Proliferation Of hES Cells Research Articles

One-Carbon Metabolism Regulates Embryonic Stem Cell Fate Through Epigenetic DNA and Histone Modifications: Implications for Transgenerational Metabolic Disorders in Adults.

Human (h) and mouse (m) embryonic stem (ES) cells need specific amino acids to proliferate. mES cells require threonine (Thr) metabolism for epigenetic histone modifications. Thr is converted to glycine and acetyl CoA, and the glycine is metabolized specifically to regulate trimethylation of lysine (Lys) residue 4 in histone H3 (H3K4me3). DNA methylation and methylation of other H3 Lys residues remain unimpaired by Thr deprivation in mES cell culture medium. Similarly, hES cells require methionine (Met) to maintain the Met-SAM (S-adenosyl methionine) cycle of 1-carbon metabolism also for H3K4me3 formation. H3K4me3 is needed specifically to regulate and maintain both mES and hES cell proliferation and their pluripotent states. Better understanding of this regulation is essential since treatment of human diseases and disorders will increasingly involve hES cells. Furthermore, since ES cells are derived from their progenitor cells in preimplantation blastocysts, they serve as models of 1-carbon metabolism in these precursors of all mammalian tissues and organs. One-carbon metabolism challenges, such as a maternal low protein diet (LPD) during preimplantation blastocyst development, contribute to development of metabolic syndrome and related abnormalities in adults. These 1-carbon metabolism challenges result in altered epigenetic DNA and histone modifications in ES progenitor cells and the tissues and organs to which they develop. Moreover, the modified histones could have extracellular as well as intracellular effects, since histones are secreted in uterine fluid and influence early embryo development. Hence, the mechanisms and transgenerational implications of these altered epigenetic DNA and histone modifications warrant concerted further study.

At Least Three Transporters Likely Mediate Threonine Uptake Needed for Mouse Embryonic Stem Cell Proliferation.

Stem cells are at the forefront of current regenerative and biomedical research. Thus, there exists an imperative and urgent need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, Threonine (Thr) is the only amino acid required in culture media for mES cell proliferation. Current research associates this need for Thr with threonine dehydrogenase (TDH), which catabolizes Thr to glycine and acetyl-CoA in mES cells. This theory depends, in part, on the ability of 3- hydroxynorvaline (3-HNV) to inhibit both TDH and mES cell proliferation. However, the concentration of 3-HNV needed to inhibit mES cell proliferation is more than an order of magnitude less than its apparent Ki for TDH inhibition. Additionally, 3-HNV inhibits human embryonic stem (hES) cell proliferation, but hES cells do not express a functional tdh gene. Such findings indicate another mechanism for Thr stimulated mES and hES cell proliferation. Since amino acid transporters may be inducers of signaling cascades, we characterized the Thr transport systems in mES cells. We found that there is a Na+-dependent and a Na+-independent component of substrate-saturable transport, with the Na+-dependent component predominating. We also found that of 20 amino acids tested, the amino acids that were the strongest inhibitors of the Na+-dependent component of radiolabeled Thr transport were Ser, Cys, 4-OH-Pro, Asn, Met, and non-radiolabeled Thr itself. Such findings are consistent with characteristics of the ASC transport system, suggesting that this ASC system is responsible for the majority of Thr transport in mES cells. We confirmed expression of mRNA encoding the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT-PCR. In conclusion, mES cells likely express at least three transporters of Thr; at least two Na+-dependent transporters and one Na+-independent one.

Glycosaminoglycan-binding hydrogels enable mechanical control of human pluripotent stem cell self-renewal.

Reaping the promise of human embryonic stem (hES) cells hinges on effective defined culture conditions. Efforts to identify chemically defined environments for hES cell propagation would benefit from understanding the relevant functional properties of the substratum. Biological materials are often employed as substrata, but their complexity obscures a molecular level analysis of their relevant attributes. Because the properties of hydrogels can be tuned and altered systematically, these materials can reveal the impact of substratum features on cell fate decisions. By tailoring the peptide displayed to cells and the substrate mechanical properties, a hydrogel was generated that binds hES cell surface glycosaminoglycans (GAGs) and functions robustly in a defined culture medium to support long-term hES cell self-renewal. A key attribute of the successful GAG-binding hydrogels is their stiffness. Only stiff substrates maintain hES cell proliferation and pluripotency. These findings indicate that cells can respond to mechanical information transmitted via GAG engagement. Additionally, we found that the stiff matrices afforded activation of the paralogous proteins YAP/TAZ, which are transcriptional coactivators implicated in mechanosensing and hES cell pluripotency. These results indicate that the substratum mechanics can be tuned to activate specific pathways linked to pluripotency. Because several different hES and induced pluripotent stem cell lines respond similarly, we conclude that stiff substrata are more effective for the long-term propagation of human pluripotent stem cells.

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Spatially Resolved Quantification of E-Cadherin on Target hES Cells

The local expression and distribution pattern of protein on a cell play essential roles in signal transduction within a cell or between cells. Here we report on the development of a spatially resolved quantification method, which was applied in the study of E-cadherin local expression in identified undifferentiated and differentiated human embryonic stem (hES) cells in their native cellular environment. This was achieved by a novel immunofluorescence assisted affinity mapping (IF-AM) method, in which immunofluorescence provides the guidance to locate a desired type of cell in a cell community for performing affinity mapping to quantify the local protein density. The results unveiled the crucial role of E-cadherin in mediating hES cell proliferation and differentiation: the expression of E-cadherin is markedly higher on undifferentiated cells, and the growth of hES cells in unique colonies is contingent on the homogeneous distribution of E-cadherin. Due to the ability of directly assessing individual proteins of a cell, the IF-AM method is shown to be a sensitive tool for resolving subtle differences in the local expression of membrane proteins even at low abundance.

Role of TGF beta and myofibroblasts in supporting the propagation of human embryonic stem cells in vitro

The feeder layer constitutes a prerequisite for the undifferentiated proliferation of human embryonic stem (hES) cells in vitro. However, a few feeders have been reported to be non-supportive in nature, suggesting that these feeders exhibit a different transcriptome and proteome, in comparison to their supportive counterparts. In an attempt to identify factors required for undifferentiated growth and many downstream applications of hES cells, transcriptomes of supportive (mouse fibroblasts derived from 13.5dpc embryos and human fetal fibroblasts) and non-supportive (mouse fibroblasts derived from 18.5dpc embryos) feeders were analyzed. Furthermore, the parallel correlation of data generated in the microarray study with the published proteome data of supportive feeder fibroblasts, helped us to focus on the proteins which seem to be likely candidates in supporting the undifferentiated expansion of ES cells in vitro. Our results indicated that TGFbeta and its associated signaling molecules facilitate the undifferentiated proliferation of hES cells in vitro. The transient differentiation of feeder fibroblasts into myofibroblasts may be the decisive factor for a feeder layer to be supportive or non-supportive in nature. We propose that the microenvironment of feeder myofibroblasts dictates TGFbeta to support proliferation and apparently plays the contradictory role of facilitating differentiation when feeder support is withdrawn, possibly by acting through different signaling mechanisms.

Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self-renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB-dependent growth control. We investigated whether self-renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220(NPAT)/HiNF-P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220(NPAT) causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220(NPAT), decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220(NPAT) are principal cell cycle regulators that determine competency for self-renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220(NPAT)/HiNF-P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis.

Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions

Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3–5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.

Cigarette smoke inhibits endometrial epithelial cell proliferation through a nitric oxide–mediated pathway

To determine the direct effects of cigarette smoke (CS) on human endometrial surface epithelial cell line proliferation. In vitro study using HES cells and primary human endometrial cells. University-based academic center. Premenopausal women in the proliferative phase of the cycle. The HES cells and primary human endometrial cells were exposed to cigarette smoke-saturated solution. Cell proliferation and expression of different isoforms of nitric oxide synthase. Cigarette smoke inhibited HES cell proliferation in a dose- and time-dependant manner. The inhibitory effect of CS was blocked by hemoglobin and enhanced by L-arginine (L-Arg). Cigarette smoking and nicotine stimulated the expression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) whereas benzo[a]pyrene (BP) only stimulated the expression of eNOS in HES cells. Cigarette smoke stimulated the expression of eNOS/iNOS in primary human endometrial cells, comprised of epithelial and stromal cells. The effect of CS on eNOS/iNOS expression in HES cells was blocked by ascorbic acid but not by glutathione. Cigarette smoke inhibits endometrial cell proliferation through a nitric oxide-mediated pathway.

Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells

Human embryonic stem (hES) cells as a renewable cell source have great prospective applications in both developmental biology research and regenerative medicine. To realize these potentials, the development of effective and safe genetic manipulation methods in hES cells is an obvious demand. We report here that baculoviral vectors were able to transduce hES cells efficiently. In transient transduction experiments, a recombinant baculoviral vector equipped with a human elongation factor 1-alpha promoter and a woodchuck hepatitis post-transcriptional regulatory element transduced up to 80% of cells in hES cell clumps and embryoid bodies. For prolonged transgene expression, hybrid baculoviral vectors that have incorporated a rep gene and inverted terminal repeat sequences from adeno-associated virus were produced. These hybrid vectors yielded stable transgene expression during the prolonged undifferentiated proliferation of hES cells and after differentiation. Baculoviral transduction did not affect the normal growth, phenotype, and pluripotency of hES cells. Thus, baculoviral vectors suitable for both transient overexpression and long-term stable expression are an attractive option for genetic manipulation of hES cells.

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.

Prostaglandin E2 induces proliferation of glandular epithelial cells of the human endometrium via extracellular regulated kinase 1/2-mediated pathway.

In this study, we investigated the effect of prostaglandin E(2) (PGE(2)) on MAPK ERK1/2 protein phosphorylation and on proliferation of epithelial cells of the human endometrium. Treatment of proliferative phase endometrium with PGE(2) induced rapid phosphorylation of ERK1/2 proteins in glandular epithelial and endothelial cells. Treatment of human endometrial tissue with PGE(2) for 24 h resulted in increased incorporation of 5-bromo-2'-deoxyuridine (a marker of cellular proliferation) in glandular epithelial cells. To investigate further the effect of PGE(2) on proliferation of epithelial cells, we used an endometrial epithelial cell line (HES). HES cells express functional EP4 (with absence of expression of EP1, EP2, and EP3) receptors and stimulate cAMP release and rapid phosphorylation of ERK1/2 proteins in response to PGE(2) or forskolin. Treatment of HES cells with PGE(2) or forskolin alone resulted in a significant increase in HES cell proliferation compared with control untreated cells (P < 0.05). Cotreatment of the cells with PGE(2) or forskolin and PD98059 abolished the increase in cellular proliferation. These data demonstrate ERK1/2 phosphorylation in response to PGE(2) in the human endometrium and suggest that PGE(2) via EP4 receptor may induce glandular epithelial cell proliferation in ERK1/2- dependent manner during the proliferative phase of the menstrual cycle.