Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells

Perfluorooctane sulfonate (PFOS) poses potential risks to reproduction and development. Mouse embryonic stem cells (mESCs) are ideal models for developmental toxicity testing of environmental contaminants in vitro. However, the mechanism by which PFOS affects early embryonic development is still unclear. In this study, mESCs were exposed to PFOS for 24 h, and then general cytotoxicity and pluripotency were evaluated. MTT assay showed that neither PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) nor control medium (0.1% DMSO) treatments affected cell viability. Furthermore, there were no significant differences in cell cycle and apoptosis between the PFOS treatment and control groups. However, we found that the mRNA and protein levels of pluripotency markers (Sox2, Nanog) in mESCs were significantly decreased following exposure to PFOS for 24 h, while there were no significant changes in the mRNA and protein levels of Oct4. Accordingly, the expression levels of miR-145 and miR-490-3p, which can regulate Sox2 and Nanog expressions were significantly increased. Chrm2, the host gene of miR-490-3p, was positively associated with miR-490-3p expression after PFOS exposure. Dual luciferase reporter assay suggests that miR-490-3p directly targets Nanog. These results suggest that PFOS can disturb the expression of pluripotency factors in mESCs, while miR-145 and miR-490-3p play key roles in modulating this effect.     

Cell growth arrest and apoptosis induced by Oct4 or Nanog knockdown in mouse embryonic stem cells: a possible role of Trp53

It has been clear that both Oct4 and Nanog play essential roles in maintaining embryonic stem cells (ESCs) undifferentiation. However, the roles of Oct4 and Nanog in ESCs growth and apoptosis have been much less explored. In this study, we systematically examined the effects of Oct4 or Nanog knockdown on mouse ESCs (mESCs) growth and apoptosis as well as potential mechanisms. Our results show that Oct4 or Nanog knockdown induces growth arrest and apoptosis in mESCs, indicating that the two genes also play important roles in mESCs survival and growth. Moreover, upregulation in Trp53 and its downstream genes expression was detected in Oct4 or Nanog knockdown mESCs, suggesting a possible role of Trp53 in Oct4 or Nanog knockdown induced mESCs growth arrest and apoptosis.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.     

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells

ABSTRACT

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3β with IC₅₀ values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.     

Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young''s modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young''s modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.     

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.     

Activin/TGF-beta signaling regulates Nanog expression in the epiblast during gastrulation

Nanog is required for the maintenance of cellular pluripotency during normal development and in cultured embryonic stem cells. A number of signaling pathways have been implicated in regulating Nanog gene expression in vitro. Using the chick model, we provide in vivo evidence for the involvement of the Activin/TGF-beta signaling pathway in regulating Nanog expression in epiblast cells during gastrulation. Nanog expression in primordial germ cells is not regulated by this pathway, indicating that these two cell types employ different mechanisms for maintaining pluripotency in early development. Furthermore, our data suggest that the bHLH factor E2A plays a role in negatively regulating Nanog expression in vivo. Overall, our data support a direct and positive role of the Smad2/3 mediated TGF-beta signaling pathway in inducing/maintaining Nanog expression.     

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.