Undifferentiated mES Cells Research Articles

Expansion of embryonic stem cells in suspension and fibrous bed bioreactors

Applications of embryonic stem (ES) cells in cellular transplantation and tissue engineering require scalable processes for mass production of these cells with controlled qualities. The main objective of this work was to evaluate two cell culture processes for long-term expansion of murine embryonic stem (mES) cells. With serial passaging, suspension cultures in spinner flasks were able to expand mES cells as aggregates for 12.5-fold in each passage of 4 days. However, extending the culturing time to 6 days in each passage caused significant loss in cell viability and induced differentiation as indicated by the reduced expression levels of SSEA-1 and Oct-4. Long-term expansion of mES cells in a fibrous bed bioreactor (FBB) was also studied for 30 days in 2 passages, 15 days in each passage. With periodically refreshing the culture medium, a high expansion fold of 60–77 was achieved in each passage. Flow cytometry and RT-PCR were used to analyze key pluripotency and differentiation markers. The results showed that the expanded cells in both suspension and FBB cultures remained in a highly pluripotent state, which was also confirmed with the embryoid body (EB) forming efficiency test. It is concluded that both the suspension and FBB cultures are suitable to support long-term expansion of undifferentiated mES cells. However, the FBB culture can sustain cell growth for a longer period without frequent passaging, requires less media and labor, and is thus more economical to use for mass production of ES cells.

CD26/Dipeptidylpeptidase IV Negatively Regulates Leukemia Inhibitory Factory Activity in Murine Embryonic Stem Cells

Abstract 4741Leukemia inhibitory factory (LIF), a member of the interleukin 6 family, promotes maintenance and self-renewal of embryonic stem (ES) cells (Smith, A.G., J.K. Heath, 1998, Nature 336(6200): 688–690). Binding of LIF to the leukemia inhibitory factory receptor (LIFR) on mouse (m) ES cells activates the Janus tyrosine associated kinase (JAK)-signal transducer activator of transcription 3 (STAT3) pathway, which positively controls mES cell self-renewal and maintenance (Cartwright, P., C. McLean, 2005, Development 132(5): 885–896). CD26/ Dipeptidylpeptidase IV (DPPIV) is a 110KD membrane associated peptidase expressed on epithelial cells of the intestine, kidney, liver, lung and prostate as well as the intestine, melanocytes and certain leukocyte subsets including hematopoietic stem and progenitor cells. Possible substrates of DPPIV include several critical cytokines, chemokines, peptide hormones as well as neuropeptides. Our lab has recently reported that a number of cytokines such as GM-CSF, G-CSF, IL-3 and Epo have DPPIV truncation sites and these truncated cytokines have greatly reduced potency compared to the full-length (FL) form of the molecule. Moreover, the truncated (T) forms blocked the full effect of the FL form. A search of two databases showed that murine LIF had a putative DPPIV truncation site. Based on this, we set up experiments that demonstrated mES cells express CD26, and then we hypothesized that CD26/DPPIV-truncation controls the potency of LIF in mES cells. CD26/DPPIV inhibitor, Diprotin A, was utilized to evaluate proliferation and the potency of mES cells. The expression of surface marker SSEA-1 and transcription factors Oct4, Nanog and KLF-4, all of which are usually used as markers for undifferentiated mES cells, were analyzed by flow cytometry after cells were pretreated with and without Diprotin A (5mM) and then placed in the absence or presence of 0.1 unit, 1 unit, 10 unit, 100 unit, and 1000 units LIF. Pretreating cells with Diprotin A 1hour prior to LIF addition greatly increased LIF potency as demonstrated by significantly increased expression of SSEA-1, Oct4, Nanog and KLF-4, although the expression levels of these four factors differed depending on LIF dose. Next, formation of embryoid bodies (EBs) was assessed to evaluate pluripotency and differentiation potential of ES cells. The cells were pretreated with Diprotin A prior to LIF addition, cultured in the absence or presence of 0.1 unit, 1 unit, 10 unit, 100 unit, and 1000 units LIF for 5 days, and then the cells were assayed for EB formation by removal of LIF. Pretreatment cells with Diprotin A resulted in significant enhancement of EB formation in cells cultured with low dose (10 unit and 100 unit) LIF, while there was no difference in other doses. To better understand signaling events in mES cells, we compared FL- and T- LIF, in varying ratios, for effect on phosphorylation of STAT3 in mES cells. FL- LIF significantly enhanced phosphorylation of STAT3 after 30 minute stimulation. T-LIF elicited decreased STAT3 phosphorylation and partially blocked the effect of FL-LIF activation of STAT3. Taken together, these results demonstrate that CD26/DPPIV negatively impacts in vitro activity of LIF for maintenance and self-renewal of mES cells, and inhibition of DPPIV greatly enhances in vitro LIF activities. The activity of T- vs. FL LIF was mechanistically linked to differing signaling capacities that are involved in maintenance and self-renewal of mES cells. Disclosures:Broxmeyer:CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.

Self-renewal of embryonic stem cells through culture on nanopattern polydimethylsiloxane substrate

Embryonic stem (ES) cells can undergo continual proliferation and differentiation into cells of all somatic cell lineages in vitro; they are an unlimited cell source for regenerative medicine. However, techniques for maintaining undifferentiated ES cells are often inefficient and result in heterogeneous cell populations. Here, we determined effects of nanopattern polydimethylsiloxane (PDMS) as a culture substrate in promoting the self-renewal of mouse ES (mES) cells, compared to commercial plastic culture dishes. After many passages, mES cells efficiently maintained their undifferentiated state on nanopattern PDMS, but randomly differentiated on commercial plastic culture dishes, as indicated by partially altered morphologies and decreases in alkaline phosphatase activity and stage-specific expression of embryonic antigen-1. Under nanopattern PDMS conditions, we found increased activities of STAT3 and Akt, important proteins involved in maintaining the self-renewal of mES cells. The substrate-cell interactions also enhanced leukemia inhibitory factor (LIF)-downstream signaling and inhibited spontaneous differentiation, concomitant with reduced focal adhesion kinase (FAK) signaling. This reduction in FAK signaling was shown to be important for promoting mES cell self-renewal. Thus, our data demonstrates that nanopattern PDMS contributes to maintaining the self-renewal of mES cells and may be applicable in the large-scale production of homogeneously undifferentiated mES cells.

Stem Cell Extracellular Matrix Interactions in Three- Dimensional System via Integrins

Stem Cell Extracellular Matrix Interactions in Three- Dimensional System via Integrins Cells of all types interact with the extracellular matrix (ECM) through integrin-mediated interactions. Adhesion of embryonic stem (ES) cells is necessary for differentiation. We have characterized integrin expression of undifferentiated mES cells and established a system of evaluating the ECM-integrin interactions in three-dimension (3D) using electrospun scaffolds. We observed a higher proliferation rate at early time points in conventional 2D culture with various ECM proteins (collagen IV, laminin, fibronectin, vitronectin) as compared to 3D conditions. At later time points, we observed higher proliferations in 3D culture conditions compared to 2D culture conditions. We also detected the importance of α5, αV, β1, β5 and αVβ5 integrin subunits for cellular adhesion in 3D conditions using a cellular adhesion assay.

Enhanced Collagen Type IV Based Differentiation of Embryonic Stem Cells Towards Flk‐1 Expressing Vascular Progenitors by the Wnt/β‐Catenin Synergist QS11

AbstractMouse embryonic stem (mES) cells when plated onto collagen type IV in the presence of serum‐containing medium differentiate into a mixed cell population that contains low levels of Flk‐1 expressing vascular progenitor cells. When isolated and re‐plated onto collagen type IV, Flk‐1+ cells further differentiate into PECAM1+ endothelial and SMA+ mural cells. However, the low abundance and transient nature by which Flk‐1+ cells are generated during embryonic stem (ES) cell differentiation presents limitations in case large numbers of Flk‐1+ cells or its derivates are to be obtained. To optimize Flk‐1 progenitor induction from undifferentiated mES cells, the effects of QS11, a small organic molecule that synergistically activates canonical Wnt signalling, were investigated. In the presence of QS11 the percentage of Flk‐1+ cells in differentiating cultures of mES cells increases 1.5 fold. Furthermore, QS11 enhances cell proliferation in differentiating mES cells that results in a 2 fold increase in cell numbers when Flk‐1 induction is maximal. The combined effects of QS11 on differentiation and proliferation increase the efficiency by which Flk‐1 progenitors can be generated by approximately 300%, thereby providing a novel tool for vascular progenitor cell production for use in fundamental research and applications such as tissue engineering.

ReProGlo: A new stem cell-based reporter assay aimed to predict embryotoxic potential of drugs and chemicals

A stem cell-based reporter assay was developed to detect drug-induced alterations in the canonical Wnt/β-catenin signaling pathway, which is involved in the regulation of early embryonic development. The so-called ReProGlo assay allows simultaneous determination of cell viability and luciferase reporter activity in a high throughput 96-well microtiter format. A clone of mouse embryonic stem (mES) cells stably expressing the SuperTopFlash reporter was established. This allows Wnt pathway activity determinations in undifferentiated mES cells and their differentiated descendants. Several test chemicals were analyzed in the new assay system. Known embryotoxicants like retinoic acid or lithium chloride induced concentration-dependent increases in reporter activity. The potency of valproic acid and a series of structural analogs to activate the Wnt pathway correlated well with their reported teratogenic activity in the mouse. Cyclophosphamide was also active but only after metabolic activation by hepatocytes. The new test may help to predict embryotoxic potential of chemicals.

Effects of Reconstituted Collagen Matrix on Fates of Mouse Embryonic Stem Cells Before and After Induction for Chondrogenic Differentiation

Embryonic stem (ES) cells are pluripotent cells with great potential in regenerative medicine. However, controlling their differentiation toward homogeneous lineages is challenging. In this study, we aim to investigate the effects of reconstituted 3D collagen matrix on the fates of mouse ES (mES) cells before and after induction for chondrogenic differentiation. Specifically, mES cells were encapsulated and cultured in 3D collagen microspheres and exposed to induction signals at different time points. Growth characteristics and differentiation status of mES cells were then evaluated. Collagen microspheres provided a suitable microenvironment supporting mES cell growth and maintaining their undifferentiated status for certain period of time. At later time points, the proportion of undifferentiated mES cells gradually decreased, accompanied by increasing proportions of mesenchymal progenitor cells. This suggests the inductive role of collagen matrix in differentiating mES cells toward mesenchymal lineages. Moreover, a lower initial collagen monomer concentration facilitated the differentiation of mES cells into chondrogenic lineages, while induction at a later time point associated with a more advanced stage of chondrogenic differentiation. This indicates that both the initial collagen concentration and the time to induce differentiation significantly affected the fates of mES cells. This study contributes to future development of ES cell-based therapies.

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Paraquat toxicity in a mouse embryonic stem cell model

The objective of this in vitro study was to use a mouse embryonic stem (mES) cell model to better understand pesticide injury that may adversely affect early pregnancy and to evaluate an antioxidant intervention. Undifferentiated D3 mES cells were incubated 24h with control, reference dose (RfD), no observed effect level (NOEL), or lowest observed effect level (LOEL) of paraquat, a commonly used, toxic agricultural herbicide. Pesticide effects were evaluated at 0 and 24h using assays for cell proliferation, total reactive oxygen species (ROS), viability, and alkaline phosphatase activity. Compared to 0 h, cell proliferation increased significantly in the 24h control treatment and was stalled in all paraquat dilutions tested. ROS production and percent necrotic and apoptotic cells were significantly increased at all paraquat concentrations examined. Alkaline phosphatase activity suggested that cells remained undifferentiated during the study period. Experiments with ascorbic acid suggested that pesticide effects on cell viability and ROS production were minimized by the recommended daily allowance (RDA) of vitamin C. Data suggest pesticide-induced injury can occur very early in development and at concentrations predicted without health consequences. Mouse ES cells may provide a useful in vitro model for rapidly screening developmental toxicants and protective interventions.

Insulin receptor substrate (IRS)‐1 regulates murine embryonic stem (mES) cells self‐renewal

Mouse embryonic stem (mES) cells are pluripotent cells that can be propagated in vitro with leukemia inhibitory factor (LIF) and serum. Intracellular signaling by LIF is principally mediated by activation of STAT-3, although additional pathways for self-renewal have been described. Here, we identified a novel role for Insulin receptor substrate-1 (IRS-1) as a critical factor in mES cells self-renewal and differentiation. IRS-1 is expressed and tyrosyl phosphorylated during mES cells self-renewal. Differentiation of mES cells, by LIF withdrawal, is associated with a marked reduction in IRS-1 expression. Targeting of IRS-1 by si-IRS-1 results in a severe reduction of Oct-4 protein expression and alkaline phosphatase activity, markers of undifferentiated mES cells. IRS-1 targeting does not interfere with LIF-induced STAT-3 phosphorylation, but negatively affects protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3beta) phosphorylation, which are downstream effectors of the LIF-mediated PI3K signaling cascade. Targeting of IRS-1 also results in a marked down regulation of Id-1 and Id-2 proteins expression, which are important components for self-renewal of ES cells. Conversely, over expression of IRS-1 inhibits mES cell differentiation. Taken together, these results suggest that expression and activity of IRS-1 are critical to the maintenance of the self-renewal program in mES cells.

Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca(2+) signals during the G(0) phase, the transition from G(1) to S, and from G(2) to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G(1) and G(2), we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca(2+) concentration during cell cycle progression. With confocal imaging of the Ca(2+)-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca(2+) oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca(2+) influx but depended on inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and the refilling of intracellular stores by a store-operated Ca(2+) influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca(2+) oscillations were confined to the G(1)/S phase ( approximately 70% oscillating cells vs. G(2)/M with approximately 15% oscillating cells) of the cell cycle. ATP induced Ca(2+) oscillations, and activation of SOC could be induced in G(1)/S and G(2)/M synchronized cells. Intracellular Ca(2+) stores were not depleted, and all three IP(3) receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP(3)-mediated Ca(2+) release is necessary for cell cycle progression through G(1)/S. Because the IP(3) receptor sensitizer thimerosal induced Ca(2+) oscillations only in G(1)/S, we propose that changes in IP(3) receptor sensitivity or basal levels of IP(3) could be the basis for the G(1)/S-confined Ca(2+) oscillations.

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

Comparison of Matrigel™ and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing

Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel™ with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel™ or 0.1% gelatin up to passage 7 (P 7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P ⩾ 0.09). In other comparisons, however, Matrigel™ supported significantly higher synthesis of alkaline phosphatase (7.7 × 10 −3 ± 0.8 vs 6.6 × 10 −3 ± 0.8 units/liter/cell, respectively, P = 0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite ( P < 0.0001), and percent development to beating cardiomyocytes at P 7 ( P = 0.01). Together, our findings suggest that Matrigel™ shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.

Na +/K + ATPase and its functional coupling with Na +/Ca 2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na +/K + ATPase (Na + pump) or Na +/Ca 2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na +/K + ATPase and Na +/Ca 2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na +] i and Na + pump current ( I p), it was shown that an ouabain-high sensitive Na +/K + ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for α1-subunit and α3-subunit of the Na +/K + ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast α2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na +/Ca 2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca 2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca 2+] i oscillations by blocking of Na +/K + ATPase, suggesting the functional coupling with Na +/Ca 2+ exchanger. From these results and RT-PCR analysis, we conclude that α2-subunit Na +/K + ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

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

Combined effects of Matrigel TM and growth factors on maintaining undifferentiated murine embryonic stem cells for embryotoxicity testing

Undifferentiated, murine embryonic stem (mES) cells have shown promise as a substrate for identifying embryotoxic chemicals and for studying mechanisms of early developmental injury. However, long-term maintenance of mES cells in an undifferentiated state is problematic. The present study evaluates the combination of Matrigel TM matrix and three growth factors for this purpose. Biomarkers of mES cell pluripotency, apoptosis, chromosome number and cardiomyocyte differentiation were monitored over 119 population doublings. D3 mES cells retained undifferentiated characteristics, including sustained expression of alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) and continued transcription of Pou5f1 ( Oct-4). Cell viability remained at ⩾95% and population-doubling times averaged 14.3 h over 10 weeks of observation. Caspase-3 activation, a marker of cellular death by apoptosis, was measured in early- and late-passage mES cells. Early-passage cells showed dose-responsive caspase-3 activation following exposure to sodium arsenite, whereas caspase-3 activation of late-passage cells dropped to background levels at toxicant dosages above 50 ppb. Aneuploidy and impaired differentiation into beating cardiomyocytes were noted for late-passage mES cells. Matrigel TM, combined with growth factors, may sustain undifferentiated mES cells. However, aneuploidy, reduced caspase-3 activation, and inability to differentiate suggests further modifications to the culture system may be needed for long-term propagation of cells for embryotoxicity endpoints.

Human embryonic stem cells--realising the potential.

Murine embryonic stem (mES) cells are permanent tissue culture cell lines isolated from explanted blastocysts. mES cells share an unusual set of properties with the stem cells of germ cell tumours (EC cells). Both cell types can be cultured under conditions in which they cycle indefinitely, or in which they terminally differentiate into a variety of specialised cells. When undifferentiated mES or EC cells are returned to the preimplantation embryo they resume a normal programme of development and give rise to chimaeric animals. However, only mES cells give rise to germ-line contributions. In mice the importance of mES cells has been predominantly as a tool to effect very precise genetic changes in transgenic animals by manipulating mES cells in vitro prior to blastocyst injection. The differentiation of mES cells in vitro also gives rise to a wide range of differentiated cell types although this occurs in a much less organised manner than in the blastocyst. In vitro differentiation of mES cells suggests obvious therapeutic applications for ES cells in the treatment of degenerative and metabolic disease.KeywordsStem CellRegenerative MedicineHuman Embryonic Stem CellAdult Stem CellEmbryoid BodyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.