We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell–differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell–differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell–based regenerative medicine.

Characterized by their capacity to differentiate into various cell lineages (pluripotency) and unlimited proliferation (self-renewal), embryonic stem cells (ESCs) hold great promise as a cell source for cell-based regenerative medicine (13). Intensive research for the past two decades developed strategies that promote ESC differentiation into various types of cells. However, ESC-differentiated cells are commonly assessed by comparison with their in vivo counterparts with regard to morphology, cell marker expression, and cell-specific function. Many other functions, such as innate immunity, cannot be assessed unless the cells are exposed to pathogens. Recent studies reported that several major tissue cell types differentiated from both human ESCs (hESCs) and mouse ESCs (mESCs) have limited innate immune response to various pathogens and cytokines (49), highlighting the potential functional deficiency of in vitro ESC-differentiated cells, as we discussed recently (10).

The immunoproperty of ESC-derived cells is an important consideration for their therapeutic application. Immunogenicity, the cause leading to the rejection of implanted cells by the host, has attracted much attention in the studies of ESCs and their differentiated cells (1113). In contrast, only limited studies investigated their immune and inflammatory responses to the host environment, despite the fact that such responses significantly impact the outcome of transplantation. The effects of the lack of innate immunity in ESC-differentiated cells remain to be evaluated; however, it is likely that this deficiency may affect their fate and functionality when used in a clinical setting, because potentially they will be placed in an inflammatory area of the patient. Although the attenuated innate immunity in ESC-differentiated cells may compromise their contribution to the tissue immunity, it could also be beneficial because the implanted cells would not potentiate the inflammatory response in the wounded area, thus avoiding further damage caused by the host’s adaptive immunity (11). Therefore, determining the molecular mechanisms that control innate immunity development and the immunoproperties of ESC-differentiated cells will provide valuable information for evaluating their therapeutic potential.

Innate immunity, presumably developed in most, if not all mammalian cells, is known as the first line of an organism’s defense and plays a critical role in mobilizing adaptive immunity. The cellular response to viral/bacterial pathogens and inflammatory cytokines is the central part of innate immunity. The lack of such function in ESC-derived cells raised concerns about their therapeutic application, and it promoted studies seeking the molecular mechanisms in ESCs from which these cells are derived. Indeed, it was demonstrated that ESCs do not show immune responses typically seen in differentiated cells infected with bacteria and viruses (14, 15). Our recent studies in mESCs (1618) and those by other investigators in hESCs and in induced pluripotent stem cells (19, 20) demonstrated that the IFN system, the central component of innate antiviral immunity in differentiated somatic cells (21), is not fully developed in these cells. Therefore, the lack of innate immune responses to bacterial and viral infection appears to be an intrinsic property of all pluripotent stem cells (10).

The cellular immune response is induced by various products from microbial pathogens. Immunostimuli are mainly detected by pattern recognition receptors that include TLRs and retinoic acid–inducible gene I–like receptors (22, 23). Although different immunostimuli are detected by distinct receptors, and the signals are transduced by different signaling pathways, the signal transduction eventually converges at the point of NF-κB activation. Activated NF-κB, alone or together with other transcription factors, directly controls the transcription of IFN, inflammatory cytokines, and many other types of inflammatory mediators (22, 24). Therefore, the activation of NF-κB plays a central role in immune and inflammatory responses. In mammals, the NF-κB family is composed of five related transcription factors: p50, p52, RelA, c-Rel, and RelB. They activate transcription of target genes through hetero- or homodimerization. The canonical NF-κB pathway involves p50/RelA (or c-Rel) and is mainly activated by pathogens and inflammatory cytokines, whereas the noncanonical pathway uses p52/RelB and is usually activated by specialized factors (25). Previous studies indicated that the lack of IFN expression in ESCs is attributable, in part, to the absence or low levels of expression of viral RNA receptors (17, 19). Our recent study further demonstrated that NF-κB is not activated in virus-infected mESCs, which explains the deficiency in IFN expression at the transcriptional level (26).

Although significant progress has been made in understanding the lack of antiviral innate immunity in mESCs and hESCs, little is known about the antibacterial and inflammatory responses in these cells. Because the NF-κB pathway is commonly activated by various pathogens and inflammatory cytokines, we reasoned that its inactive state in ESCs could also account for their lack of antibacterial and inflammatory responses. However, a limited number of published studies reported different results with inconsistent conclusions (27). In this study, we demonstrate that mESCs and hESCs are deficient in mounting inflammatory responses to LPS (a bacterial endotoxin that strongly induces inflammatory response), TNF-α (a prototype of inflammatory cytokines), and viral infection. In vitro differentiation can induce, but only partially, the development of the inflammatory response mechanism in mESC-differentiated cells (mESC-DCs). In this article, we provide the molecular basis underlying these observations.

Two mESC lines, D3 and DBA252 (DBA) mESCs, were used in this study. They were maintained in standard mESC medium, as previously described (17). The hESC line H9 (WA09) was obtained from the WiCell Research Institute (Madison, WI). hESCs were seeded on Matrigel (Corning) and fed daily with mTeSR media (STEMCELL Technologies). mESC-differentiated fibroblasts (mESC-FBs), C3H10T1/2 (10T1/2) cells (a line of mouse embryonic fibroblasts; American Type Culture Collection [ATCC]), RAW264.7 cells (RAW; ATCC), and HeLa Cells (ATCC) were cultured in DMEM with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The culture of HUVECs was described previously (4). All cells were maintained at 37°C in a humidified incubator with 5% CO2.

In vitro differentiation of mESCs was carried out using two methods that we described previously, with minor modifications. The first method is ESC differentiation through embryoid body (EB) formation (28). Briefly, mESCs (1 × 105 cells per milliliter) were suspended in a bacterial culture dish where they formed EBs and were differentiated for 5 d. The EBs were transferred to gelatin-coated cell culture dishes where the cells within the EBs differentiate into a mixed cell population (mESC-DCs). Among mESC-DCs, a distinctive cell population has large, flattened bodies and expresses α–smooth muscle actin (α-SMA). These cells were purified from the mixture based on their high adherence. For this purpose, mESC-DCs were trypsinized. The resulting single-cell suspension was reseeded in an uncoated cell culture dish, to which the large, flattened α-SMA+ cells quickly attached within ∼30 min. After removal of unattached cells from the medium, the attached cells were used for further analysis in other experiments.

In the second method of differentiation, mESCs grown in a monolayer were directly induced with retinoic acid. The differentiation, purification, and characterization of mESC-FBs were described in our earlier reports (18, 26). mESC-FBs between passages 10 and 30 were used for the experiments. D3 and DBA mESCs and their differentiated fibroblasts (i.e., D3-fibroblasts [D3-FBs] and DBA-fibroblasts) displayed similar properties as previously characterized (18, 26). Most experiments were performed with D3 cells and D3-FBs because D3 cells are an mESC line commonly used in the literature. Some key experiments were confirmed with DBA cells.

ESCs (30–50% confluence), mESC-FBs, and other cells (70–80% confluence) were treated with LPS (1 μg/ml; Sigma), human or mouse TNF-α or IL-1β (20 ng/ml; PeproTech), or actinomycin D (ActD; 0.4 μg/ml; Sigma) under the conditions specified in the individual experiments.

Immunochemistry and Western blot analyses were performed according to our published methods, with some modifications (28). The Abs used in this study were purchased from Santa Cruz Biotechnology (RelA subunit of NF-κB, IκB, and α-SMA) and BD Biosciences (PECAM1). The cells were examined under a LSM 510 laser-scanning confocal microscope (Zeiss), a Leica DM IL LED inverted fluorescence microscope, or an Olympus CKX31 phase-contrast microscope. The images were acquired with a digital camera mounted on the microscope.

Total RNA was extracted using TRI Reagent (Sigma). cDNA was prepared using Moloney murine leukemia virus reverse transcriptase (Sigma). Real-time quantitative PCR (RT-qPCR) was performed using SYBR Green Ready Mix (Bio-Rad) on a MX3000P RT-PCR system (Stratagene), as previously described (28). The mRNA levels from RT-qPCR were calculated using the comparative Ct method (29). β-actin was used as a calibrator for the calculation of relative mRNA of the tested genes. In the experiments with viruses, 18S rRNA was used because of the degradation of β-actin caused by viral infection.

Mouse cells.

The following primer sets were used: β-actin: forward, 5′-CATGTACGTAGCCATCCAGGC-3′ and reverse, 5′-CTCTTTGATGTCACGCACGAT-3′; 18s rRNA: forward, 5′-GTAACCCGTTGAACCCCATT-3′ and reverse, 5′-CCATCCAATCGGTAGTAGCG-3′; CD14: forward, 5′-CTCTGTCCTTAAAGCGGCTTAC-3′ and reverse, 5′-GTTGCGGAGGTTCAAGATGTT-3′; TLR4: forward, 5′-TGCACTGAGCTTTAGTGGTTGC-3′ and reverse, 5′-GACCCATGAAATTGGCACTCAT-3′; TNFR1: forward, 5′-CCGGGAGAAGAGGGATAGCTT-3′ and reverse, 5′-TCGGACAGTCACTCACCAAGT-3′; ICAM1: forward, 5′-GGCATTGTTCTCTAATGTCTCCG-3′ and reverse, 5′-GCTCCAGGTATATCCGAGCTTC-3′; IL-6: forward, 5′-TAGTCCTTCCTACCCCAATTTCC-3′ and reverse, 5′-TTGGTCCTTAGCCACTCCTTC-3′; and MCP-5: forward, 5′-ATTTCCACACTTCTATGCCTCCT-3′ and reverse, 5′-ATCCAGTATGGTCCTGAAGATCA-3′.

Human cells.

The following primer sets were used: β-actin: forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′; ICAM1: forward, 5′-AGAGGTCTCAGAAGGGACCG-3′ and reverse, 5′-GGGCCATACAGGACACGAAG-3′; and IL-6: forward, 5′-AACCTGAACCTTCCAAAGATGG-3′ and reverse, 5′-TCTGGCTTGTTCCTCACTACT-3′.

Protein analysis by flow cytometry was performed according to our published method (17). Anti-TLR4 and CD14 Abs were preconjugated with PE (BioLegend). Anti-RelA Abs (Santa Cruz Biotechnology) were detected with secondary Abs that were conjugated with FITC. The Ab-labeled cells were analyzed with an Accuri C6 flow cytometer (BD Biosciences). Isotype Abs or cell samples without primary Ab incubation were used as negative controls and were used as controls for fluorescence gating. The fluorescence intensity, which correlates with the protein level detected with its specific Ab, was determined and analyzed with CFlow software.

The cells were treated with TNF-α or ActD alone, or they were treated with ActD for 30 min, followed by treatment with TNF-α for the specified times. The cells were fixed with methanol and stained with toluidine blue. The absorbance (630 nm) of stained cells was measured with a BioTek ELx800 microtiter plate reader. The absorbance values, which correlate with the amount of cellular content (proteins/DNA), were used as an indirect measurement of viable cells, as previously described (17).

Chikungunya virus (CHIKV; LR 2006 OPY1 strain) was propagated in Vero cells as previously described (30). The cells were infected with CHIKV at a multiplicity of infection = 1 and analyzed under the conditions specified in individual experiments.

Data are presented as the mean ± SD. Statistical analyses were performed using two-tailed and paired Student t tests. Differences are considered statistically significant when p < 0.05.

We first investigated the response of NF-κB to LPS, TNF-α, and IL-1β, three well-known agents that induce the expression of inflammatory genes through the activation of NF-κB. In resting cells, NF-κB is retained in the cytoplasm by binding to IκB. Upon cell activation, IκB is degraded, and NF-κB translocates to the nucleus where it activates transcription of target genes. Therefore, nuclear translocation is commonly used as an indicator of NF-κB activation (31). As shown in Fig. 1A, mESCs (DBA and D3) are characterized by their small size with a large nucleus and clonal growth. In control cells, NF-κB was detected in the cytoplasm of mESCs, as expected. However, treatment with LPS, TNF-α, or IL-1β did not cause a detectable change (Fig. 1A). In contrast, TNF-α induced clear translocation of NF-κB from the cytoplasm to the nucleus in HUVECs, which were used as a positive control (4) (Fig. 1B, upper panels), concurrent with complete degradation of IκB, which did not take place in DBA cells (Fig. 1B, lower panels) or D3 cells (data not shown). The three agents did not cause detectable changes in NF-κB cellular location with 15–90 min of treatment, indicating that none of them could activate the NF-κB pathway in mESCs.

FIGURE 1.

NF-κB is not activated by TNF-α, IL-1β, or LPS in mESCs. (A) mESCs (D3 and DBA) were treated with TNF-α, IL-1β, or LPS for 20 min or were left untreated (CON). The cellular location of NF-κB was analyzed with an Ab against NF-κB (RelA subunit) under a fluorescence microscope. (B) TNF-α–induced NF-κB nuclear translocation in HUVECs (upper panels). Cells were treated and analyzed for the cellular location of NF-κB under the conditions described in (A). HUVECs and DBA cells were treated with TNF-α for 20 min, and the level of IκB was analyzed by Western blot (lower panels). The lower portions of the blots show the proteins stained with Ponceau S to show the loading of the protein samples. Scale bars, 20 μm. Arrows indicate the location of a representative nucleus. The images are from representative experiments performed at least two times.

FIGURE 1.

NF-κB is not activated by TNF-α, IL-1β, or LPS in mESCs. (A) mESCs (D3 and DBA) were treated with TNF-α, IL-1β, or LPS for 20 min or were left untreated (CON). The cellular location of NF-κB was analyzed with an Ab against NF-κB (RelA subunit) under a fluorescence microscope. (B) TNF-α–induced NF-κB nuclear translocation in HUVECs (upper panels). Cells were treated and analyzed for the cellular location of NF-κB under the conditions described in (A). HUVECs and DBA cells were treated with TNF-α for 20 min, and the level of IκB was analyzed by Western blot (lower panels). The lower portions of the blots show the proteins stained with Ponceau S to show the loading of the protein samples. Scale bars, 20 μm. Arrows indicate the location of a representative nucleus. The images are from representative experiments performed at least two times.

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We hypothesized that the lack of NF-κB activation could be an intrinsic property of mESCs. Thus, differentiation should turn on the NF-κB pathway and the developmental program that controls the inflammatory response. The pluripotency of mESCs is maintained by LIF. Removal of LIF from the culture medium triggers spontaneous differentiation (32). When mESCs are cultured in suspension, they grow in aggregates and form EBs, three-dimensional structures that resemble an early embryo (28). As shown in Fig. 2Aa, when EBs (inset) were allowed to attach to the surface of a cell culture dish, the cells in EBs differentiated into a round patch-like structure in which cells undergo further differentiation to different cell types (collectively designated as ESC-DCs) (28). Fig. 2Aa illustrates a section of this patch structure in which cells can be roughly divided into three zones: the cells in the center zone (Z1) are highly compacted with small size, representing the least differentiated cells; the cells in the middle zone (Z2) are heterogeneous in morphology; and the cells at the peripheral zone (Z3) are large, flattened cells forming a relatively uniform monolayer.

FIGURE 2.

Differentiation of mESCs and the effects of TNF-α and LPS on NF-κB activation in mESCs and their differentiated cells. (A) Differentiation of mESC-DCs through EB formation. (Aa) The morphology of mESC-DCs generated from an EB (inset) located in different zones (Z1, Z2, and Z3). (Ab) Immunolocalization of NF-κB (green), endothelial cells (PECAM1, red), and α-SMA+ cells (SMA, red) in different zones. (Ac) Purified SMA+ cells were double stained with Abs against NF-κB (green) and SMA (red). In (Ab) and (Ac), the cells were treated with TNF-α for 15 min or LPS for 30 min. An arrow indicates the location of a representative nucleus. (B) TNF-α induced activation of NF-κB in mESC-FBs but not in mESCs. (Ba) D3-FBs and 10T1/2 cells were treated with TNF-α for 20 min. The cellular location of NF-κB was analyzed with Abs against RelA subunit. An arrow indicates the location of a representative nucleus. (Bb) D3 cells and D3-FBs (or 10T1/2 cells, not shown) were grown in a coculture in which D3 cells were identified by their clonal growth (circled area) and D3-FBs were identified by their flattened large cell bodies under a phase-contrast microscope. (Bc) The cells in the coculture were treated with TNF-α and analyzed for the cellular location of NF-κB, as described in (Ba). In CON, NF-κB is mainly detected in the cytoplasm of all cells tested. TNF-α–induced NF-κB nuclear translocation took place in D3-FBs and 10T1/2 cells [(Ba) and (Bc), arrows] but not in D3 cells [(Bc), arrowheads]. Scale bars, 20 μm. The images are from representative experiments performed at least two times. CON, control cells that were not treated.

FIGURE 2.

Differentiation of mESCs and the effects of TNF-α and LPS on NF-κB activation in mESCs and their differentiated cells. (A) Differentiation of mESC-DCs through EB formation. (Aa) The morphology of mESC-DCs generated from an EB (inset) located in different zones (Z1, Z2, and Z3). (Ab) Immunolocalization of NF-κB (green), endothelial cells (PECAM1, red), and α-SMA+ cells (SMA, red) in different zones. (Ac) Purified SMA+ cells were double stained with Abs against NF-κB (green) and SMA (red). In (Ab) and (Ac), the cells were treated with TNF-α for 15 min or LPS for 30 min. An arrow indicates the location of a representative nucleus. (B) TNF-α induced activation of NF-κB in mESC-FBs but not in mESCs. (Ba) D3-FBs and 10T1/2 cells were treated with TNF-α for 20 min. The cellular location of NF-κB was analyzed with Abs against RelA subunit. An arrow indicates the location of a representative nucleus. (Bb) D3 cells and D3-FBs (or 10T1/2 cells, not shown) were grown in a coculture in which D3 cells were identified by their clonal growth (circled area) and D3-FBs were identified by their flattened large cell bodies under a phase-contrast microscope. (Bc) The cells in the coculture were treated with TNF-α and analyzed for the cellular location of NF-κB, as described in (Ba). In CON, NF-κB is mainly detected in the cytoplasm of all cells tested. TNF-α–induced NF-κB nuclear translocation took place in D3-FBs and 10T1/2 cells [(Ba) and (Bc), arrows] but not in D3 cells [(Bc), arrowheads]. Scale bars, 20 μm. The images are from representative experiments performed at least two times. CON, control cells that were not treated.

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To test the activation of NF-κB, ESC-DCs were exposed to TNF-α and LPS under the conditions described in Fig. 1. Treated cells were double stained with an Ab against NF-κB and an Ab against PECAM1 (an endothelial marker) or an Ab against α-SMA (a marker for smooth muscle cells/fibroblasts). TNF-α or LPS did not induce NF-κB nuclear translocation in the cells in zone 1 (Fig. 2Ab, zone 1, a representative nucleus is denoted by an arrow) or the cells in zone 2, including the endothelial cells that were assembled into prototypes of vessel-like structures (Fig. 2Ab, zone 2). However, TNF-α, but not LPS, induced detectable NF-κB nuclear translocation in the cells in zone 3, where the majority of the cells are large, flattened α-SMA+ cells (Fig. 2Ab, zone 3). These cells can be easily separated from other cell types in mESC-DCs based on their plastic-adhering property. TNF-α–induced nuclear translocation of NF-κB was confirmed in the purified α-SMA+ cells (Fig. 2Ac).

We then examined NF-κB activation in mESC-FBs, which were derived from mESCs through retinoic acid–induced differentiation. They share extensive similarities with 10T1/2 cells (fibroblasts isolated from a 14-d-old embryo) (33, 34) with regard to cell marker expression, growth pattern, and morphology, as we described previously (18, 26). TNF-α–induced NF-κB nuclear translocation is clearly demonstrated in D3-FBs, although the fluorescence intensity of NF-κB in their nuclei is substantially lower than in 10T1/2 cells (Fig. 2Ba). This result indicates that NF-κB has undergone the transition from an inactive status in mESCs to an active status in mESC-FBs. For a direct comparison, we used a coculture model of D3 and D3-FBs (or 10T1/2 cells) where D3 colonies could be easily distinguished from the large, flattened D3-FBs or 10T1/2 cells under either a phase-contrast or fluorescence microscope (Fig. 2Bb, Fig. 2Bc, respectively, circled cells). In control cells, NF-κB was detected in the cytoplasm of D3 cells and D3-FBs or 10T1/2 cells. TNF-α–induced NF-κB nuclear translocation took place in D3-FBs and 10T1/2 cells but not in D3 cells (Fig. 2Bc, indicated by an arrow in D3-FBs or 10T1/2 cells and by an arrowhead in D3 cells). The exclusive detection of NF-κB in the nucleus of D3-FBs clearly demonstrated that the NF-κB pathway is activated only after differentiation. A similar observation was made in cells that were treated with IL-1β, but LPS did not induce NF-κB nuclear translocation in D3 cells, D3-FBs, or 10T1/2 cells (data not shown).

We further analyzed the responsiveness of mESCs and their differentiated cells to LPS and TNF-α by determining the expression of ICAM1 and IL-6, two genes that are known to be under the transcriptional control of NF-κB (35, 36). As shown in Fig. 3A, neither LPS nor TNF-α induced the expression of the two genes in D3 cells. TNF-α induced a slight increase in ICAM1 and IL-6 in mESC-DCs (2–5-fold) after 12 h of incubation. The effect of TNF-α was notably increased in mESC-FBs, in which the expression of ICAM1 and IL-6 was induced ∼4- and 20-fold, respectively, but it was substantially lower than the effect of TNF-α in 10T1/2 cells at all time points tested (Fig. 3A, 3B). LPS failed to induce expression of the two genes in D3 cells, D3-FBs, and even 10T1/2 cells (Fig. 3A, 3B). Similar results were obtained when the cells were treated with TNF-α (up to 100 ng/ml) and LPS (up to 10 μg/ml) (data not shown). To confirm this observation, we tested the effect of LPS on RAW cells and HUVECs as positive controls. LPS induced the expression of ICAM1 and IL-6 in both cells, as expected (Fig. 3C). The expression levels of ICAM1 and IL-6 in D3 cells, D3-DCs, D3-FBs, and 10T1/2 cells correlated well with NF-κB nuclear translocation in response to LPS and TNF-α (Figs. 1, 2). Together, our data suggest that the differentiation process induced, but only partially, the development of the molecular mechanisms that mediate the inflammatory response.

FIGURE 3.

TNF-α− and LPS-induced expression of ICAM1 and IL-6 in mESCs and mESC-DCs. Cells were treated with TNF-α and LPS for 12 h (A) or for 24 and 48 h (B). 10T1/2 cells were used for comparison. To confirm the effect of LPS, RAW cells and HUVECs were treated with LPS (5 and 12 h, respectively). (C) The expression of ICAM1 and IL-6 was determined by RT-qPCR. The results are expressed as fold-activation; the mRNA level in the control of each set of experiments is designated as 1 (not shown). The values are mean ± SD of representative experiments performed in triplicate that were repeated at least three times.

FIGURE 3.

TNF-α− and LPS-induced expression of ICAM1 and IL-6 in mESCs and mESC-DCs. Cells were treated with TNF-α and LPS for 12 h (A) or for 24 and 48 h (B). 10T1/2 cells were used for comparison. To confirm the effect of LPS, RAW cells and HUVECs were treated with LPS (5 and 12 h, respectively). (C) The expression of ICAM1 and IL-6 was determined by RT-qPCR. The results are expressed as fold-activation; the mRNA level in the control of each set of experiments is designated as 1 (not shown). The values are mean ± SD of representative experiments performed in triplicate that were repeated at least three times.

Close modal

To determine whether the observations that we made in mESCs also apply to hESCs, we analyzed the effects of LPS and TNF-α on hESCs under similar conditions as described for mESCs. As shown in Fig. 4 (images), TNF-α strongly induced NF-κB nuclear translocation in HeLa cells as a positive control, but it failed to induce any detectable change in hESCs. A similar observation was made in hESCs that were treated with LPS (data not shown). In response to LPS and TNF-α, HeLa cells, but not hESCs, expressed ICAM1 and IL-6 (Fig. 4, graph). These results are basically the same as the observations made in mESCs (Figs. 1, 3), indicating that the lack of an inflammatory response to LPS and TNF-α is a common feature of mESCs and hESCs.

FIGURE 4.

NF-κB is not activated by TNF-α or LPS in hESCs. hESCs and HeLa cells were treated with TNF-α or LPS for 20 min or were left untreated (CON). The cellular location of NF-κB was analyzed with an Ab against NF-κB (RelA subunit) under a fluorescence microscope (inset). An arrow indicates the location of a representative nucleus. Scale bar, 20 μm. The bar graph shows hESCs and HeLa cells that were treated with TNF-α and LPS for 12 and 24 h. The expression of ICAM1 and IL-6 was determined by RT-qPCR. The results are expressed as fold activation; the mRNA level in untreated control cells (CON) is designated as 1. The values are mean ± SD of a representative experiment performed in triplicate that was repeated two times.

FIGURE 4.

NF-κB is not activated by TNF-α or LPS in hESCs. hESCs and HeLa cells were treated with TNF-α or LPS for 20 min or were left untreated (CON). The cellular location of NF-κB was analyzed with an Ab against NF-κB (RelA subunit) under a fluorescence microscope (inset). An arrow indicates the location of a representative nucleus. Scale bar, 20 μm. The bar graph shows hESCs and HeLa cells that were treated with TNF-α and LPS for 12 and 24 h. The expression of ICAM1 and IL-6 was determined by RT-qPCR. The results are expressed as fold activation; the mRNA level in untreated control cells (CON) is designated as 1. The values are mean ± SD of a representative experiment performed in triplicate that was repeated two times.

Close modal

TNF-α is a pleiotropic cytokine that is involved in inflammation, as well as causes cytotoxicity that leads to apoptosis or necrosis of certain tumor cells or infected cells (37). We took a different approach to further investigate the responsiveness of mESCs and mESC-FBs to TNF-α. Normal tissue cells are usually resistant to the cytotoxicity of TNF-α, but they become susceptible when they are exposed to TNF-α in the presence of transcription or translation inhibitors (31, 38). TNF-α alone (10–20 ng/ml) did not show detectable toxicity in D3 cells, D3-FBs, or 10T1/2 for up to a 3-d incubation period, but it caused significant cell death of L929 cells (a fibroblast cell line that is sensitive to TNF-α toxicity) within a 24-h incubation (data not shown). However, when the cells were treated with TNF-α in the presence of the transcription inhibitor ActD, 10T1/2 cells quickly lost viability after 5–6 h of incubation, and ∼90% of cells lost viability by 12 h. D3-FBs were susceptible to the cytotoxic effect but with less sensitivity (Fig. 5). ActD alone caused apparent cell death of 10T1/2 and D3-FBs at 12 h. D3 cells were more sensitive to ActD than D3-FBs and 10T1/2 cells; toxicity was apparent as early as 6 h. However, a major difference is that, unlike in 10T1/2 and D3-FBs, TNF-α did not exacerbate the toxicity caused by ActD in D3 cells (Fig. 5B, ActD versus ActD+TNF). These results provide additional evidence that the mechanisms to detect and mediate the effects of TNF-α are not developed in mESCs but are promoted by the process of differentiation. The higher sensitivity of D3 cells to ActD toxicity is likely due to their rapid cell proliferation and metabolic rate that depend on the transcriptional activity.

FIGURE 5.

mESCs are insensitive to TNF-α cytotoxicity. D3 cells, D3-FBs, and 10T1/2 cells were incubated with ActD for 30 min, followed by treatment with TNF-α for the indicated times. (A) Cell viability was determined by toluidine blue staining. Data are mean ± SD of a representative experiment performed in triplicate that was repeated at least three times. (B) The morphology of cells was examined at 12 h of treatment and photographed under a phase-contrast microscope (original magnification ×400). *p < 0.05.

FIGURE 5.

mESCs are insensitive to TNF-α cytotoxicity. D3 cells, D3-FBs, and 10T1/2 cells were incubated with ActD for 30 min, followed by treatment with TNF-α for the indicated times. (A) Cell viability was determined by toluidine blue staining. Data are mean ± SD of a representative experiment performed in triplicate that was repeated at least three times. (B) The morphology of cells was examined at 12 h of treatment and photographed under a phase-contrast microscope (original magnification ×400). *p < 0.05.

Close modal

The IFN response is the central part of the antiviral mechanism, but the inflammatory response is also a prominent event associated with viral infection. We showed previously that mESCs do not express IFN when infected with several viruses (18, 26), including CHIKV, which causes an inflammatory response in a mouse model and in cultured cells, as we reported recently (39). We infected D3 cells, D3-FBs, and 10T1/2 cells with CHIKV and analyzed the expression of several inflammatory molecules. As illustrated in Fig. 6A, CHIKV infection induced the expression of TNF-α, ICAM1, and MCP5 in 10T1/2 cells but not in D3 cells. CHIKV-infected D3-FBs expressed increased levels of these molecules compared with D3 cells, but at levels much lower than in 10T1/2 cells. These results resemble the pattern of IFN expression in those cells that were infected with CHIKV (18, 26).

FIGURE 6.

Viral infection–induced expression of inflammatory molecules in mESCs and mESC-FBs and the effect of LPS and TNF-α. (A) D3 cells, D3-FBs, and 10T1/2 cells were infected with CHIKV (multiplicity of infection = 1) for 20 h. The mRNA levels of tested genes were determined by RT-qPCR and compared with their mRNA levels in cells without infection (Con, designated as 1). (B) D3 cells, D3-FBs, and 10T1/2 cells were incubated with TNF-α (upper panel) or LPS (lower panel) for 3 h, followed by CHIKV infection for 20 h. Cell viability was determined by toluidine blue staining. Data are mean ± SD of a representative experiment performed in triplicate that was repeated two times. *p < 0.05.

FIGURE 6.

Viral infection–induced expression of inflammatory molecules in mESCs and mESC-FBs and the effect of LPS and TNF-α. (A) D3 cells, D3-FBs, and 10T1/2 cells were infected with CHIKV (multiplicity of infection = 1) for 20 h. The mRNA levels of tested genes were determined by RT-qPCR and compared with their mRNA levels in cells without infection (Con, designated as 1). (B) D3 cells, D3-FBs, and 10T1/2 cells were incubated with TNF-α (upper panel) or LPS (lower panel) for 3 h, followed by CHIKV infection for 20 h. Cell viability was determined by toluidine blue staining. Data are mean ± SD of a representative experiment performed in triplicate that was repeated two times. *p < 0.05.

Close modal

Because inflammatory cytokines play important roles in the regulation of antiviral responses, we further tested the effects of LPS and TNF-α on the viability of CHIKV-infected cells. D3 cells, D3-FBs, and 10T1/2 cells were treated with LPS and TNF-α under the conditions described in Fig. 5, followed by infection with CHIKV. As shown in Fig. 6B, CHIKV infection caused apparent cell death in all three types of cells; however, TNF-α augmented CHIKV-induced cell death in D3-FBs and 10T1/2 cells but not in D3 cells (Fig. 6B, upper panel), whereas LPS did not show an additional effect on CHIKV-induced cell death in any cells tested (Fig. 6B, lower panel). These results further support the conclusion that mESCs are unable to mount an inflammatory response. Differentiated mESC-FBs have partially developed the mechanism to respond to TNF-α, but they lack the mechanism to respond to LPS.

To define the molecular basis underlying the attenuated inflammatory responses in mESCs and mESC-FBs, we first analyzed the expression levels of TLR4 and CD14, the receptor and coreceptor for LPS, respectively, by flow cytometry. TLR4 was not detected in D3 cells and D3-FBs, whereas the expression of CD14 in D3-FBs was slightly higher than in D3 cells (Fig. 7A). For comparison, we analyzed TLR4 and CD14 in RAW cells, which were shown to respond to LPS (Fig. 3C). TLR4 was expressed at a low level in RAW cells, but the difference is that its expression, as well as that of CD14, was significantly stimulated by LPS in RAW cells but not in D3 cells and D3-FBs (Fig. 7B). These results explain the unresponsiveness of D3 cells and D3-FBs to LPS. We also determined the relative expression of TLR4, CD14, and TNFR1 (the receptor that mediates the inflammatory response of TNF-α) by RT-qPCR. The mRNA of the three molecules tested was expressed at the lowest levels in D3 cells, but it increased several fold in D3-FBs to levels comparable to 10T1/2 cells (Fig. 7D, upper panel). It is interesting that the mRNA of TLR4 was not translated to functional TLR4 in D3-FBs. In the case of NF-κB, its protein is readily detected in undifferentiated mESCs, as indicated by immunofluorescence microscopy. This was confirmed by flow cytometry. The expression of NF-κB was further upregulated in D3-FBs (Fig. 7C, indicated by the increased fluorescence intensity of the profile, D3 versus D3-FBs) to a level comparable to 10T1/2 cells. α-SMA as a marker of fibroblasts was extensively expressed in D3-FBs and 10T1/2 cells (Fig. 7C). Therefore, the transition of NF-κB from an inactive state in mESCs to an active state during differentiation is evident by its nuclear translocation and its increased expression.

FIGURE 7.

Differentiation-induced upregulation of signaling molecules that regulate inflammatory responses. (A) Expression levels of TLR4 and CD14 in D3 cells, D3-FBs, and RAW cells determined by flow cytometry (the lines denoted by arrows). The lines denoted by arrowheads are negative controls. (B) The effect of LPS on the expression of TLR4 and CD14. The expression levels of TLR4 and CD14 in control cells and in cells that were treated with LPS for 20 h were determined by flow cytometry. The lines denoted by arrowheads represent negative controls. (C) Expression levels of NF-κB and α-SMA in D3 cells, D3-FBs, and 10T1/2 cells determined by flow cytometry (denoted by arrows). The lines denoted by arrowheads are negative controls. (D) Relative mRNA levels of the indicated genes in D3 cells, D3-FBs, and 10T1/2 cells were determined by RT-qPCR (upper panel). The mRNA level of each gene was normalized to β-actin in each cell type. Long-term culture–induced upregulation of the indicated genes in D3-FBs was determined by RT-qPCR and compared with their mRNA levels in the cells before the long-term culture (0 d, designated as 1) (lower panel). Data are mean ± SD of representative experiments performed in triplicate that were repeated at least two times. *p < 0.05 versus D3 cells.

FIGURE 7.

Differentiation-induced upregulation of signaling molecules that regulate inflammatory responses. (A) Expression levels of TLR4 and CD14 in D3 cells, D3-FBs, and RAW cells determined by flow cytometry (the lines denoted by arrows). The lines denoted by arrowheads are negative controls. (B) The effect of LPS on the expression of TLR4 and CD14. The expression levels of TLR4 and CD14 in control cells and in cells that were treated with LPS for 20 h were determined by flow cytometry. The lines denoted by arrowheads represent negative controls. (C) Expression levels of NF-κB and α-SMA in D3 cells, D3-FBs, and 10T1/2 cells determined by flow cytometry (denoted by arrows). The lines denoted by arrowheads are negative controls. (D) Relative mRNA levels of the indicated genes in D3 cells, D3-FBs, and 10T1/2 cells were determined by RT-qPCR (upper panel). The mRNA level of each gene was normalized to β-actin in each cell type. Long-term culture–induced upregulation of the indicated genes in D3-FBs was determined by RT-qPCR and compared with their mRNA levels in the cells before the long-term culture (0 d, designated as 1) (lower panel). Data are mean ± SD of representative experiments performed in triplicate that were repeated at least two times. *p < 0.05 versus D3 cells.

Close modal

We noticed that the expression levels of TLR4 and TNF-α receptors in D3-FBs increase slightly at higher passages, indicating that the signaling mechanisms undergo further development during subculturing. This is better demonstrated by the experiments in which cells were cultured for a prolonged period of time without splitting. As shown in Fig. 7D (lower panel), the mRNA levels of TLR4, CD14, TNFR1, and NF-κB all were progressively upregulated in the cells that were continuously cultured for 10 and 20 d. These cells showed increased induction of ICAM1 and IL-6 by TNF-α, but they still lack a response to LPS (data not shown).

A previous study demonstrated that mESCs were susceptible to bacterial infection, but they did not exhibit the immune and inflammatory responses typically displayed by somatic cells (14). This observation echoes the lack of IFN expression in virus-infected ESCs (16, 19). Because NF-κB is a key transcription factor that mediates a broad spectrum of immune and inflammatory responses induced by numerous pathogens and cytokines (40), we reasoned that the inactive status of NF-κB in ESCs, as noted in virus-infected cells, could also account for the lack of immune and inflammatory responses in bacteria-infected ESCs. However, studies with LPS, which is widely used to replicate many aspects of bacterial infection, reported different results in ESCs. For example, Lee et al. (41) reported that LPS increased cell proliferation, with its receptor TLR4 mRNA positively detected in mESCs. In contrast, Taylor et al. (42) showed that LPS inhibited mESC proliferation, but its conventional receptor TLR4 was not expressed. They proposed that the effect of LPS was mediated by TLR2 in the absence of TLR4 (42). In contrast, the lack of response to LPS in mESCs and hESCs, and even their differentiated cells, was also reported (5, 6). The above-mentioned studies also disagreed on whether NF-κB was activated by LPS. The only two studies that we are aware of that investigated the effect of TNF-α also reported different results. One study suggests that mESCs and their differentiated vascular cells lack a response to TNF-α (8), whereas the other indicates that TNF-α negatively impacts mESC proliferation, viability, and differentiation (43). Although the reasons for these discrepancies are not clear, the current study, based on the data obtained from mESCs and their differentiated cells via multiple experimental approaches, clearly demonstrates that mESCs have a deficient inflammatory response and provide the molecular basis for such deficiency in these cells.

Although best characterized as a key transcription factor in immune and inflammatory responses, NF-κB also regulates a variety of cellular events (25). Not surprisingly, the initial interest in NF-κB in ESCs was its role in the regulation of their stem cell properties: pluripotency and differentiation. RelA and p50, the two subunits in the canonical NF-κB pathway, are expressed at low levels in mESCs and hESCs, but they are upregulated upon differentiation (4447). Furthermore, the activity of NF-κB is repressed by Nanog, one of the key pluripotency genes in hESCs (44), and by miR-290 cluster in mESCs (48). These studies suggest that NF-κB activity in ESCs is repressed as a mechanism to maintain pluripotency. Although none of the aforementioned studies investigated the role of NF-κB in the context of innate immunity, their results support our conclusions. It is interesting that a recent study reported that hESCs were unable to respond to LPS, like we described in mESCs in this study, but the NF-κB pathway seemed to be functional (5), which is in contrast to another study showing that NF-κB is not activated by TNF-α in hESCs (47). The reasons for these different results are not clear, because the two studies used different hESC lines and assays. Our data from hESCs clearly illustrate that they are similar to mESCs in the lack of NF-κB activation and inflammatory response to LPS and TNF-α. These results are also in accordance with the fact that mESCs and hESCs are deficient in the expression of type I IFN in the antiviral response, which is an NF-κB–dependent process (10). Considering the highly conserved role of NF-κB in innate immunity among different species of vertebrates (40), our findings suggest that the lack of NF-κB activation in response to immune and inflammatory stimuli is likely an intrinsic property of hESCs and mESCs.

In addition to the inactive status of NF-κB, the absent or low-level expression of LPS and TNF-α receptors seems to be another factor that contributes to the deficiency of the inflammatory response in mESCs. The differentiation process promoted the transition of NF-κB to a functional status along with increased expression of NF-κB. The results from experiments with TNF-α provide a correlation among the expression levels of ICAM1 and IL-6, NF-κB nuclear translocation, and the expression levels of TNFR1 in mESCs, mESC-DCs, and ESC-FBs. Zampetaki et al. (8) reported that mESCs and even mESC-differentiated endothelial cells generated through EB differentiation were not responsive to TNF-α. This result is similar to our observation in EB-differentiated endothelial cells. However, we are able to demonstrate that mESC-FBs are responsive to TNF-α. Therefore, the inflammatory response mechanism is a developmentally regulated process that is affected by differentiation format, duration, and cell type. Although this study mainly used TNF-α, the selected experiments performed with IL-1β showed similar results, indicating that the conclusions from TNF-α could apply to other inflammatory cytokines whose actions depend on the activation of NF-κB.

mESCs, mESC-DCs, and mESC-FBs all failed to show detectable responses to LPS, even in mESC-FBs that have a functional NF-κB. This result can be explained by the lack of functional TLR4 expression in mESCs and mESC-FBs. It is intriguing that the mRNA of TLR4 was expressed, but it is not translated into functional proteins. The lack of LPS-induced gene expression is also noted in hESCs and their differentiated vascular cells (5). Studies showed that primary mouse embryonic fibroblasts are responsive to LPS (49, 50). We are uncertain whether the lack of response of mESC-FBs to LPS is related to in vitro differentiation, because a similar observation was made in 10T1/2 cells. In addition to the results obtained from LPS and TNF-α, the lack of an inflammatory response in mESCs was also demonstrated in response to viral infection, similar to the lack of IFN expression in CHIKV-infected mESCs (18, 26).

Although the biological implications for the lack of an IFN-based antiviral response and an inflammatory response remain to be fully appreciated, it is not entirely surprising considering the fact that inflammatory cytokines and IFN are primarily produced for the purpose of defense and are negative regulators of growth and development (51). It is logical for ESCs not to produce these molecules at a developmental stage when cell proliferation and differentiation are major events, especially when ESCs reside in the womb where they could be protected by the mother’s immune system (52). In this view, the innate immunity is not innate in ESCs but rather is developmentally acquired during differentiation by somatic cells, at least in cases of IFN-based antiviral and inflammatory mechanisms. Recent studies on this subject led to the notion that ESCs and differentiated somatic cells may have adapted distinct defense mechanisms at different stages of organismal development (27, 53).

Together with previous studies (49, 26), our data demonstrate that commonly used in vitro–differentiation methods only partially promote the development of innate immune response mechanisms. Questions remain as to how and to what degree the attenuated innate immunity may affect their therapeutic potential. To fully understand these questions, it will be essential to have a complete characterization of ESC-derived cells by in vitro and in vivo studies. Eventually, human cells will have to be used for therapeutic applications. However, like in many other areas of biomedical research, the knowledge derived from mouse models has been instrumental in understanding human physiology and diseases. The knowledge derived from studies of mESCs and mESC-DCs will be valuable for developing strategies to obtain clinically usable cells from human pluripotent stem cells.

We thank the Mississippi-IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by National Institute of General Medical Sciences Grant P20 GM103476-11).

This work was supported by National Institutes of Health Grant 1R15GM109299-01A1 (to Y.-L.G.).

Abbreviations used in this article:

ActD

actinomycin D

ATCC

American Type Culture Collection

CHIKV

Chikungunya virus

DBA

DBA252

EB

embryoid body

D3-FB

D3-fibroblast

ESC

embryonic stem cell

hESC

human ESC

mESC

mouse ESC

mESC-DC

mESC-differentiated cell

mESC-FB

mESC-differentiated fibroblast

RAW

RAW264.7

RT-qPCR

real-time quantitative PCR

α-SMA

α–smooth muscle actin

10T1/2

C3H10T1/2.

1
Wobus
A. M.
,
Boheler
K. R.
.
2005
.
Embryonic stem cells: prospects for developmental biology and cell therapy.
Physiol. Rev.
85
:
635
678
.
2
Keller
G.
2005
.
Embryonic stem cell differentiation: emergence of a new era in biology and medicine.
Genes Dev.
19
:
1129
1155
.
3
Tabar
V.
,
Studer
L.
.
2014
.
Pluripotent stem cells in regenerative medicine: challenges and recent progress.
Nat. Rev. Genet.
15
:
82
92
.
4
Rajan
S.
,
Ye
J.
,
Bai
S.
,
Huang
F.
,
Guo
Y. L.
.
2008
.
NF-kappaB, but not p38 MAP kinase, is required for TNF-α-induced expression of cell adhesion molecules in endothelial cells.
J. Cell. Biochem.
105
:
477
486
.
5
Földes
G.
,
Liu
A.
,
Badiger
R.
,
Paul-Clark
M.
,
Moreno
L.
,
Lendvai
Z.
,
Wright
J. S.
,
Ali
N. N.
,
Harding
S. E.
,
Mitchell
J. A.
.
2010
.
Innate immunity in human embryonic stem cells: comparison with adult human endothelial cells.
PLoS One
5
:
e10501
.
6
Zampetaki
A.
,
Xiao
Q.
,
Zeng
L.
,
Hu
Y.
,
Xu
Q.
.
2006
.
TLR4 expression in mouse embryonic stem cells and in stem cell-derived vascular cells is regulated by epigenetic modifications.
Biochem. Biophys. Res. Commun.
347
:
89
99
.
7
Sidney
L. E.
,
Kirkham
G. R.
,
Buttery
L. D.
.
2014
.
Comparison of osteogenic differentiation of embryonic stem cells and primary osteoblasts revealed by responses to IL-1β, TNF-α, and IFN-γ.
Stem Cells Dev.
23
:
605
617
.
8
Zampetaki
A.
,
Zeng
L.
,
Xiao
Q.
,
Margariti
A.
,
Hu
Y.
,
Xu
Q.
.
2007
.
Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-α is rescued by HDAC inhibitor trichostatin A.
Am. J. Physiol. Cell Physiol.
293
:
C1226
C1238
.
9
Glaser
D. E.
,
Gower
R. M.
,
Lauer
N. E.
,
Tam
K.
,
Blancas
A. A.
,
Shih
A. J.
,
Simon
S. I.
,
McCloskey
K. E.
.
2011
.
Functional characterization of embryonic stem cell-derived endothelial cells.
J. Vasc. Res.
48
:
415
428
.
10
Guo
Y. L.
,
Carmichael
G. G.
,
Wang
R.
,
Hong
X.
,
Acharya
D.
,
Huang
F.
,
Bai
F.
.
2015
.
Attenuated innate immunity in embryonic stem cells and its implications in developmental biology and regenerative medicine.
Stem Cells
33
:
3165
3173
.
11
English
K.
,
Wood
K. J.
.
2011
.
Immunogenicity of embryonic stem cell-derived progenitors after transplantation.
Curr. Opin. Organ Transplant.
16
:
90
95
.
12
de Almeida
P. E.
,
Ransohoff
J. D.
,
Nahid
A.
,
Wu
J. C.
.
2013
.
Immunogenicity of pluripotent stem cells and their derivatives.
Circ. Res.
112
:
549
561
.
13
Tan
Y.
,
Ooi
S.
,
Wang
L.
.
2014
.
Immunogenicity and tumorigenicity of pluripotent stem cells and their derivatives: genetic and epigenetic perspectives.
Curr. Stem Cell Res. Ther.
9
:
63
72
.
14
Yu
J.
,
Rossi
R.
,
Hale
C.
,
Goulding
D.
,
Dougan
G.
.
2009
.
Interaction of enteric bacterial pathogens with murine embryonic stem cells. [Published erratum appears in 2009 Infect. Immun. 77: 2239.]
Infect. Immun.
77
:
585
597
.
15
Wash
R.
,
Calabressi
S.
,
Franz
S.
,
Griffiths
S. J.
,
Goulding
D.
,
Tan
E.-P.
,
Wise
H.
,
Digard
P.
,
Haas
J.
,
Efstathiou
S.
,
Kellam
P.
.
2012
.
Permissive and restricted virus infection of murine embryonic stem cells.
J. Gen. Virol.
93
:
2118
2130
.
16
Wang
R.
,
Teng
C.
,
Spangler
J.
,
Wang
J.
,
Huang
F.
,
Guo
Y.-L.
.
2014
.
Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of mRNA-mediated gene expression strategy.
Stem Cells Dev.
23
:
594
604
.
17
Wang
R.
,
Wang
J.
,
Paul
A. M.
,
Acharya
D.
,
Bai
F.
,
Huang
F.
,
Guo
Y. L.
.
2013
.
Mouse embryonic stem cells are deficient in type I interferon expression in response to viral infections and double-stranded RNA.
J. Biol. Chem.
288
:
15926
15936
.
18
Wang
R.
,
Wang
J.
,
Acharya
D.
,
Paul
A. M.
,
Bai
F.
,
Huang
F.
,
Guo
Y. L.
.
2014
.
Antiviral responses in mouse embryonic stem cells: differential development of cellular mechanisms in type I interferon production and response.
J. Biol. Chem.
289
:
25186
25198
.
19
Chen
L. L.
,
Yang
L.
,
Carmichael
G. G.
.
2010
.
Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells.
Cell Cycle
9
:
3552
3564
.
20
Chen
G. Y.
,
Hwang
S. M.
,
Su
H. J.
,
Kuo
C. Y.
,
Luo
W. Y.
,
Lo
K. W.
,
Huang
C. C.
,
Chen
C. L.
,
Yu
S. H.
,
Hu
Y. C.
.
2012
.
Defective antiviral responses of induced pluripotent stem cells to baculoviral vector transduction.
J. Virol.
86
:
8041
8049
.
21
Samuel
C. E.
2001
.
Antiviral actions of interferons.
Clin. Microbiol. Rev.
14
:
778
809
.
22
Kawai
T.
,
Akira
S.
.
2011
.
Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.
Immunity
34
:
637
650
.
23
Yoneyama
M.
,
Kikuchi
M.
,
Natsukawa
T.
,
Shinobu
N.
,
Imaizumi
T.
,
Miyagishi
M.
,
Taira
K.
,
Akira
S.
,
Fujita
T.
.
2004
.
The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.
Nat. Immunol.
5
:
730
737
.
24
Kato
H.
,
Takahasi
K.
,
Fujita
T.
.
2011
.
RIG-I-like receptors: cytoplasmic sensors for non-self RNA.
Immunol. Rev.
243
:
91
98
.
25
Hayden
M. S.
,
Ghosh
S.
.
2012
.
NF-κB, the first quarter-century: remarkable progress and outstanding questions.
Genes Dev.
26
:
203
234
.
26
D’Angelo
W.
,
Acharya
D.
,
Wang
R.
,
Wang
J.
,
Gurung
C.
,
Chen
B.
,
Bai
F.
,
Guo
Y. L.
.
2016
.
Development of antiviral innate immunity during in vitro differentiation of mouse embryonic stem cells.
Stem Cells Dev.
25
:
648
659
.
27
Guo
Y. L.
2017
.
Utilization of different anti-viral mechanisms by mammalian embryonic stem cells and differentiated cells.
Immunol. Cell Biol.
95
:
17
23
.
28
Guo
Y. L.
,
Ye
J.
,
Huang
F.
.
2007
.
p38α MAP kinase-deficient mouse embryonic stem cells can differentiate to endothelial cells, smooth muscle cells, and neurons.
Dev. Dyn.
236
:
3383
3392
.
29
Pfaffl
M. W.
2001
.
A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res.
29
:
e45
.
30
Bai
F.
,
Wang
T.
,
Pal
U.
,
Bao
F.
,
Gould
L. H.
,
Fikrig
E.
.
2005
.
Use of RNA interference to prevent lethal murine West Nile virus infection.
J. Infect. Dis.
191
:
1148
1154
.
31
Guo
Y. L.
,
Kang
B.
,
Yang
L. J.
,
Williamson
J. R.
.
1999
.
Tumor necrosis factor-alpha and ceramide induce cell death through different mechanisms in rat mesangial cells.
Am. J. Physiol.
276
:
F390
F397
.
32
Niwa
H.
,
Burdon
T.
,
Chambers
I.
,
Smith
A.
.
1998
.
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.
Genes Dev.
12
:
2048
2060
.
33
Pinney
D. F.
,
Emerson
C. P.
 Jr.
1989
.
10T1/2 cells: an in vitro model for molecular genetic analysis of mesodermal determination and differentiation.
Environ. Health Perspect.
80
:
221
227
.
34
Reznikoff
C. A.
,
Brankow
D. W.
,
Heidelberger
C.
.
1973
.
Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division.
Cancer Res.
33
:
3231
3238
.
35
Ledebur
H. C.
,
Parks
T. P.
.
1995
.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-κB site and p65 homodimers.
J. Biol. Chem.
270
:
933
943
.
36
Libermann
T. A.
,
Baltimore
D.
.
1990
.
Activation of interleukin-6 gene expression through the NF-κB transcription factor.
Mol. Cell. Biol.
10
:
2327
2334
.
37
Sedger
L. M.
,
McDermott
M. F.
.
2014
.
TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants - past, present and future.
Cytokine Growth Factor Rev.
25
:
453
472
.
38
Guo
Y. L.
,
Baysal
K.
,
Kang
B.
,
Yang
L. J.
,
Williamson
J. R.
.
1998
.
Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor-alpha in rat mesangial cells.
J. Biol. Chem.
273
:
4027
4034
.
39
Acharya
D.
,
Paul
A. M.
,
Anderson
J. F.
,
Huang
F.
,
Bai
F.
.
2015
.
Loss of glycosaminoglycan receptor binding after mosquito cell passage reduces chikungunya virus infectivity.
PLoS Negl. Trop. Dis.
9
:
e0004139
.
40
Baeuerle
P. A.
,
Henkel
T.
.
1994
.
Function and activation of NF-κB in the immune system.
Annu. Rev. Immunol.
12
:
141
179
.
41
Lee
S. H.
,
Hong
B.
,
Sharabi
A.
,
Huang
X. F.
,
Chen
S. Y.
.
2009
.
Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.
Stem Cells
27
:
1604
1615
.
42
Taylor
T.
,
Kim
Y. J.
,
Ou
X.
,
Derbigny
W.
,
Broxmeyer
H. E.
.
2010
.
Toll-like receptor 2 mediates proliferation, survival, NF-kappaB translocation, and cytokine mRNA expression in LIF-maintained mouse embryonic stem cells.
Stem Cells Dev.
19
:
1333
1341
.
43
Wuu
Y. D.
,
Pampfer
S.
,
Vanderheyden
I.
,
Lee
K. H.
,
De Hertogh
R.
.
1998
.
Impact of tumor necrosis factor alpha on mouse embryonic stem cells.
Biol. Reprod.
58
:
1416
1424
.
44
Torres
J.
,
Watt
F. M.
.
2008
.
Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NFkappaB and cooperating with Stat3.
Nat. Cell Biol.
10
:
194
201
.
45
Armstrong
L.
,
Hughes
O.
,
Yung
S.
,
Hyslop
L.
,
Stewart
R.
,
Wappler
I.
,
Peters
H.
,
Walter
T.
,
Stojkovic
P.
,
Evans
J.
, et al
.
2006
.
The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis.
Hum. Mol. Genet.
15
:
1894
1913
.
46
Kim
Y. E.
,
Kang
H. B.
,
Park
J. A.
,
Nam
K. H.
,
Kwon
H. J.
,
Lee
Y.
.
2008
.
Upregulation of NF-kappaB upon differentiation of mouse embryonic stem cells.
BMB Rep.
41
:
705
709
.
47
Kang
H. B.
,
Kim
Y. E.
,
Kwon
H. J.
,
Sok
D. E.
,
Lee
Y.
.
2007
.
Enhancement of NF-kappaB expression and activity upon differentiation of human embryonic stem cell line SNUhES3.
Stem Cells Dev.
16
:
615
623
.
48
Lüningschrör
P.
,
Stöcker
B.
,
Kaltschmidt
B.
,
Kaltschmidt
C.
.
2012
.
miR-290 cluster modulates pluripotency by repressing canonical NF-κB signaling.
Stem Cells
30
:
655
664
.
49
Kurt-Jones
E. A.
,
Sandor
F.
,
Ortiz
Y.
,
Bowen
G. N.
,
Counter
S. L.
,
Wang
T. C.
,
Finberg
R. W.
.
2004
.
Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity.
J. Endotoxin Res.
10
:
419
424
.
50
Sacre
S. M.
,
Lundberg
A. M. C.
,
Andreakos
E.
,
Taylor
C.
,
Feldmann
M.
,
Foxwell
B. M.
.
2007
.
Selective use of TRAM in lipopolysaccharide (LPS) and lipoteichoic acid (LTA) induced NF-kappaB activation and cytokine production in primary human cells: TRAM is an adaptor for LPS and LTA signaling.
J. Immunol.
178
:
2148
2154
.
51
Hertzog
P. J.
,
Hwang
S. Y.
,
Kola
I.
.
1994
.
Role of interferons in the regulation of cell proliferation, differentiation, and development.
Mol. Reprod. Dev.
39
:
226
232
.
52
Levy
O.
2007
.
Innate immunity of the newborn: basic mechanisms and clinical correlates.
Nat. Rev. Immunol.
7
:
379
390
.
53
Pare
J. M.
,
Sullivan
C. S.
.
2014
.
Distinct antiviral responses in pluripotent versus differentiated cells.
PLoS Pathog.
10
:
e1003865
.

The authors have no financial conflicts of interest.