deposited by plasma polymerization. In addition, tissue-treated polystyrene and control glass cover slips were also examined. Some samples were fixed three days after plating and immunofluorescence stained with platelet endothelial-cell adhesion molecule, while the others were fixed seven days after plating and immunofluorescence stained with von Willebrand Factor. Positive results seen by ES cell derivatives precociously expressing the vWF and PECAM genetic markers on the plasma-polymerized tetraglyme treated surfaces suggest that the plasma-polymerized surfaces direct differentiation of ES cells into endothelial cells. Research goals of this dissertation include: characterization of the material properties of the plasma-polymerized tetraglyme surfaces that induce directed differentiation of ES cells into endothelial cells, optimization of the plasma-polymerization process to maximize the number of endothelial cells derived from R1 ES cells, and biological experimentation to characterize properties of the mechanism of directed differentiation. A potential application of this work is in the design and construction of an artificial blood vessel. Current small-scale arterial substitutes have proved inadequate because of thrombogenicity and infection. Moreover, the lower blood flow velocities of smaller vessels pose a different set of design criteria and introduce new problems not encountered in large arterial substitutes. By utilizing a tissue engineering approach that incorporates embryonic stem cell-derived endothelial cells, the longevity of the prosthesis can be ensured.

  18. Generation of KCL031 clinical grade human embryonic stem cell line

    PubMed Central

    Jacquet, Laureen; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  19. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  20. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    PubMed

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs. PMID:24180743

  1. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

    PubMed Central

    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  2. Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages

    PubMed Central

    Gasimli, Leyla; Hickey, Anne M.; Yang, Bo; Li, Guoyun; Rosa, Mitche dela; Nairn, Alison V.; Kulik, Michael J.; Dordick, Jonathan S.; Moremen, Kelley W.; Dalton, Stephen; Linhardt, Robert J.

    2014-01-01

    Background Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly change the proteoglycanome General Significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. PMID:24412195

  3. Femtosecond laser pulses for chemical-free embryonic and mesenchymal stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; Dholakia, Kishan; Gunn-Moore, Frank

    2011-10-01

    Owing to their self renewal and pluripotency properties, stem cells can efficiently advance current therapies in tissue regeneration and/or engineering. Under appropriate culture conditions in vitro, pluripotent stem cells can be primed to differentiate into any cell type some examples including neural, cardiac and blood cells. However, there still remains a pressing necessity to answer the biological questions concerning how stem cell renewal and how differentiation programs are operated and regulated at the genetic level. In stem cell research, an urgent requirement on experimental procedures allowing non-invasive, marker-free observation of growth, proliferation and stability of living stem cells under physiological conditions exists. Femtosecond (fs) laser pulses have been reported to non-invasively deliver exogenous materials, including foreign genetic species into both multipotent and pluripotent stem cells successfully. Through this multi-photon facilitated technique, directly administering fs laser pulses onto the cell plasma membrane induces transient submicrometer holes, thereby promoting cytosolic uptake of the surrounding extracellular matter. To display a chemical-free cell transfection procedure that utilises micro-litre scale volumes of reagents, we report for the first time on 70 % transfection efficiency in ES-E14TG2a cells using the enhanced green fluorescing protein (EGFP) DNA plasmid. We also show how varying the average power output during optical transfection influences cell viability, proliferation and cytotoxicity in embryonic stem cells. The impact of utilizing objective lenses of different numerical aperture (NA) on the optical transfection efficiency in ES-E14TG2a cells is presented. Finally, we report on embryonic and mesenchymal stem cell differentiation. The produced specialized cell types could thereafter be characterized and used for cell based therapies.

  4. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

    PubMed Central

    Zhang, Rong; Mjoseng, Heidi K.; Hoeve, Marieke A.; Bauer, Nina G.; Pells, Steve; Besseling, Rut; Velugotla, Srinivas; Tourniaire, Guilhem; Kishen, Ria E. B.; Tsenkina, Yanina; Armit, Chris; Duffy, Cairnan R. E.; Helfen, Martina; Edenhofer, Frank; de Sousa, Paul A.; Bradley, Mark

    2013-01-01

    Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2–6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications. PMID:23299885

  5. 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 Central

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

    2014-01-01

    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. PMID:24384367

  6. 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. PMID:24384367

  7. Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

    SciTech Connect

    Theunissen, P.T.; Robinson, J.F.; Pennings, J.L.A.; Herwijnen, M.H. van; Kleinjans, J.C.S.; Piersma, A.H.

    2012-08-01

    Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

  8. Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina

    PubMed Central

    Centanin, Lázaro; Ander, Janina-J.; Hoeckendorf, Burkhard; Lust, Katharina; Kellner, Tanja; Kraemer, Isabel; Urbany, Cedric; Hasel, Eva; Harris, William A.; Simons, Benjamin D.; Wittbrodt, Joachim

    2014-01-01

    The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution. PMID:25142461

  9. Effects of Polymer Surfaces on Proliferation and Differentiation of Embryonic Stem Cells and Bone Marrow Stem Cells

    NASA Astrophysics Data System (ADS)

    Qin, Sisi; Liao, Wenbin; Ma, Yupo; Simon, Marcia; Rafailovich, Miriam; Stony Brook Medical Center Collaboration; Stony Brook Dental Schoo Collaboration

    2013-03-01

    Currently, proliferation and differentiation of stem cell is usually accomplished either in vivo, or on chemical coated tissue culture petri dish with the presence of feeder cells. Here we investigated whether they can be directly cultured on polymeric substrates, in the absence of additional factors. We found that mouse embryonic stem cells did not require gelatin and could remain in the undifferentiated state without feeder cells at least for four passages on partially sulfonated polystyrene. The modulii of cells was measured and found to be higher for cells plated directly on the polymer surface than for those on the same surface covered with gelatin and feeder cells. When plated with feeder cells, the modulii was not sensitive to gelatin. Whereas the differentiation properties of human bone marrow stem cells, which are not adherent, are less dependent on either chemical or mechanical properties of the substrate. However, they behave differently on different toughness hydrogels as oppose to on polymer coated thin films.

  10. Enhanced Generation of Myeloid Lineages in Hematopoietic Differentiation from Embryonic Stem Cells by Silencing Transcriptional Repressor Twist-2

    PubMed Central

    Sharabi, Andrew B.; Lee, Sung-Hyung; Goodell, Margaret A.; Huang, Xue F.

    2009-01-01

    Abstract The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1+ and F4/80+ cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor. PMID:20025523

  11. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes.

    PubMed

    Lee, Desy S; Chen, Jyh-Hong; Lundy, David J; Liu, Chung-Hung; Hwang, Shiaw-Min; Pabon, Lil; Shieh, Ru-Chi; Chen, Chien-Chang; Wu, Sheng-Nan; Yan, Yu-Ting; Lee, Sho-Tone; Chiang, Po-Min; Chien, Shu; Murry, Charles E; Hsieh, Patrick C H

    2015-09-29

    Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling. PMID:26365191

  12. Human BCAS3 Expression in Embryonic Stem Cells and Vascular Precursors Suggests a Role in Human Embryogenesis and Tumor Angiogenesis

    PubMed Central

    Siva, Kavitha; Venu, Parvathy; Mahadevan, Anita; S. K., Shankar; Inamdar, Maneesha S.

    2007-01-01

    Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker. PMID:18030336

  13. Transcriptional expression profile of cultured human embryonic stem cells in vitro and in vivo.

    PubMed

    Keil, Marlen; Siegert, Antje; Eckert, Klaus; Gerlach, Jörg; Haider, Wolfram; Fichtner, Iduna

    2012-03-01

    The aims of this study were to analyze the spontaneous differentiation of human embryonic stem cells in vitro and in vivo and to investigate the influence of in vitro partial differentiation on in vivo teratoma formation in immunodeficient mice. Standardized methods are needed for long-term cultivation of undifferentiated stem cells and the multilineage cells that spontaneously differentiate from them. Accordingly, SA002 human embryonic stem cells were cultured on irradiated mouse embryonic fibroblasts cells, on irradiated human foreskin fibroblasts, or were cultured feeder-free using matrigel. Expression of marker protein transcripts was analyzed in undifferentiated and differentiated stem cells using real-time PCR, and both types of stem cells were transplanted subcutaneously into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice to test for teratoma formation. Teratoma histology and expression profiles were subsequently characterized. Cells cultured using different conditions and morphologically undifferentiated cells had comparable marker expression profiles, showing high expression levels of markers for pluripotency and low-to-moderate expression levels of germ layer markers. Cells showing spontaneous differentiation that were cultured in feeder-free conditions in the absence of basic fibroblast growth factor demonstrated slight upregulation of sex determining region Y-box 17, connexin 32, and albumin expression at early time points, as well as expression of octamer-binding transcription factor 4, proteoglycan epitopes on podocalyxin (Trafalgar), and alkaline phosphatase. At later time points, expression of hepatocyte nuclear factor-3-beta, and hepatocyte nuclear factor-4-alpha and alpha fetoprotein was upregulated, whereas beta-3-tubulin, chemokine receptor, nestin, sex-determining region Y-box 17, and connexin 32 were downregulated. Expression of pluripotency markers remained high, and hematopoetic markers were not expressed. SA002 cells that showed

  14. Mechanistic Target of Rapamycin (Mtor) Is Essential for Murine Embryonic Heart Development and Growth

    PubMed Central

    Zhu, Yi; Pires, Karla M. P.; Whitehead, Kevin J.; Olsen, Curtis D.; Wayment, Benjamin; Zhang, Yi Cheng; Bugger, Heiko; Ilkun, Olesya; Litwin, Sheldon E.; Thomas, George; Kozma, Sara C.; Abel, E. Dale

    2013-01-01

    Mechanistic target of rapamycin (Mtor) is required for embryonic inner cell mass proliferation during early development. However, Mtor expression levels are very low in the mouse heart during embryogenesis. To determine if Mtor plays a role during mouse cardiac development, cardiomyocyte specific Mtor deletion was achieved using α myosin heavy chain (α-MHC) driven Cre recombinase. Initial mosaic expression of Cre between embryonic day (E) 10.5 and E11.5 eliminated a subset of cardiomyocytes with high Cre activity by apoptosis and reduced overall cardiac proliferative capacity. The remaining cardiomyocytes proliferated and expanded normally. However loss of 50% of cardiomyocytes defined a threshold that impairs the ability of the embryonic heart to sustain the embryo’s circulatory requirements. As a result 92% of embryos with cardiomyocyte Mtor deficiency died by the end of gestation. Thus Mtor is required for survival and proliferation of cardiomyocytes in the developing heart. PMID:23342106

  15. Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells.

    PubMed

    Enomoto, Kei; Watanabe-Susaki, Kanako; Kowno, Megumi; Takada, Hitomi; Intoh, Atsushi; Yamanaka, Yuko; Hirano, Hisashi; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

    2015-01-01

    Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells. PMID:26399336

  16. Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics

    SciTech Connect

    Zhao Yong . E-mail: yongzhao@uic.edu; 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, 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.

  17. Parthenogenetic dopamine neurons from primate embryonic stem cells restore function in experimental Parkinson's disease

    PubMed Central

    Lee, Hyojin; Patterson, Michaela; Reske-Nielsen, Casper; Yoshizaki, Takahito; Sonntag, Kai C.; Studer, Lorenz; Isacson, Ole

    2008-01-01

    The identity and functional potential of dopamine neurons derived in vitro from embryonic stem cells are critical for the development of a stem cell-based replacement therapy for Parkinson's disease. Using a parthenogenetic primate embryonic stem cell line, we have generated dopamine neurons that display persistent expression of midbrain regional and cell-specific transcription factors, which establish their proper identity and allow for their survival. We show here that transplantation of parthenogenetic dopamine neurons restores motor function in hemi-parkinsonian, 6-hydroxy-dopamine-lesioned rats. Exposure to Wnt5a and fibroblast growth factors (FGF) 20 and 2 at the final stage of in vitro differentiation enhanced the survival of dopamine neurons and, correspondingly, the extent of motor recovery of transplanted animals. Importantly for future development of clinical applications, dopamine neurons were post-mitotic at the time of transplantation and there was no tumour formation. These data provide proof for the concept that parthenogenetic stem cells are a suitable source of functional neurons for therapeutic applications. PMID:18669499

  18. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  19. Bioimaging: An Useful Tool to Monitor Differentiation of Human Embryonic Stem Cells into Chondrocytes.

    PubMed

    Suchorska, Wiktoria M; Lach, Michał S; Richter, Magdalena; Kaczmarczyk, Jacek; Trzeciak, Tomasz

    2016-05-01

    To improve the recovery of damaged cartilage tissue, pluripotent stem cell-based therapies are being intensively explored. A number of techniques exist that enable monitoring of stem cell differentiation, including immunofluorescence staining. This simple and fast method enables changes to be observed during the differentiation process. Here, two protocols for the differentiation of human embryonic stem cells into chondrocytes were used (monolayer cell culture and embryoid body formation). Cells were labeled for markers expressed during the differentiation process at different time points (pluripotent: NANOG, SOX2, OCT3/4, E-cadherin; prochondrogenic: SOX6, SOX9, Collagen type II; extracellular matrix components: chondroitin sulfate, heparan sulfate; beta-catenin, CXCR4, and Brachyury). Comparison of the signal intensity of differentiated cells to control cell populations (articular cartilage chondrocytes and human embryonic stem cells) showed decreased signal intensities of pluripotent markers, E-cadherin and beta-catenin. Increased signal intensities of prochondrogenic markers and extracellular matrix components were observed. The changes during chondrogenic differentiation monitored by evaluation of pluripotent and chondrogenic markers signal intensity were described. The changes were similar to several studies over chondrogenesis. These results were confirmed by semi-quantitative analysis of IF signals. In this research we indicate a bioimaging as a useful tool to monitor and semi-quantify the IF pictures during the differentiation of hES into chondrocyte-like. PMID:26354117

  20. Small molecule-mediated directed differentiation of human embryonic stem cells toward ventricular cardiomyocytes.

    PubMed

    Karakikes, Ioannis; Senyei, Grant D; Hansen, Jens; Kong, Chi-Wing; Azeloglu, Evren U; Stillitano, Francesca; Lieu, Deborah K; Wang, Jiaxian; Ren, Lihuan; Hulot, Jean-Sebastien; Iyengar, Ravi; Li, Ronald A; Hajjar, Roger J

    2014-01-01

    The generation of human ventricular cardiomyocytes from human embryonic stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and growth factors, we developed a fully chemically defined, directed differentiation system to generate ventricular-like cardiomyocytes (VCMs) from human embryonic stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the differentiation recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-1 plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies. PMID:24324277

  1. CD133 Is a Marker For Long-Term Repopulating Murine Epidermal Stem Cells

    PubMed Central

    Charruyer, A; Strachan, LR; Yue, L; Toth, AS; Mancianti, ML; Ghadially, R

    2012-01-01

    Maintenance, repair and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells. Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine epidermal stem populations. Putative epidermal stem cell populations were isolated by FACS sorting. The CD133+ population and the subpopulation of CD133+ cells that exhibits high mitochondrial membrane potential (DΨmhi), were enriched for long-term repopulating epidermal stem cells vs. unfractionated cells (3.9 and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133+ and CD133+ΔΨmhi keratinocytes. CD133+ keratinocytes were multipotent and produced significantly more hair follicles than CD133− cells. CD133+ cells were a subset of the previously described integrin α6+CD34+ bulge cell population and 28.9±8.6% were label retaining cells. Thus, murine keratinocytes within the CD133+ and CD133+ΔΨmhi populations contain epidermal stem cells that regenerate epidermis for the long-term, are self-renewing, multipotent, and label-retaining cells. PMID:22763787

  2. Imaging of murine embryonic cardiovascular development using optical coherence tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Huang, Yongyang; Degenhardt, Karl R.; Astrof, Sophie; Zhou, Chao

    2016-03-01

    We have demonstrated the capability of spectral domain optical coherence tomography (SDOCT) system to image full development of mouse embryonic cardiovascular system. Monitoring morphological changes of mouse embryonic heart occurred in different embryonic stages helps identify structural or functional cardiac anomalies and understand how these anomalies lead to congenital heart diseases (CHD) present at birth. In this study, mouse embryo hearts ranging from E9.5 to E15.5 were prepared and imaged in vitro. A customized spectral domain OCT system was used for imaging, with a central wavelength of 1310nm, spectral bandwidth of ~100nm and imaging speed of 47kHz A-scans/s. Axial resolution of this system was 8.3µm in air, and transverse resolution was 6.2 µm with 5X objective. Key features of mouse embryonic cardiovascular development such as vasculature remodeling into circulatory system, separation of atria and ventricles and emergence of valves could be clearly seen in three-dimensional OCT images. Optical clearing was applied to overcome the penetration limit of OCT system. With high resolution, fast imaging speed, 3D imaging capability, OCT proves to be a promising biomedical imaging modality for developmental biology studies, rivaling histology and micro-CT.

  3. Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

    PubMed Central

    Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

    2012-01-01

    Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

  4. Embryonic stem cells derived neuron transplantation recovery in models of parkinsonism in relation to severity of the disorder in rats.

    PubMed

    Haobam, Reena; Tripathy, Debasmita; Kaidery, Navneet A; Mohanakumar, Kochupurackal P

    2015-04-01

    6-Hydroxydopamine (6-OHDA)- and 1-methyl-4-phenylpyridinium (MPP(+))-induced hemi-parkinsonism was investigated in relation to the severity of the disorder in terms of behavioral disability and nigral neuronal loss and recovery regarding the number of stem cell-derived neurons transplanted in the striatum. Intra-median forebrain bundle infusion of the parkinsonian neurotoxins and intra-striatal transplantation of differentiated embryonic stem cells (ESCs) were carried out by rat brain stereotaxic surgery. The severity of the disease was determined using the number of amphetamine- or apomorphine-induced rotations, striatal dopamine levels as estimated by high-performance liquid chromatography (HPLC)-electrochemistry, and the number of surviving tyrosine hydroxylase immunoreactive dopaminergic neurons in the substantia nigra pars compacta. Rats that received unilateral infusion of 6-OHDA or MPP(+) responded with dose-dependent, unilateral bias in turning behavior when amphetamine or apomorphine was administered. Rotational asymmetry in both models correlated significantly well with the loss in the number of nigral dopaminergic neurons and striatal dopamine depletion. Transplantation of 2×10(5) differentiated murine ESCs revealed remarkably similar kinds of recovery in both animal models. The survival of the grafted dopaminergic cells in the striatum was better in animals with low-severity parkinsonism, but poor in the animals with severe parkinsonism. Amphetamine-induced rotational recovery correlated positively with an increasing number of cells transplanted in animals with uniform nigral neuronal lesion. These results suggest that disease severity is an important factor for determining the number of cells to be transplanted in parkinsonian rats for desirable recovery, which may be true in clinical conditions too. PMID:25546608

  5. Embryonic Stem Cells Derived Neuron Transplantation Recovery in Models of Parkinsonism in Relation to Severity of the Disorder in Rats

    PubMed Central

    Haobam, Reena; Tripathy, Debasmita; Kaidery, Navneet A.

    2015-01-01

    Abstract 6-Hydroxydopamine (6-OHDA)- and 1-methyl-4-phenylpyridinium (MPP+)-induced hemi-parkinsonism was investigated in relation to the severity of the disorder in terms of behavioral disability and nigral neuronal loss and recovery regarding the number of stem cell–derived neurons transplanted in the striatum. Intra-median forebrain bundle infusion of the parkinsonian neurotoxins and intra-striatal transplantation of differentiated embryonic stem cells (ESCs) were carried out by rat brain stereotaxic surgery. The severity of the disease was determined using the number of amphetamine- or apomorphine-induced rotations, striatal dopamine levels as estimated by high-performance liquid chromatography (HPLC)-electrochemistry, and the number of surviving tyrosine hydroxylase immunoreactive dopaminergic neurons in the substantia nigra pars compacta. Rats that received unilateral infusion of 6-OHDA or MPP+ responded with dose-dependent, unilateral bias in turning behavior when amphetamine or apomorphine was administered. Rotational asymmetry in both models correlated significantly well with the loss in the number of nigral dopaminergic neurons and striatal dopamine depletion. Transplantation of 2×105 differentiated murine ESCs revealed remarkably similar kinds of recovery in both animal models. The survival of the grafted dopaminergic cells in the striatum was better in animals with low-severity parkinsonism, but poor in the animals with severe parkinsonism. Amphetamine-induced rotational recovery correlated positively with an increasing number of cells transplanted in animals with uniform nigral neuronal lesion. These results suggest that disease severity is an important factor for determining the number of cells to be transplanted in parkinsonian rats for desirable recovery, which may be true in clinical conditions too. PMID:25546608

  6. Competitive Interactions Eliminate Unfit Embryonic Stem Cells at the Onset of Differentiation

    PubMed Central

    Sancho, Margarida; Di-Gregorio, Aida; George, Nancy; Pozzi, Sara; Sánchez, Juan Miguel; Pernaute, Barbara; Rodríguez, Tristan A.

    2013-01-01

    Summary A fundamental question in developmental biology is whether there are mechanisms to detect stem cells with mutations that, although not adversely affecting viability, would compromise their ability to contribute to further development. Here, we show that cell competition is a mechanism regulating the fitness of embryonic stem cells (ESCs). We find that ESCs displaying defective bone morphogenetic protein signaling or defective autophagy or that are tetraploid are eliminated at the onset of differentiation by wild-type cells. This elimination occurs in an apoptosis-dependent manner and is mediated by secreted factors. Furthermore, during this process, we find that establishment of differential c-Myc levels is critical and that c-Myc overexpression is sufficient to induce competitive behavior in ESCs. Cell competition is, therefore, a process that allows recognition and elimination of defective cells during the early stages of development and is likely to play important roles in tissue homeostasis and stem cell maintenance. PMID:23867226

  7. Instructing an Embryonic Stem Cell-Derived Oocyte Fate: Lessons from Endogenous Oogenesis

    PubMed Central

    Nicholas, Cory R.; Chavez, Shawn L.; Baker, Valerie L.; Reijo Pera, Renee A.

    2009-01-01

    Female reproductive potential is limited in the majority of species due to oocyte depletion. Because functional human oocytes are restricted in number and accessibility, a robust system to differentiate oocytes from stem cells would enable a thorough investigation of the genetic, epigenetic, and environmental factors affecting human oocyte development. Also, the differentiation of functional oocytes from stem cells may permit the success of human somatic cell nuclear transfer for reprogramming studies and for the production of patient-specific embryonic stem cells (ESCs). Thus, ESC-derived oocytes could ultimately help to restore fertility in women. Here, we review endogenous and ESC-derived oocyte development, and we discuss the potential and challenges for differentiating functional oocytes from ESCs. PMID:19366753

  8. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  9. RNA Genes: Retroelements and Virally Retroposable microRNAs in Human Embryonic Stem Cells

    PubMed Central

    Fujii, Yoichi R.

    2010-01-01

    Embryonic stem cells (ESCs) are capable of undergoing self-renewal, and their developmental ability is known as the stemness. Recently, microRNAs (miRNAs) as regulators have been isolated from ESCs. Although Dicer and DiGeorge syndrome critical region gene 8 (DGCR8) are essential factors for the biogeneration of miRNA, Dicer-knockout (KO) ESCs have showed to fail to express differentiation markers and DGCR8-KO ESCs have showed to be arrest in the G1 phase. Furthermore, Dicer-KO ESCs lost the ability to epigenetically silence retroelemtns (REs). REs are expressed and transposed in ESCs, whose transcripts control expression of miRNAs, and their transposable retroelement (TE) expression is, therefore related to ESC proliferation and differentiation, suggesting that the interplay between miRNAs and REs may have a deep responsibility for the stemness including a short G1/S transition and for RE regulation in ESCs. PMID:20835360

  10. Embryonic stem (ES) cell-derived cardiomyocytes: a good candidate for cell therapy applications.

    PubMed

    Pal, Rajarshi

    2009-03-01

    During the last decade, embryonic stem cells (ESC) have unleashed new avenues in the field of developmental biology and emerged as a potential tool to understand the molecular mechanisms taking place during the process of differentiation from the embryonic stage to adult phenotype. Their uniqueness lies in retaining the capacity of unlimited proliferation and to differentiate into all somatic cells. Together with promising results from rodent models, ESC has raised great hope among for human ESC-based cell replacement therapy. ESC could potentially revolutionize medicine by providing a powerful and renewable cell source capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as Parkinson's disease, myocardial infarction (MI) and diabetes. Somatic stem cells are an attractive option to explore for transplantation because they are autologous, but their differentiation potential is very limited. Currently, the major sources of somatic cells used for basic research and clinical trials come from bone marrow. But their widespread acceptability has not been gained because many of the results are confusing and inconsistent. The focus here is on human embryonic stem cells (hESCs), using methods to induce their differentiation to cardiomyocytes in vitro. Their properties in relation to primary human cardiomyocytes and their ability to integrate into host myocardium have been investigated into how they can enhance cardiac function. However, important aspects of stem cell biology and the transplantation process remain unresolved. In summary, this review updates the recent progress of ES cell research in cell therapy, discusses the problems in the practical utility of ESC, and evaluates how far this adjunctive experimental approach can be successful. PMID:19121644

  11. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. PMID:26006729

  12. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis.

    PubMed

    Zhang, Juan; Jiao, Jianwei

    2015-01-01

    The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis. PMID:26421301

  13. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis

    PubMed Central

    Zhang, Juan; Jiao, Jianwei

    2015-01-01

    The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis. PMID:26421301

  14. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal.

    PubMed

    Holm, Frida; Hellqvist, Eva; Mason, Cayla N; Ali, Shawn A; Delos-Santos, Nathaniel; Barrett, Christian L; Chun, Hye-Jung; Minden, Mark D; Moore, Richard A; Marra, Marco A; Runza, Valeria; Frazer, Kelly A; Sadarangani, Anil; Jamieson, Catriona H M

    2015-12-15

    Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination. PMID:26621726

  15. Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells.

    PubMed

    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Virutamasen, Pramuan; Pruksananonda, Kamthorn

    2016-04-22

    Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line. PMID:26821869

  16. Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells

    PubMed Central

    RUNGSIWIWUT, Ruttachuk; NUMCHAISRIKA, Pranee; AHNONKITPANIT, Vichuda; VIRUTAMASEN, Pramuan; PRUKSANANONDA, Kamthorn

    2016-01-01

    Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line. PMID:26821869

  17. Lack of Rybp in Mouse Embryonic Stem Cells Impairs Cardiac Differentiation.

    PubMed

    Ujhelly, Olga; Szabo, Viktoria; Kovacs, Gergo; Vajda, Flora; Mallok, Sylvia; Prorok, Janos; Acsai, Karoly; Hegedus, Zoltan; Krebs, Stefan; Dinnyes, Andras; Pirity, Melinda Katalin

    2015-09-15

    Ring1 and Yy1 binding protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1, it has an important function in regulating pluripotency and differentiation of embryonic stem cells (ESCs). Earlier, we had proved that Rybp plays an essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development. Prominent Rybp expression persists during all embryonic stages, and Rybp marks differentiated cell types of the heart. By utilizing rybp null ESCs in an in vitro cardiac differentiation assay, we found that rybp null ESCs do not form rhythmically beating cardiomyocytes (CMCs). Gene expression profiles revealed a downregulation of cardiac terminal and upregulation of germline-specific markers in the rybp null CMCs. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp. Among these are several that are important in cardiac development and contractility such as Plagl1, Isl1, and Tnnt2. Importantly, forced expression of rybp in rybp-deficient ESCs by a lentiviral vector was able to rescue the mutant phenotype. Our data provide evidence for a previously unrecognized function of Rybp in heart development and point out the importance of germ cell lineage gene silencing during somatic differentiation. PMID:26110923

  18. Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues.

    PubMed

    Jebeniani, Imen; Leschik, Julia; Puceat, Michel

    2016-01-01

    Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription. PMID:27285123

  19. A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

    SciTech Connect

    Sauerzweig, Steven Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger

    2009-01-01

    The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.

  20. Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

    PubMed

    Bhartiya, Deepa; Hinduja, Indira; Patel, Hiren; Bhilawadikar, Rashmi

    2014-01-01

    The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing

  1. Very small embryonic-like stem-cell optimization of isolation protocols: an update of molecular signatures and a review of current in vivo applications

    PubMed Central

    Shin, Dong-Myung; Suszynska, Malwina; Mierzejewska, Kasia; Ratajczak, Janina; Ratajczak, Mariusz Z

    2013-01-01

    As the theory of stem cell plasticity was first proposed, we have explored an alternative hypothesis for this phenomenon: namely that adult bone marrow (BM) and umbilical cord blood (UCB) contain more developmentally primitive cells than hematopoietic stem cells (HSCs). In support of this notion, using multiparameter sorting we were able to isolate small Sca1+Lin−CD45− cells and CD133+Lin−CD45− cells from murine BM and human UCB, respectively, which were further enriched for the detection of various early developmental markers such as the SSEA antigen on the surface and the Oct4 and Nanog transcription factors in the nucleus. Similar populations of cells have been found in various organs by our team and others, including the heart, brain and gonads. Owing to their primitive cellular features, such as the high nuclear/cytoplasm ratio and the presence of euchromatin, they are called very small embryonic-like stem cells (VSELs). In the appropriate in vivo models, VSELs differentiate into long-term repopulating HSCs, mesenchymal stem cells (MSCs), lung epithelial cells, cardiomyocytes and gametes. In this review, we discuss the most recent data from our laboratory and other groups regarding the optimal isolation procedures and describe the updated molecular characteristics of VSELs. PMID:24232255

  2. Microgravity Reduces the Differentiation and Regenerative Potential of Embryonic Stem Cells.

    PubMed

    Blaber, Elizabeth A; Finkelstein, Hayley; Dvorochkin, Natalya; Sato, Kevin Y; Yousuf, Rukhsana; Burns, Brendan P; Globus, Ruth K; Almeida, Eduardo A C

    2015-11-15

    Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth's gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth's gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth. PMID:26414276

  3. Microgravity Reduces the Differentiation and Regenerative Potential of Embryonic Stem Cells

    PubMed Central

    Blaber, Elizabeth A.; Finkelstein, Hayley; Dvorochkin, Natalya; Sato, Kevin Y.; Yousuf, Rukhsana; Burns, Brendan P.; Globus, Ruth K.

    2015-01-01

    Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including inhibition of regenerative stem cell differentiation. To address this hypothesis, we investigated the effects of microgravity on early lineage commitment of mouse embryonic stem cells (mESCs) using the embryoid body (EB) model of tissue differentiation. We found that exposure to microgravity for 15 days inhibits mESC differentiation and expression of terminal germ layer lineage markers in EBs. Additionally, microgravity-unloaded EBs retained stem cell self-renewal markers, suggesting that mechanical loading at Earth's gravity is required for normal differentiation of mESCs. Finally, cells recovered from microgravity-unloaded EBs and then cultured at Earth's gravity showed greater stemness, differentiating more readily into contractile cardiomyocyte colonies. These results indicate that mechanical unloading of stem cells in microgravity inhibits their differentiation and preserves stemness, possibly providing a cellular mechanistic basis for the inhibition of tissue regeneration in space and in disuse conditions on earth. PMID:26414276

  4. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells.

    PubMed

    Needham, Karina; Hyakumura, Tomoko; Gunewardene, Niliksha; Dottori, Mirella; Nayagam, Bryony A

    2014-01-01

    In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons. PMID:24280418

  5. Distinct allelic patterns of nanog expression impart embryonic stem cell population heterogeneity.

    PubMed

    Wu, Jincheng; Tzanakakis, Emmanuel S

    2013-01-01

    Nanog is a principal pluripotency regulator exhibiting a disperse distribution within stem cell populations in vivo and in vitro. Increasing evidence points to a functional role of Nanog heterogeneity on stem cell fate decisions. Allelic control of Nanog gene expression was reported recently in mouse embryonic stem cells. To better understand how this mode of regulation influences the observed heterogeneity of NANOG in stem cell populations, we assembled a multiscale stochastic population balance equation framework. In addition to allelic control, gene expression noise and random partitioning at cell division were considered. As a result of allelic Nanog expression, the distribution of Nanog exhibited three distinct states but when combined with transcriptional noise the profile became bimodal. Regardless of their allelic expression pattern, initially uniform populations of stem cells gave rise to the same Nanog heterogeneity within ten cell cycles. Depletion of NANOG content in cells switching off both gene alleles was slower than the accumulation of intracellular NANOG after cells turned on at least one of their Nanog gene copies pointing to Nanog state-dependent dynamics. Allelic transcription of Nanog also raises issues regarding the use of stem cell lines with reporter genes knocked in a single allelic locus. Indeed, significant divergence was observed in the reporter and native protein profiles depending on the difference in their half-lives and insertion of the reporter gene in one or both alleles. In stem cell populations with restricted Nanog expression, allelic regulation facilitates the maintenance of fractions of self-renewing cells with sufficient Nanog content to prevent aberrant loss of pluripotency. Our findings underline the role of allelic control of Nanog expression as a prime determinant of stem cell population heterogeneity and warrant further investigation in the contexts of stem cell specification and cell reprogramming. PMID:23874182

  6. PHENOTYPE AND HEMATOPOIETIC POTENTIAL OF SIDE POPULATION CELLS THROUGHOUT EMBRYONIC DEVELOPMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adult murine bone marrow hematopoietic stem cells (HSCs) can be purified by sorting Hoechst 33342-extruding side population (SP) cells. Herein we investigated whether SP cells reside within embryonic tissues and exhibit hematopoietic progenitor activity. We isolated yolk sac (YS) and embryonic tissu...

  7. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation

    PubMed Central

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2012-01-01

    Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and insitu immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research. PMID:20541472

  8. 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

  9. REST Regulates Distinct Transcriptional Networks in Embryonic and Neural Stem Cells

    PubMed Central

    Kunarso, Galih; Wong, Kee Yew; Srinivasan, Gopalan; Cooper, Megan L; Volta, Manuela; Chan, Sarah Su-ling; Lipovich, Leonard; Pollard, Steven M; Karuturi, R. Krishna Murthy; Wei, Chia-lin; Buckley, Noel J; Stanton, Lawrence W

    2008-01-01

    The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype. PMID:18959480

  10. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  11. Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.

    PubMed

    Gertow, Karin; Przyborski, Stefan; Loring, Jeanne F; Auerbach, Jonathan M; Epifano, Olga; Otonkoski, Timo; Damjanov, Ivan; Ahrlund-Richter, Lars

    2007-10-01

    This unit describes protocols on how to assess the developmental potency of human embryonic stem cells (hESCs) by performing xenografting into immunodeficient mice to induce teratoma formation. hESCs can be injected under the testis capsule, or alternatively into the kidney or subcutaneously. Teratomas that develop from grafted hESCs are surgically removed, fixed in formaldehyde, and paraffin embedded. The tissues in the teratoma are analyzed histologically to determine whether the hESCs are pluripotent and form tissues derived from of all three embryonic germ layers (ectoderm, mesoderm, and endoderm). Teratomas can also be fixed in Bouin's or cryosectioned for analysis, and they can be analyzed by immunohistochemistry for tissue markers. Methods for these procedures are included in this unit. PMID:18785162

  12. Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

    PubMed

    Szade, Krzysztof; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Jozef

    2016-01-01

    The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC. PMID:27138010

  13. Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

    PubMed Central

    Krais, Annette M.; Mühlbauer, Karl-Rudolf; Kucab, Jill E.; Chinbuah, Helena; Cornelius, Michael G.; Wei, Quan-Xiang; Hollstein, Monica; Phillips, David H.; Arlt, Volker M.; Schmeiser, Heinz H.

    2015-01-01

    We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by 32P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells. PMID:25230394

  14. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

    PubMed

    Novo, Clara Lopes; Tang, Calvin; Ahmed, Kashif; Djuric, Ugljesa; Fussner, Eden; Mullin, Nicholas P; Morgan, Natasha P; Hayre, Jasvinder; Sienerth, Arnold R; Elderkin, Sarah; Nishinakamura, Ryuichi; Chambers, Ian; Ellis, James; Bazett-Jones, David P; Rugg-Gunn, Peter J

    2016-05-01

    An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells. PMID:27125671

  15. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells

    PubMed Central

    Thakore-Shah, Kaushali; Koleilat, Tasneem; Jan, Majib; John, Alan; Pyle, April D.

    2015-01-01

    REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down’s syndrome and Huntington’s disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells. PMID:26690059

  16. BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos

    PubMed Central

    Liu, W; Stein, P; Cheng, X; Yang, W; Shao, N-Y; Morrisey, E E; Schultz, R M; You, J

    2014-01-01

    Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant-negative approach suppresses Nanog expression, and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)), a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers. PMID:25146928

  17. The culture of human embryonic stem cells in microchannel perfusion bioreactors

    NASA Astrophysics Data System (ADS)

    Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

    2007-12-01

    The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.

  18. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells

    PubMed Central

    Novo, Clara Lopes; Tang, Calvin; Ahmed, Kashif; Djuric, Ugljesa; Fussner, Eden; Mullin, Nicholas P.; Morgan, Natasha P.; Hayre, Jasvinder; Sienerth, Arnold R.; Elderkin, Sarah; Nishinakamura, Ryuichi; Chambers, Ian; Ellis, James; Bazett-Jones, David P.; Rugg-Gunn, Peter J.

    2016-01-01

    An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells. PMID:27125671

  19. Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Xie, Xiaoyan; Fu, Ji-Dong; Drukker, Micha; Lee, Andrew; Li, Ronald A.; Gambhir, Sanjiv S.; Weissman, Irving L.; Robbins, Robert C.; Wu, Joseph C.

    2008-01-01

    Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies. PMID:18941512

  20. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    PubMed Central

    Abouzaripour, Morteza; Pasbakhsh, Parichehr; Atlasi, Nader; Shahverdi, Abdol Hossein; Mahmoudi, Reza; Kashani, Iraj Ragerdi

    2016-01-01

    Objective Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion Our study clearly demonstrates the potential of SSEA-1 positive

  1. Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

    PubMed Central

    Dukhovny, Anna; Sloutskin, Anna; Markus, Amos; Yee, Michael B.; Kinchington, Paul R.

    2012-01-01

    Pluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of “neurospheres,” immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication. PMID:22238301

  2. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis.

    PubMed

    Zhang, M; Zhu, X; Zhang, Y; Cai, Y; Chen, J; Sivaprakasam, S; Gurav, A; Pi, W; Makala, L; Wu, J; Pace, B; Tuan-Lo, D; Ganapathy, V; Singh, N; Li, H

    2015-12-01

    The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. PMID:25952549

  3. Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes.

    PubMed

    Meyer, S; Nolte, J; Opitz, L; Salinas-Riester, G; Engel, W

    2010-11-01

    DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs). PMID:20624824

  4. MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

    PubMed

    Zhang, Jie Ting; Weng, Zhi Hui; Tsang, Kam Sze; Tsang, Lai Ling; Chan, Hsiao Chang; Jiang, Xiao Hua

    2016-01-01

    The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on MycN have been conducted in human tumor cells, thus, the role of MycN in normal human neural crest development is completely unknown. In the present study, we determined the role of MycN in hNCSCs isolated from in vitro-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of MycN in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of MycN in hNCSCs increases the expression of Cdkn1a, Cdkn2a and Cdkn2b, which encodes the cyclin-dependent kinases p21CIP1, p16 INK4a and p15INK4b. In addition, MycN is involved in the regulation of human sympathetic neurogenesis, as knockdown of MycN enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including Phox2a, Phox2b, Mash1, Hand2 and Gata3. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases. PMID:26815535

  5. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos.

    PubMed

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  6. 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

  7. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    PubMed Central

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

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

    PubMed

    Jin, Li-Fang; Li, Jin-Song

    2016-07-18

    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

  9. Laser-induced fusion of human embryonic stem cells with optical tweezers

    NASA Astrophysics Data System (ADS)

    Chen, Shuxun; Cheng, Jinping; Kong, Chi-Wing; Wang, Xiaolin; Han Cheng, Shuk; Li, Ronald A.; Sun, Dong

    2013-07-01

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  10. Laser-induced fusion of human embryonic stem cells with optical tweezers

    SciTech Connect

    Chen Shuxun; Wang Xiaolin; Sun Dong; Cheng Jinping; Han Cheng, Shuk; Kong, Chi-Wing; Li, Ronald A.

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  11. Derivation of Huntington Disease affected Genea091 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Schaft, Julia; McKernan, Robert; Hu, Jesselyn; Schmidt, Uli

    2016-03-01

    The Genea091 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 40 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 98% SSEA4 and gave a Pluritest pluripotency score of 38.36, Novelty of 1.35. The cell line was negative for Mycoplasma and visible contamination. PMID:27346013

  12. Derivation of DM1 affected human embryonic stem cell line Genea067.

    PubMed

    Dumevska, Biljana; Main, Heather; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea067 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying expansion of CTG repeats in the DMPK gene, indicative of Myotonic Dystrophy Type 1 (DM1). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 97% Oct4, 73% Tra1-60 and 98% SSEA4 and gave a Pluritest Pluripotency score of 25.75, Novelty of 1.46. The cell line was negative for Mycoplasma and visible contamination. PMID:27346009

  13. Derivation of the human embryonic stem cell line RCe012-A (RC-8).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; Bradburn, H; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346021

  14. Derivation of the clinical grade human embryonic stem cell line RCe020-a (RC-16).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe020-A (RC-16) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe020-A (RC-16) shows normal pluripotency marker expression and differentiates to mesoderm and potentially ectoderm in vitro. It has an abnormal 47XX, +14, i(20)(q10) female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346205

  15. Derivation of the human embryonic stem cell line RCe009-A (RC-5).

    PubMed

    De Sousa, P A; Tye, B; Bruce, K; Dand, P; Russell, G; Collins, D M; Gardner, J; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe009-A (RC-5) was derived from a frozen and thawed Day 2 embryo voluntarily donated as unsuitable and surplus to requirement for fertility treatment following informed consent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic fibrosis trans-membrane regulator gene associated with the disease cystic fibrosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available. PMID:27346004

  16. Derivation of DM2 affected human embryonic stem cell line Genea066.

    PubMed

    Dumevska, Biljana; Schaft, Julia; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea066 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying expansion of CCTG repeats in exon 1 of the ZNF9 gene, indicative of Myotonic Dystrophy Type 2 (DM2). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 88% of cells expressed Nanog, 97% Oct4, 80% Tra1-60 and 99% SSEA4 and gave a Pluritest Pluripotency score of 31.3, Novelty of 1.22. The cell line was negative for Mycoplasma and visible contamination. PMID:27346023

  17. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1-60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination. PMID:27346012

  18. Derivation of Huntington Disease affected Genea089 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; McKernan, Robert; Hu, Jesselyn; Schmidt, Uli

    2016-03-01

    The Genea089 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 41 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 91% of cells expressed Nanog, 95% Oct4, 90% Tra1-60 and 100% SSEA4 and gave a PluriTest Pluripotency score of 39.28, Novelty of 1.2. The cell line was negative for Mycoplasma and visible contamination. PMID:27346008

  19. Derivation of the clinical grade human embryonic stem cell line RCe018-A (RC-14).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood group typing data are available. PMID:27346202

  20. Derivation of the human embryonic stem cell line RCe006-A (RC-2).

    PubMed

    De Sousa, P A; Tye, B; Bruce, K; Dand, P; Russell, G; Gardner, J; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Microsatellite DNA marker identity and HLA and blood group typing data are available. PMID:27346014

  1. Derivation of Huntington Disease affected Genea090 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Schaft, Julia; McKernan, Robert; Hu, Jesselyn; Schmidt, Uli

    2016-03-01

    The Genea090 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female allele pattern. The hESC line had pluripotent cell morphology, 91% of cells expressed Nanog, 95% Oct4, 90% Tra1-60 and 100% SSEA4 and gave a pluritest pluripotency score of 30.91, novelty of 1.23. The cell line was negative for Mycoplasma and visible contamination. PMID:27346026

  2. Derivation of Huntington disease affected Genea020 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea020 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 48 repeats, indicative of Huntington disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female allele pattern. The hESC line had pluripotent cell morphology, 89% of cells expressed Nanog, 95% Oct4, 29% Tra1-60 and 99% SSEA4, gave a Pluritest pluripotency score of 27.51, novelty of 1.43 and demonstrated alkaline phosphatase activity. The cell line was negative for Mycoplasma and visible contamination. PMID:27346007

  3. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

    PubMed Central

    Prajumwongs, Piya; Weeranantanapan, Oratai; Jaroonwitchawan, Thiranut; Noisa, Parinya

    2016-01-01

    Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs) recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation. PMID:27239201

  4. Generation of KCL034 clinical grade human embryonic stem cell line

    PubMed Central

    Devito, Liani; Jacquet, Laureen; Petrova, Anastasia; Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL034 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility, specific and non-specific human pathogens.

  5. Generation of KCL033 clinical grade human embryonic stem cell line

    PubMed Central

    Devito, Liani; Petrova, Anastasia; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility and specific and non-specific human pathogens. PMID:27345988

  6. Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe017-A (RC-13) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe017-A (RC-13) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data are available. PMID:27346201

  7. Derivation of the human embryonic stem cell line RCe014-A (RC-10).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; Bradburn, H; Downie, J M; Bateman, M; Courtney, A

    2016-03-01

    The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346029

  8. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP) standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346204

  9. Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15).

    PubMed

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Downie, J M; Bateman, M; Courtney, A

    2016-05-01

    The human embryonic stem cell line RCe019-A (RC-15) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346200

  10. Patenting, morality and human embryonic stem cell science: bioethics and cultural politics in Europe.

    PubMed

    Salter, Brian

    2007-05-01

    As the recent experience of the European Patent Office graphically demonstrates, there is an inherent political tension between the individual ownership rights necessary for the operation of an international market in human embryonic stem cell science and the communal values of the many cultures in which such markets operate. This report examines the basis of the conflict between patenting and morality at national and international levels, the manifestation of those tensions in European patenting policy, and the contribution of bioethics to the attempt by European institutions to develop a governance response. PMID:17511566

  11. Derivation of Trisomy 21 affected human embryonic stem cell line Genea053.

    PubMed

    Dumevska, Biljana; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea053 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analysis demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology and expressed pluripotent cell markers including 83% Nanog positive, 87% Oct4, 88% Tra1-60 and 98% SSEA4. The cell line was negative for Mycoplasma and visible contamination. PMID:27346024

  12. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Main, Heather; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1-60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346003

  13. Expanding the mouse embryonic stem cell proteome: Combining three proteomic approaches

    PubMed Central

    Gundry, Rebekah L.; Tchernyshyov, Irina; Sheng, Shijun; Tarasova, Yelena; Raginski, Kimberly; Boheler, Kenneth R.; Van Eyk, Jennifer E.

    2010-01-01

    The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole-cell lysates were subjected either to 2-D-LC, or 1-DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non-redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform-specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins. PMID:20512790

  14. 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

  15. Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

    PubMed

    Ursu, Andrei; Illich, Damir J; Takemoto, Yasushi; Porfetye, Arthur T; Zhang, Miao; Brockmeyer, Andreas; Janning, Petra; Watanabe, Nobumoto; Osada, Hiroyuki; Vetter, Ingrid R; Ziegler, Slava; Schöler, Hans R; Waldmann, Herbert

    2016-04-21

    The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs. PMID:27049670

  16. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance

    PubMed Central

    Fedorov, Oleg; Castex, Josefina; Tallant, Cynthia; Owen, Dafydd R.; Martin, Sarah; Aldeghi, Matteo; Monteiro, Octovia; Filippakopoulos, Panagis; Picaud, Sarah; Trzupek, John D.; Gerstenberger, Brian S.; Bountra, Chas; Willmann, Dominica; Wells, Christopher; Philpott, Martin; Rogers, Catherine; Biggin, Philip C.; Brennan, Paul E.; Bunnage, Mark E.; Schüle, Roland; Günther, Thomas; Knapp, Stefan; Müller, Susanne

    2015-01-01

    Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5′-triphosphate)–driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine–dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes. PMID:26702435

  17. Decoded calreticulin-deficient embryonic stem cell transcriptome resolves latent cardiophenotype.

    PubMed

    Faustino, Randolph S; Chiriac, Anca; Niederlander, Nicolas J; Nelson, Timothy J; Behfar, Atta; Mishra, Prasanna K; Macura, Slobodan; Michalak, Marek; Terzic, Andre; Perez-Terzic, Carmen

    2010-07-01

    Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype. PMID:20506533

  18. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.

    PubMed

    Fedorov, Oleg; Castex, Josefina; Tallant, Cynthia; Owen, Dafydd R; Martin, Sarah; Aldeghi, Matteo; Monteiro, Octovia; Filippakopoulos, Panagis; Picaud, Sarah; Trzupek, John D; Gerstenberger, Brian S; Bountra, Chas; Willmann, Dominica; Wells, Christopher; Philpott, Martin; Rogers, Catherine; Biggin, Philip C; Brennan, Paul E; Bunnage, Mark E; Schüle, Roland; Günther, Thomas; Knapp, Stefan; Müller, Susanne

    2015-11-01

    Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes. PMID:26702435

  19. 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

  20. The Maintenance of Pluripotency Following Laser Direct-Write of Mouse Embryonic Stem Cells

    PubMed Central

    Raof, Nurazhani Abdul; Schiele, Nathan R; Xie, Yubing; Chrisey, Douglas B; Corr, David T

    2010-01-01

    The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment. PMID:21168910

  1. Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin.

    PubMed

    Forni, Maria Fernanda; Ramos Maia Lobba, Aline; Pereira Ferreira, Alexandre Hamilton; Sogayar, Mari Cleide

    2015-01-01

    The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft. PMID:26462205

  2. Functional screen identifies regulators of murine hematopoietic stem cell repopulation.

    PubMed

    Holmfeldt, Per; Ganuza, Miguel; Marathe, Himangi; He, Bing; Hall, Trent; Kang, Guolian; Moen, Joseph; Pardieck, Jennifer; Saulsberry, Angelica C; Cico, Alba; Gaut, Ludovic; McGoldrick, Daniel; Finkelstein, David; Tan, Kai; McKinney-Freeman, Shannon

    2016-03-01

    Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host. PMID:26880577

  3. Transcriptional repression of p27 is essential for murine embryonic development

    PubMed Central

    Teratake, Youichi; Kuga, Chisa; Hasegawa, Yuta; Sato, Yoshiharu; Kitahashi, Masayasu; Fujimura, Lisa; Watanabe-Takano, Haruko; Sakamoto, Akemi; Arima, Masafumi; Tokuhisa, Takeshi; Hatano, Masahiko

    2016-01-01

    The Nczf gene has been identified as one of Ncx target genes and encodes a novel KRAB zinc-finger protein, which functions as a sequence specific transcriptional repressor. In order to elucidate Nczf functions, we generated Nczf knockout (Nczf−/−) mice. Nczf−/− mice died around embryonic day 8.5 (E8.5) with small body size and impairment of axial rotation. Histopathological analysis revealed that the cell number decreased and pyknotic cells were occasionally observed. We examined the expression of cell cycle related genes in Nczf−/− mice. p27 expression was increased in E8.0 Nczf−/− mice compared to that of wild type mice. Nczf knockdown by siRNA resulted in increased expression of p27 in mouse embryonic fibroblasts (MEFs). Furthermore, p27 promoter luciferase reporter gene analysis confirmed the regulation of p27 mRNA expression by Nczf. Nczf−/−; p27−/− double knockout mice survived until E11.5 and the defect of axial rotation was restored. These data suggest that p27 repression by Nczf is essential in the developing embryo. PMID:27196371

  4. Transcriptional repression of p27 is essential for murine embryonic development.

    PubMed

    Teratake, Youichi; Kuga, Chisa; Hasegawa, Yuta; Sato, Yoshiharu; Kitahashi, Masayasu; Fujimura, Lisa; Watanabe-Takano, Haruko; Sakamoto, Akemi; Arima, Masafumi; Tokuhisa, Takeshi; Hatano, Masahiko

    2016-01-01

    The Nczf gene has been identified as one of Ncx target genes and encodes a novel KRAB zinc-finger protein, which functions as a sequence specific transcriptional repressor. In order to elucidate Nczf functions, we generated Nczf knockout (Nczf-/-) mice. Nczf-/- mice died around embryonic day 8.5 (E8.5) with small body size and impairment of axial rotation. Histopathological analysis revealed that the cell number decreased and pyknotic cells were occasionally observed. We examined the expression of cell cycle related genes in Nczf-/- mice. p27 expression was increased in E8.0 Nczf-/- mice compared to that of wild type mice. Nczf knockdown by siRNA resulted in increased expression of p27 in mouse embryonic fibroblasts (MEFs). Furthermore, p27 promoter luciferase reporter gene analysis confirmed the regulation of p27 mRNA expression by Nczf. Nczf-/-; p27-/- double knockout mice survived until E11.5 and the defect of axial rotation was restored. These data suggest that p27 repression by Nczf is essential in the developing embryo. PMID:27196371

  5. [Ideological conflicts leading to regulation of investigation with embryonic stem cells].

    PubMed

    Brena, Ingrid

    2015-01-01

    Human stem cells, particularly embryonic, have huge therapeutic potential to many degenerative diseases, so they are the subject of intense research in many countries. Because obtaining human stem cells involves the use of zygotes obtained by in vitro fertilization, when they arrive in the blastocyst stage, ethical issues arise that some groups considered insurmountable; in Mexico to date it has not been possible to established a law or rule that regulates the issue. The purpose of this paper is to discuss the ideological conflicts that have led to this situation, and about the light a judgment delivered by the Inter-American Court of Human Rights may shed on a democratic and secular legislation. PMID:25946541

  6. Modeling the Initiation of Ewing Sarcoma Tumorigenesis in Differentiating Human Embryonic Stem Cells

    PubMed Central

    Gordon, David J.; Motwani, Mona; Pellman, David

    2015-01-01

    Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However, despite the well-established role of EWS-FLI1 in tumor initiation, the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark Ewing sarcoma tumors. PMID:26455317

  7. Conversion of embryonic stem cells into extraembryonic lineages by CRISPR-mediated activators

    PubMed Central

    Wei, Shu; Zou, Qingjian; Lai, Sisi; Zhang, Quanjun; Li, Li; Yan, Quanmei; Zhou, Xiaoqing; Zhong, Huilin; Lai, Liangxue

    2016-01-01

    The recently emerged CRISPR/Cas9 technique has opened a new perspective on readily editing specific genes. When combined with transcription activators, it can precisely manipulate endogenous gene expression. Here, we enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus mouse embryonic stem cells (ESCs) were directly converted into two extraembryonic lineages, i.e., typical trophoblast stem cells (TSCs) and extraembryonic endoderm cells (XENCs), which exhibited characters of TSC or XENC derived from the blastocyst extraembryonic lineages such as cell morphology, specific gene expression, and differentiation ability in vitro and in vivo. This study demonstrates that the cell fate can be effectively manipulated by directly activating of specific endogenous gene expression with CRISPR-mediated activator. PMID:26782778

  8. Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells.

    PubMed

    Kalaszczynska, Ilona; Geng, Yan; Iino, Tadafumi; Mizuno, Shin-ichi; Choi, Yoon; Kondratiuk, Ilona; Silver, Daniel P; Wolgemuth, Debra J; Akashi, Koichi; Sicinski, Piotr

    2009-07-23

    Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation. PMID:19592082

  9. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    PubMed Central

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-01

    Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. PMID:26771356

  10. Subaltern biology? Local biologies, Indian odysseys, and the pursuit of human embryonic stem cell therapies.

    PubMed

    Bharadwaj, Aditya

    2013-01-01

    As an emerging life form, stem cells are viewed as global biological entities. This extends Margaret Lock's categorization of local biological or local biologies as emerging from debates about menopause, brain death, and organ transplant. I seek to reconceptualize the local, shape-shifting nature of the biological form and the ontological mutations as inherently subaltern. I draw on a multisited ethnography and a spectrum of global publics from more than 22 countries in search of contentious human embryonic stem cell therapies (hESC) in India. In so doing, I show that the subaltern local biological experience seldom finds utterance and acknowledgement in the face of hegemonic, universalized, epistemic discourse on human body and biological form. PMID:23768220

  11. Dissecting the heterogeneity of gene expressions in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Zou, Ling-Nan; Thomson, Matt; Liu, S. John; Ramanathan, Sharad

    2011-03-01

    A population of genetically identical cells, of the same nominal cell type, and cultured in the same petri dish, will nevertheless often exhibit varying patterns of gene expression. Taking mouse embryonic stem (ES) cells as a model system, we use immunofluorescence and flow cytometry to examine in detail the distribution of expression levels for various transcription factors key to the maintenance of the ES cell identity. We find the population-level distribution of many proteins, once rescaled by the average expression level, have very similar shapes. This suggest the largest component of observed heterogeneity comes from a single source. More subtly, we find the expression many of genes appears to modulate with the cell cycle. This may suggest that the program for maintaining ES cell identity is tightly coupled to the cell cycle machinery. This work is supported by the Harvard Stem Cell Institute and the Jane Coffin Childs Memorial Fund for Medical Research.

  12. The role of oxygen availability in embryonic development and stem cell function

    PubMed Central

    Simon, M. Celeste; Keith, Brian

    2010-01-01

    Low levels of oxygen (O2) occur naturally in developing embryos. Cells respond to their hypoxic microenvironment by stimulating several hypoxia-inducible factors (and other molecules that mediate O2 homeostasis), which then coordinate the development of the blood, vasculature, placenta, nervous system, and other organs. Furthermore, embryonic stem and progenitor cells frequently occupy hypoxic ‘niches’ and low O2 regulates their differentiation. Recent work has revealed an important link between factors involved in regulating stem/progenitor cell behaviour and hypoxia-inducible factors, which provides a molecular framework for hypoxic control of differentiation and cell fate. These findings have important implications for the development of therapies for tissue regeneration and disease. PMID:18285802

  13. DNA methyltransferase-3-dependent nonrandom template segregation in differentiating embryonic stem cells.

    PubMed

    Elabd, Christian; Cousin, Wendy; Chen, Robert Y; Chooljian, Marc S; Pham, Joey T; Conboy, Irina M; Conboy, Michael J

    2013-10-14

    Asymmetry of cell fate is one fundamental property of stem cells, in which one daughter cell self-renews, whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms, such as plants, fungi, and mammals, has already been shown. However, before this current work, asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs), and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing, differentiating human and mouse ESCs. Moreover, we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3), indicating a molecular mechanism that regulates this phenomenon. Furthermore, our data support the hypothesis that retention of chromatids with the "old" template DNA preserves the epigenetic memory of cell fate, whereas localization of "new" DNA strands and de novo DNA methyltransferase to the lineage-destined daughter cell facilitates epigenetic adaptation to a new cell fate. PMID:24127215

  14. Stimulation of human embryonic stem cell-derived cardiomyocytes on thin-film microelectrodes.

    PubMed

    Viitanen, Jouko; Heimala, Päivi; Hokkanen, Ari; Iljin, Kristiina; Kerkelä, Erja; Kolari, Kai; Kattelus, Hannu

    2011-05-01

    We describe successful long-term stimulation of human embryonic stem cell-derived cardiomyocyte clusters on thin-film microelectrode structures in vitro. Interdigitated electrode structures were constructed using plain titanium on glass as the electrode material. Titanium rapidly oxidizes in atmospheric conditions to produce an insulating TiO(χ) layer with high relative permittivity. Capacitive coupling to the incubation medium and to the cells adherent to the electrodes was still efficient, and the dielectric layer prevented electrolysis, allowing a wider window of possible stimulation amplitudes to be used, relative to conducting surfaces. A common hypothesis suggests that to achieve proper differentiation of electroactive cells from the stem cells electrical stimuli are also needed. Spontaneously beating cardiomyocyte clusters were seeded on the glass-electrode surfaces, and we successfully altered and resynchronized a clearly different beat interval. The new pace was reliably maintained for extended periods of several tens of minutes. PMID:21416608

  15. Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc.

    SciTech Connect

    Finney, L.; Vogt, S.; Wolford, J. L.; Chishti, Y.; Jin, Q.; Ward, J.; Chen, L.

    2010-01-01

    The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

  16. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates.

    PubMed

    Keefer, C L; Pant, D; Blomberg, L; Talbot, N C

    2007-03-01

    Embryonic stem (ES) cell lines provide an invaluable research tool for genetic engineering, developmental biology and disease models. These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. While mouse ES cell lines were first established over two decades ago and primate ES cells in the 1990 s, validated ES cell lines have yet to be established in ungulates. Why competent, pluripotent ES cells can be established from certain strains of mice and from primates, and not from cows, sheep, goats or pigs is an on-going topic of interest to animal reproduction scientists. The identification of appropriate stem cell markers, functional cytokine pathways, and key pluripotency-maintaining factors along with the release of more comprehensive bovine and porcine genomes, provide encouragement for establishment of ungulate ES cell lines in the near future. PMID:17097839

  17. Reasonable magic and the nature of alchemy: Jewish reflections on human embryonic stem cell research.

    PubMed

    Zoloth, Laurie

    2002-03-01

    The controversy about research on human embryonic stem cells both divides and defines us, raising fundamental ethical and religious questions about the nature of the self and the limits of science. This article uses Jewish sources to articulate fundamental concerns about the forbiddenness of knowledge in general and of knowledge thought of as magical creation. Alchemy, and the turning of elements into gold and into substances for longevity, and magic used for the creation of living beings was at stake in various Talmudic texts. Since contemporary discourse calls regenerative science magical, and makes claims about its restorative power, careful reflection on when magic is forbidden and when it is responsible allows a novel understanding of ethical questions in stem cell research. PMID:12211267

  18. Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

    PubMed Central

    Bais, Manish; McLean, Jody; Sebastiani, Paola; Young, Megan; Wigner, Nathan; Smith, Temple; Kotton, Darrell N.; Einhorn, Thomas A.; Gerstenfeld, Louis C.

    2009-01-01

    Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture

  19. 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.

  20. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

    PubMed Central

    Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

    2013-01-01

    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 the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the 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 an 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 the 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. PMID:24311969

  1. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells.

    PubMed

    Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z; Gimzewski, James K; Nakano, Atsushi

    2013-08-01

    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 the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the 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 an 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 the 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. PMID:24311969

  2. Organoids and the genetically encoded self‐assembly of embryonic stem cells

    PubMed Central

    Baillie‐Johnson, Peter

    2015-01-01

    Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to “self‐organization,” as often suggested, but rather to a process of genetically encoded self‐assembly where genetic programs encode and control the emergence of biological structures. PMID:26666846

  3. Folic Acid Supplementation Stimulates Notch Signaling and Cell Proliferation in Embryonic Neural Stem Cells

    PubMed Central

    Liu, Huan; Huang, Guo-wei; Zhang, Xu-mei; Ren, Da-lin; X. Wilson, John

    2010-01-01

    The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14–16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system. PMID:20838574

  4. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. PMID:22952932

  5. 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

  6. Murine Hematopoietic Stem cells and Progenitors Express Adrenergic Receptors

    PubMed Central

    Muthu, Kuzhali; Iyer, Sivaraman; He, L-K.; Szilagyi, Andrea; Gamelli, Richard L; Shankar, Ravi; Jones, Stephen B

    2007-01-01

    Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the β-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of α1-, α2- and β2- AR in enriched populations of ER-MP20+ and ER-MP12+ myeloid progenitors, CD117+ and CD34+ multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development. PMID:17428548

  7. Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells.

    PubMed

    Qiu, Juhui; Fan, Xiaoying; Wang, Yixia; Jin, Hongbin; Song, Yixiao; Han, Yang; Huang, Shenghong; Meng, Yaping; Tang, Fuchou; Meng, Anming

    2016-08-01

    Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM. PMID:27252540

  8. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells

    PubMed Central

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-01-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5–100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  9. Efficient differentiation of human embryonic stem cells into functional cerebellar-like cells.

    PubMed

    Erceg, Slaven; Ronaghi, Mohammad; Zipancic, Ivan; Lainez, Sergio; Roselló, Mireia Gárcia; Xiong, Chen; Moreno-Manzano, Victoria; Rodríguez-Jiménez, Fernando Javier; Planells, Rosa; Alvarez-Dolado, Manuel; Bhattacharya, Shom Shanker; Stojkovic, Miodrag

    2010-11-01

    The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum-related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell differentiation into cerebellar-like cells such as granule neurons, Purkinje cells, interneuron, and glial cells. Neurons derived using our protocol showed a T-shaped polarity phenotype and express similar markers to the developed human cerebellum. Electrophysiological measurements confirmed functional electrical properties compatible with these cells. In vivo implantation of differentiated human embryonic stem cells transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular and the Purkinje cell layers and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in the development of cerebellum can be efficiently recapitulated in vitro, which enables the design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases. PMID:20521974

  10. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  11. Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species.

    PubMed

    Serena, Elena; Figallo, Elisa; Tandon, Nina; Cannizzaro, Christopher; Gerecht, Sharon; Elvassore, Nicola; Vunjak-Novakovic, Gordana

    2009-12-10

    Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H(2)O(2). Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS. PMID:19720058

  12. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    PubMed Central

    Ha, Misook; Hong, Soondo

    2016-01-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac. PMID:27075878

  13. The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

    PubMed Central

    Adler, Priit; Aksoy, Irène; Anastassiadis, Konstantinos; Bader, Michael; Billon, Nathalie; Boeuf, Hélène; Bourillot, Pierre-Yves; Buchholz, Frank; Dani, Christian; Doss, Michael Xavier; Forrester, Lesley; Gitton, Murielle; Henrique, Domingos; Hescheler, Jürgen; Himmelbauer, Heinz; Hübner, Norbert; Karantzali, Efthimia; Kretsovali, Androniki; Lubitz, Sandra; Pradier, Laurent; Rai, Meena; Reimand, Jüri; Rolletschek, Alexandra; Sachinidis, Agapios; Savatier, Pierre; Stewart, Francis; Storm, Mike P.; Trouillas, Marina; Vilo, Jaak; Welham, Melanie J.; Winkler, Johannes; Wobus, Anna M.; Hatzopoulos, Antonis K.

    2009-01-01

    Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells. PMID:19727443

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

    PubMed Central

    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. PMID:26091100

  15. STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

    PubMed Central

    Wongtrakoongate, Patompon; Jones, Mark; Gokhale, Paul J.; Andrews, Peter W.

    2013-01-01

    Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells. PMID:23457636

  16. DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

    PubMed

    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

    2016-04-21

    It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-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 N(6)-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, N(6)-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 N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. PMID:27027282

  17. Efficient reprogramming of human and mouse primary extra-embryonic cells to pluripotent stem cells.

    PubMed

    Nagata, Shogo; Toyoda, Masashi; Yamaguchi, Shinpei; Hirano, Kunio; Makino, Hatsune; Nishino, Koichiro; Miyagawa, Yoshitaka; Okita, Hajime; Kiyokawa, Nobutaka; Nakagawa, Masato; Yamanaka, Shinya; Akutsu, Hidenori; Umezawa, Akihiro; Tada, Takashi

    2009-12-01

    Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications. PMID:19912344

  18. Treating cancer with embryonic stem cells: rationale comes from aging studies.

    PubMed

    Ukraintseva, Svetlana V; Yashin, Anatoly I

    2005-01-01

    In an earlier poster paper (1) we proposed that cancer can be viewed not only as a fatal disease but also as a local aberrant, rejuvenation, in an organism, and this fact can be useful for developing new anti-aging and anti-cancer treatments. In this paper we provide additional evidence from human and experimental animal studies in support of this view. First, we discuss cancer genes as candidate targets for anti-aging interventions. We review examples in which the life of experimental animals has been prolonged in situations of increased activity of proto-oncogenes - or decreased activity of tumor suppressors - in normal (non-cancerous) cells in vivo. Studies of genetic polymorphisms revealed similar effects on longevity in humans. Second, we discuss the possibility of treating cancer with embryonic stem cells. The fact that cancer cells do not, age, means that these cells overcome aging host cells. However, cancer cells can be suppressed by young and quickly proliferating non-cancer cells, such as embryonic stem cells. The grafting of these cells in the tumor environment could be a prospective non-toxic anti-cancer treatment. We discuss recent evidence in support of this view. PMID:15569600

  19. Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells.

    PubMed

    Yamane, Junko; Aburatani, Sachiyo; Imanishi, Satoshi; Akanuma, Hiromi; Nagano, Reiko; Kato, Tsuyoshi; Sone, Hideko; Ohsako, Seiichiroh; Fujibuchi, Wataru

    2016-07-01

    Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development. PMID:27207879

  20. Whole transcriptome data analysis of mouse embryonic hematopoietic stem and progenitor cells that lack Geminin expression.

    PubMed

    L Patmanidi, Alexandra; Kanellakis, Nikolaos I; Karamitros, Dimitris; Papadimitriou, Christos; Lygerou, Zoi; Taraviras, Stavros

    2016-06-01

    We performed cDNA microarrays (Affymetrix Mouse Gene 1.0 ST Chip) to analyze the transcriptome of hematopoietic stem and progenitor cells (HSPCs) from E15.5dpc wild type and Geminin (Gmnn) knockout embryos. Lineage negative cells from embryonic livers were isolated using fluorescence activated cell sorting. RNA samples were used to examine the transcriptional programs regulated by Geminin during embryonic hematopoiesis. The data sets were analyzed using the GeneSpring v12.5 platform (Agilent). The list of differentially expressed genes was filtered in meta-analyses to investigate the molecular basis of the phenotype observed in the knockout embryos, which exhibited defective hematopoiesis and death. The data from this study are related to the research article "Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors" (Karamitros et al., 2015) [1]. The microarray dataset has been deposited at the Gene Expression Omnibus (GEO) under accession GEO: GSE53056. PMID:27077091

  1. Transcriptional Profiles of Imprinted Genes in Human Embryonic Stem Cells During In vitro Differentiation

    PubMed Central

    Park, Sang-Wook; Do, Hyo-Sang; Kim, Dongkyu; Ko, Ji-Yun; Lee, Sang-Hun; Han, Yong-Mahn

    2014-01-01

    Background and Objectives: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. Methods and Results: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. Conclusions: Transcriptional expression of imprinted genes is regulated in a cell type- specific manner in hESCs during in vitro differentiation. PMID:25473448

  2. Transforming growth factor-{beta}2 enhances differentiation of cardiac myocytes from embryonic stem cells

    SciTech Connect

    Kumar, Dinender . E-mail: Dinender.Kumar@uvm.edu; Sun, Baiming

    2005-06-24

    Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-{beta} (TGF-{beta}) -{beta}1, -{beta}2, and -{beta}3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-{beta}1, or -{beta}3, treatment of mouse ES cells with TGF-{beta}2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-{beta}2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and {alpha}-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-{beta}2 but not TGF-{beta}1, or -{beta}3 promotes cardiac myocyte differentiation from ES cells.

  3. Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells

    PubMed Central

    Sritanaudomchai, Hathaitip; Ma, Hong; Clepper, Lisa; Gokhale, Sumita; Bogan, Randy; Hennebold, Jon; Wolf, Don; Mitalipov, Shoukhrat

    2010-01-01

    BACKGROUND Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression. METHODS AND RESULTS We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele. CONCLUSION Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes. PMID:20522441

  4. One dimensional self-assembly of mouse embryonic stem cells using an array of hydrogel microstrands

    PubMed Central

    Raof, Nurazhani Abdul; Padgen, Michael R.; Gracias, Alison; Bergkvist, Magnus; Xie, Yubing

    2011-01-01

    The ability of embryonic stem (ES) cells to self-renew indefinitely and to differentiate into multiple cell lineages hold promise for advances in modeling disease progression, screening drugs and treating diseases. To realize these potentials, it is imperative to study self-assembly in an embryonic microenvironment, as this may increase our understanding of ES cell maintenance and differentiation. In this study, we synthesized an array of one-dimensional alginate gel microstrands and aqueous microstrands through an SU-8 filter device by means of capillary action. Furthermore, we investigated self-assembly behaviors and differentiation potentials of mouse ES cells cultured in microstrands of varying diameters. We found that microstrands with an aqueous interior facilitated high density cell culture and formed compact microtissue structures, while microstrands with gelled interiors promote smaller cell-aggregate structures. In particular, we noticed that ES cells collected from one dimensional aqueous microstrands favored the differentiation towards cell lineages of endoderm and mesoderm, whereas those from gelled microstrands preferred to differentiate into ectoderm and mesoderm lineages. In addition to providing a “liquid-like” tubular microenvironment to understand one dimensional self-assembly process of ES cells, this alginate hydrogel microstrand system also offers an alternative way to manipulate the stem cell fate-decision using bioengineered microenvironments. PMID:21459438

  5. Bone scaffold architecture modulates the development of mineralized bone matrix by human embryonic stem cells

    PubMed Central

    Marcos-Campos, Ivan; Marolt, Darja; Petridis, Petros; Bhumiratana, Sarindr; Schmidt, Daniel; Vunjak-Novakovic, Gordana

    2012-01-01

    Decellularized bone has been widely used as a scaffold for bone formation, due to its similarity to the native bone matrix and excellent osteoinductive and biomechanical properties. We have previously shown that human mesenchymal and embryonic stem cells form functional bone matrix on such scaffolds, without the use of growth factors. In this study, we focused on differences in bone matrix that exist even among identical harvesting sites, and the effects of the matrix architecture and mineral content on bone formation by human embryonic stem cells (hESC). Mesenchymal progenitors derived from hESCs were cultured for 5 weeks in decellularized bone scaffolds with three different densities: low (0.281 ± 0.018 mg/mm3), medium (0.434 ± 0.015 mg/mm3) and high (0.618 ± 0.027 mg/mm3). The medium-density group yielded highest densities of cells and newly assembled bone matrix, presumably due to the best balance between the transport of nutrients and metabolites to and from the cells, space for cell infiltration, surface for cell attachment and the mechanical strength of the scaffolds, all of which depend on the scaffold density. Bone mineral was beneficial for the higher expression of bone markers in cultured cells and more robust accumulation of the new bone matrix. PMID:22901965

  6. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid.

    PubMed

    Kashyap, Vasundhra; Gudas, Lorraine J; Brenet, Fabienne; Funk, Patricia; Viale, Agnes; Scandura, Joseph M

    2011-02-01

    Retinoic acid (RA) regulates clustered Hox gene expression during embryogenesis and is required to establish the anterior-posterior body plan. Using mutant embryonic stem cell lines deficient in the RA receptor γ (RARγ) or Hoxa1 3'-RA-responsive element, we studied the kinetics of transcriptional and epigenomic patterning responses to RA. RARγ is essential for RA-induced Hox transcriptional activation, and deletion of its binding site in the Hoxa1 enhancer attenuates transcriptional and epigenomic activation of both Hoxa and Hoxb gene clusters. The kinetics of epigenomic reorganization demonstrate that complete erasure of the polycomb repressive mark H3K27me3 is not necessary to initiate Hox transcription. RARγ is not required to establish the bivalent character of Hox clusters, but RA/RARγ signaling is necessary to erase H3K27me3 from activated Hox genes during embryonic stem cell differentiation. Highly coordinated, long range epigenetic Hox cluster reorganization is closely linked to transcriptional activation and is triggered by RARγ located at the Hoxa1 3'-RA-responsive element. PMID:21087926

  7. Very Small Embryonic-Like Stem Cells: Biology and Therapeutic Potential for Heart Repair

    PubMed Central

    Zuba-Surma, Ewa K.; Wojakowski, Wojciech; Ratajczak, Mariusz Z.

    2011-01-01

    Abstract Very small embryonic-like stem cells (VSELs) represent a population of extremely small nonhematopoietic pluripotent cells that are negative for lineage markers and express Sca-1 in mice and CD133 in humans. Their embryonic-like characteristics include the expression of markers of pluripotency; the ability to give rise to cellular derivatives of all three germ-layers; and the ability to form embryoid-like bodies. Indeed, quiescent VSELs may represent the remnants of epiblast-derived cells in adult organs. After tissue injury, including acute myocardial infarction (MI), bone marrow–derived VSELs are mobilized into the peripheral blood and home to the damaged organ. Given the ability of VSELs to differentiate into cardiomyocytes and endothelial cells, and their ability to secrete various cardioprotective growth factors/cytokines, VSELs may serve as an ideal cellular source for cardiac repair. Consistently, transplantation of VSELs after an acute MI improves left ventricular (LV) structure and function, and these benefits remain stable during long-term follow-up. Although the mechanisms remain under investigation, effects of secreted factors, regeneration of cellular constituents, and stimulation of endogenous stem/progenitors may play combinatorial roles. The purpose of this review is to summarize the current evidence regarding the biologic features of VSELs, and to discuss their potential as cellular substrates for therapeutic cardiac repair. Antioxid. Redox Signal. 15, 1821–1834. PMID:21194389

  8. A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development

    PubMed Central

    Ellery, Paul E. R.; Maroney, Susan A.; Cooley, Brian C.; Luyendyk, James P.; Zogg, Mark; Weiler, Hartmut

    2015-01-01

    Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi−/−) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi+/− mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4−/−), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi−/− embryos, but >40% of expected Tfpi−/−:Par4−/− offspring survived to adulthood. Adult Tfpi−/−:Par4−/− mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi−/−:Par4−/− mice have platelet and fibrin accumulation similar to Par4−/− mice following venous electrolytic injury but were more susceptible than Par4−/− mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi−/−:Par4−/− mice were born with short tails. Tfpi−/−:Par4−/− mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation. PMID:25954015

  9. Embryonic Stem Cell Grafting in Normal and Infarcted Myocardium: Serial Assessment with MR Imaging and PET Dual Detection

    PubMed Central

    Qiao, Hui; Zhang, Hualei; Zheng, Yuanjie; Ponde, Datta E.; Shen, Dinggang; Gao, Fabao; Bakken, Ashley B.; Schmitz, Alexander; Kung, Hank F.; Ferrari, Victor A.; Zhou, Rong

    2009-01-01

    Purpose: To use magnetic resonance (MR) imaging and positron emission tomography (PET) dual detection of cardiac-grafted embryonic stem cells (ESCs) to examine (a) survival and proliferation of ESCs in normal and infarcted myocardium, (b) host macrophage versus grafted ESC contribution to serial MR imaging signal over time, and (c) cardiac function associated with the formation of grafts and whether improvement in cardiac function is related to cardiac differentiation of ESCs. Materials and Methods: All animal procedures were approved by the institutional animal care and use committee. Murine ESCs were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase, HSV1-sr39tk, and also were labeled with superparamagnetic iron oxide (SPIO) particles. Cells were injected directly in the border zone of the infarcted heart or in corresponding regions of normal hearts in athymic rats. PET and MR imaging were performed longitudinally for 4 weeks in the same animals. Results: ESCs survived and underwent proliferation in the infarcted and normal hearts, as demonstrated by serial increases in 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine PET signals. In parallel, the hypointense areas on MR images at the injection sites decreased over time. Double staining for host macrophages and SPIO particles revealed that the majority of SPIO-containing cells were macrophages at week 4 after injection. Left ventricular ejection fraction increased in the ESC-treated rats but decreased in culture media–treated rats, and border-zone function was preserved in ESC-treated animals; however, cardiac differentiation of ESCs was less than 0.5%. Conclusion: Dual-modality imaging permits complementary information in regard to cell survival and proliferation, graft formation, and effects on cardiac function. © RSNA, 2009 PMID:19244049

  10. Influence of in vitro and in vivo oxygen modulation on β cell differentiation from human embryonic stem cells.

    PubMed

    Cechin, Sirlene; Alvarez-Cubela, Silvia; Giraldo, Jaime A; Molano, Ruth D; Villate, Susana; Ricordi, Camillo; Pileggi, Antonello; Inverardi, Luca; Fraker, Christopher A; Domínguez-Bendala, Juan

    2014-03-01

    The possibility of using human embryonic stem (hES) cell-derived β cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional β cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells. We have previously determined that oxygenation is a powerful driver of murine PP differentiation along the endocrine lineage of the pancreas. We hypothesized that targeting physiological oxygen partial pressure (pO2) levels seen in mature islets would help the differentiation of PP cells along the β-cell lineage. This hypothesis was tested both in vivo (by exposing PP-transplanted immunodeficient mice to a daily hyperbaric oxygen regimen) and in vitro (by allowing PP cells to mature in a perfluorocarbon-based culture device designed to carefully adjust pO2 to a desired range). Our results show that oxygen modulation does indeed contribute to enhanced maturation of PP cells, as evidenced by improved engraftment, segregation of α and β cells, body weight maintenance, and rate of diabetes reversal in vivo, and by elevated expression of pancreatic endocrine makers, β-cell differentiation yield, and insulin production in vitro. Our studies confirm the importance of oxygen modulation as a key variable to consider in the design of β-cell differentiation protocols and open the door to future strategies for the transplantation of fully mature β cells. PMID:24375542

  11. Proteomic Shifts in Embryonic Stem Cells with Gene Dose Modifications Suggest the Presence of Balancer Proteins in Protein Regulatory Networks

    PubMed Central

    Mao, Lei; Zabel, Claus; Herrmann, Marion; Nolden, Tobias; Mertes, Florian; Magnol, Laetitia; Chabert, Caroline; Hartl, Daniela; Herault, Yann; Delabar, Jean Maurice; Manke, Thomas; Himmelbauer, Heinz; Klose, Joachim

    2007-01-01

    Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of “balancer” proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the “elasticity” of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions. PMID:18043732

  12. Detection of embryonic stem cell lysate biomarkers by surface plasmon resonance with reduced nonspecific adsorption.

    PubMed

    Tyagi, Deependra; Perez, Javier Batista; Nand, Amita; Zhiqiang, Cheng; Wang, Peizhe; Na, Jie; Zhu, Jingsong

    2015-02-15

    Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study. PMID:25447493

  13. Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

    PubMed

    Feng, Lingling; Cook, Brandoch; Tsai, Su-Yi; Zhou, Ting; LaFlamme, Brooke; Evans, Todd; Chen, Shuibing

    2016-05-31

    Sorely missing from the "toolkit" for directed differentiation of stem/progenitor cells are agonists of the BMP-signaling pathway. Using a high-throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1) inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using single guide RNA (sgRNA) confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/transforming growth factor beta (TGF-β) signaling pathways and a useful pharmacological reagent for directed differentiation of stem cells. PMID:27210748