Inhibition of MAGEA2 regulates pluripotency,proliferation, apoptosis,and differentiation in mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.     

CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing

Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G₁ phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G₁ phase, although the details of this mechanism are not well understood. Similarly, the role of 3′ end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3′ end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G₁ phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3′ end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3′ end processing.     

The effect of potassium on the cell membrane potential and the passage of synchronized cells through the cell cycle

The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the G₁ phase. A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K⁺. Cells placed directly into high K⁺ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K⁺ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase in isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early G₁ which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.     

The effect of extracellular matrix on the differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are promising research materials to investigate cell fate determination since they have the capability to differentiate. Stem cell differentiation has been extensively studied with various microenvironment mimicking structures to modify cellular dynamics associated with the cell-extracellular matrix (ECM) interactions and cell-cell communications. In the current study, our aim was to determine the effect of microenvironmental proteins with different concentrations on the capacity and differentiation capability of mouse ESCs (mESCs), combining the biochemical assays, imaging techniques, Fourier transform infrared (FTIR) spectroscopy, and unsupervised multivariate analysis. Based on our data, coating the surface of mESCs with Matrigel, used as an acellular matrix substrate, resulted in morphological and biochemical changes. mESCs exhibited alterations in their phenotype after growing on the Matrigel-coated surfaces, including their differentiation capacity, cell cycle phase pattern, membrane fluidity, and metabolic activities. In conclusion, mESCs can be stimulated physiologically, chemically, or mechanically to convert them a new phenotype. Thus, identification of ESCs’ behavior in the acellular microenvironment could be vital to elucidate the mechanism of diseases. It might also be promising to control the cell fate in the field of tissue engineering.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.     

Three‐dimensional culture systems for the expansion of pluripotent embryonic stem cells

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two‐dimensional (2D) static culturing techniques are inadequate for large‐scale production. The culture of mammalian cells in three‐dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher‐S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher‐S out‐performed the Solohill microcarriers. When cultured for successive passages on Cultispher‐S microcarriers, mESCs maintained their pluripotency, demonstrated by self‐renewal, expression of pluripotency markers and the ability to undergo multi‐lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher‐S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra‐1–60, NANOG, and OCT‐4. Our study highlights the importance of optimization of initial seeding parameters and provides proof‐of‐concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695. © 2010 Wiley Periodicals, Inc.     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10⁹ cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.     

Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) have the capability to undergo unlimited cell division and differentiate into derivatives of all three embryonic germ layers. These fundamental features enable mESCs to potentially be appropriate, efficient models for biological and medical research. Therefore, it is essential to produce high-performance mESCs. In the current study, we have produced mESCs from blastocysts that developed from fertilized oocytes of 2 (2-C57)-, 4 (4-C57)-, and 6 (6-C57)-month-old C57BL/6 mice. A comparison of isolated stem cells was done from the viewpoint of the efficiency of mESC derivation, self-renewal, and their differentiation capacity. All generated mESCs showed a similar expression of the molecular markers protein of pluripotency and AP activity. In the 3i medium, there was a significant decrease in undifferentiated marker genes expression in the 2-C57 cells compared with the other two groups ( P < 0.05) but developmental genes significantly increased in the 4-C57 and 6-C57 cells compared with the 2-C57 cells ( P < 0.05). The differentiation capacity into three germ layers through the embryoid body formation and percentage of cell lines with normal numbers of chromosomes reduced with increased maternal age. The highest DT and highest percentage of cells in the S phase belonged to 2-C57 cells. These data demonstrated that blastocysts which developed from fertilized oocytes of 2-, 4-, and 6-month-old C57BL/6 mice can generate pluripotent stem cells, and suggested that both the efficiency of mESC isolation and the behavior of these isolated mESCs including pluripotency, self-renewal, cell cycle, and DT changed with increasing maternal age.     

MicroRNA对胚胎干细胞的多能性网络调控

胚胎干细胞(Embryonic stem cells, ESCs)是一类能够无限增殖和诱导分化为多种类型细胞的干细胞。MicroRNA(miRNA)是一类内源性具有调控基因表达功能的非编码RNA, 在ESCs增殖和分化过程中起重要作用。MiRNA可以通过对ESCs多能性网络中的转录因子、细胞周期、表观遗传学、信号转导等方面调控, 促使ESCs维持多能性状态。文章重点综述了miRNA的生成过程、调控ESCs多能性的主要miRNA家族以及miRNA对ESCs多能性网络调控作用等内容。     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.