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PubMed2001

Enrichment of neurons and neural precursors from human embryonic stem cells.

Human embryonic stem (hES) cells proliferate and maintain their pluripotency for over a year in vitro (M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P., Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson. 2000. Dev. Biol. 227: 271-278) and may therefore provide a cell source for cell therapies. hES cells were maintained for over 6 months in vitro (over 100 population doublings) before their ability to differentiate into the neural lineage was evaluated. Differentiation was induced by the formation of embryoid bodies that were subsequently plated onto appropriate substrates in defined medium containing mitogens. These populations contained cells that showed positive immunoreactivity to nestin, polysialylated neural cell adhesion molecule (PS-NCAM) and A2B5. After further maturation, these cells expressed additional neuron-specific antigens (such as MAP-2, synaptophysin, and various neurotransmitters). Calcium imaging demonstrated that these cells responded to neurotransmitter application. Electrophysiological analyses showed that cell membranes contained voltage-dependent channels and that action potentials were triggered by current injection. PS-NCAM and A2B5 immunoselection or culture conditions could be used to produce enriched populations (60-90%) which could be further differentiated into mature neurons. The properties of the hES-derived progenitors and neurons were found to be similar to those of cells derived from primary tissue. These data indicate that hES cells could provide a cell source for the neural progenitor cells and mature neurons for therapeutic and toxicological uses.

Calcium↗

PubMed1991

Establishment of a germ-line competent C57BL/6 embryonic stem cell line.

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.

Animals↗

PubMed2003

Expressions of hepatobiliary organic anion transporters and bilirubin-conjugating enzyme in differentiating embryonic stem cells.

Mouse embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. We previously reported that ES cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (HNF-3beta). In the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugt1a1) in undifferentiated and differentiating HNF-3beta-transfected ES (HNF-3beta-ES) cells. The expression of organic anion transporting polypeptide 1 (oatp1), multidrug resistance-associated protein 1 (mrp1), mrp2, mrp3, and ugt1a1 was not seen in the undifferentiated HNF-3beta-ES cells by RT-PCR, whereas all were expressed in differentiating HNF-3beta-ES cells. Protein expression for oatp1, mrp1, mrp2, mrp3, and ugt1a1 was also observed in the differentiating HNF-3beta-ES cells by Western blotting. An immunofluorescence examination revealed that oatp1 was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with CD26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes.

Animals↗

PubMed2004

Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (mES) cells have the potential to differentiate into all types of cells, but the physiological properties of undifferentiated mES cells, including Ca2+ signaling systems, are not fully understood. In this study, we investigated Ca2+ signaling pathways in mES cells by using confocal Ca2+ imaging systems, patch clamp techniques and RT-PCR. The stimulations with ATP and histamine (His) induced a transient increase of intracellular Ca2+ concentration ([Ca2+]i), which were prevented by the pretreatment of 2-amino-ethoxydiphenyl borate (2-APB), a blocker for inositol-1,4,5-triphosphate receptors (InsP3Rs). The application of caffeine (Caff) or ryanodine (Ry) did not change [Ca2+]i. When stores were depleted with Ca2+ -ATPase blocker, thapsigargin (TG), or histamine, the capacitative Ca2+ entry (CCE) was observed. In whole cell patch clamp mode, store-operated Ca2+ currents could be recorded in cells treated with histamine and thapsigargin. On the other hand, voltage-operated Ca2+ channels (VOCCs) could not be elicited. The application of blockers for plasma membrane Ca2+ pump (PMCAs) (carboxeosin or caloxin2A1) induced a large increase of [Ca2+]i. When the Na+/Ca2+ exchangers (NCXs) were blocked by Na+ free solution or KBR7943, [Ca2+]i was also elevated. Using RT-PCR, mRNAs for InsP3Rs type-1, -2, and -3, PMCA-1 and -4, NCX-1, -2, and -3 could be detected. From these results, we conclude that Ca2+ release from ER is mediated by InsP3Rs in mES cells before differentiation and Ca2+ entry through plasma membrane is mainly mediated by the store-operated Ca2+ channels (SOCs). For the Ca2+ extrusion systems, both NCXs and PMCAs play important roles for maintaining the low level of [Ca2+]i.

Animals↗

PubMed2005

Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells.

Human embryonic stem (hES) cells may generate all major somatic cell types, yet no neuronal subtype has yet been specifically generated in useful purity from hES culture. We report here the selective induction and isolation of functional spinal motor neurons (MNs) from human ES cells. hES cells of the H1 line were transfected with plasmids encoding GFP placed under the control of an MN-specifying enhancer within the 5'-regulatory region of the gene encoding the transcription factor Hb9 and treated with sonic hedgehog (Shh) and retinoic acid (RA). As MNs were induced under the influence of Shh and RA, they activated Hb9-driven GFP expression, permitting their isolation by fluorescence-activated cell sorting (FACS). The MNs thereby generated and isolated became cholinergic and achieved functional maturation in vitro, as evidenced by their fast sodium currents and action potentials on whole-cell patch-clamp and alpha-bungarotoxin-identified clustering of AChR receptors on co-cultured skeletal myoblasts. The serial combination of these two approaches, motor neuron phenotypic induction followed by Hb9 enhancer-based FACS, permitted the high-efficiency induction and isolation of functional motor neurons from hES cells. These results suggest the utility of promoter/enhancer-based FACS for the isolation of specific phenotypes from hES cell populations as a means of purifying clinically appropriate vectors for cell therapy.

Acetylcholinesterase↗

PubMed2001

Induced neuronal differentiation of human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells capable of forming differentiated embryoid bodies (EBs) in culture. We examined the ability of growth factors under controlled conditions to increase the number of human ES cell-derived neurons. Retinoic acid (RA) and nerve growth factor (betaNGF) were found to be potent enhancers of neuronal differentiation, eliciting extensive outgrowth of processes and the expression of neuron-specific molecules. Our findings show that human ES cells have great potential to become an unlimited cell source for neurons in culture. These cells may then be used in transplantation therapies for neural pathologies.

Antineoplastic Agents↗

PubMed2000

MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.

Acetyltransferases↗

PubMed2003

Pharmacological potential of embryonic stem cells.

Established embryonic stem (ES) cell lines have been at the forefront of approaches to understand gene function during embryogenesis and in adult vertebrate organisms, principally due to exploitation of two essential attributes; their pluripotency or ability to contribute to all three germinal layers and germ line in mice and their ease of genetic modification. Endeavours to routinely establish ES cells from species other than mice have met with limited success, although with rapid progress being made in our understanding of their basic cell biology, the regular derivation of lines from pre-implantation embryos will become easier for many species including humans. With a recent growing awareness of how these cells can be made to grow in an unlimited, but regulated manner plus how their fate can be directed or manipulated into diverse, mature phenotypes in culture, it has become clear that the biological resource offers additional attractive features applicable for future biomedical research and therapy. Advanced mouse ES-based technologies are being used in the industry for pharmaceutical discovery and development, while it is also anticipated that human ES cell reagents will revolutionise aspects of regenerative medicine. This review will summarise the advantages, potential and great hope for ES cell based systems in these contexts.

Animals↗

PubMed2004

Immobilization of leukemia inhibitory factor (LIF) to culture murine embryonic stem cells.

Murine embryonic stem (ES) cells were cultured on a material containing immobilized leukemia inhibitory factor (LIF). To immobilize LIF, we synthesized photoreactive gelatin mixed with LIF and cast the mixture on a polystyrene plate, which was then dried. LIF was immobilized by photoirradiation in the presence or absence of a photo mask. The plate was washed until LIF was no longer released. Murine ES cells were cultured on the immobilized LIF. Activation of STAT3 was maintained on the immobilized LIF for 6 d even after removing soluble LIF. Oct-3/4 was also expressed in the cells cultured on the immobilized LIF. As a result, the mouse ES cells were cultured without differentiating on the immobilized LIF for 6 d. It was possible to culture murine ES cells without adding soluble LIF at each medium change. We conclude that our material containing immobilized LIF might be useful in the culture of murine ES cells.

Journal Article↗

PubMed2005

Efficient gene delivery in differentiated human embryonic stem cells.

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Actins↗

PubMed2006

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

Animals↗

PubMed2002

Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells are known to express D-type cyclins at very low levels and these levels increase dramatically during in vitro and in vivo differentiation. Here, we investigate some of the signalling pathways regulating expression of cyclin D1 and progression to S phase, the Ras/Extracellular signal-regulated protein kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. We demonstrate that ERK phosphorylation is fully dispensable for the regulation of cyclin D1 level and for the progression from G1 to S phase in ES cells. By contrast, PI3-kinase activity is required for both. Differentiation induced by retinoic acid results in the gain of ERK-dependent control of cyclin D1 expression and of S phase progression. Differentiation is also paralleled by an increase in PI3-kinase activity. This leads (a) to an increase in the p70 S6 kinase-dependent regulation of the steady-state level of cyclin D1, and (b) to a concomitant decrease in the GSK3beta-dependent rate of cyclin D1 degradation. Altogether, these multiple pathways account for the dramatic increase in the level of cyclin D1 protein which parallels ES cell differentiation. Our studies suggest that PI3-kinase is an important regulator of the ES cell cycle and that its activity is not regulated by mitogen stimulation.

Animals↗

PubMed1998

Production of medakafish chimeras from a stable embryonic stem cell line.

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Animals↗

PubMed2001

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Animals↗

PubMed2006

Strategies for preventing immunologic rejection of transplanted human embryonic stem cells.

Human embryonic stem alls (hESC) have an unlimited capacity of proliferation and self-renewal resulting in a promise for future applications in regenerative medicine. One major problem derived from their use in cellular therapy protocols is the immunological rejection due to HLA incompatibility. Currently, there are four strategies to prevent allograft rejection of hESC; the development of a ''universal hESC line'' with lack of HLA class 1 expression; the creation of nuclear transfer hESC line; the development of hESC line banks; and the generation of haemopoietic chimerism.

Embryonic Stem Cells↗

PubMed1994

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.

Animals↗

PubMed2001

Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells.

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

Animals↗

PubMed2004

Propagation and maintenance of undifferentiated human embryonic stem cells.

Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clinical uses have not yet been realized from the study of hES cells. Anticipation that these cells would be immediately useful for creating models of human disease has not yet been fulfilled. However, because of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for many areas of research and medicine. A major problem complicating developments in hES cell research is the difficulty of propagating and maintaining these cells in vitro without differentiation. This review addresses this problem and potential solutions in detail. In addition, the current state of research regarding the growth and maintenance of hES cells is summarized, along with basic protocols utilized by our laboratory for the successful propagation, characterization, and investigation of hES cells.

Animals↗