Embryonic stem cells (ESCs) represent a unique cell population in the blastocyst stage embryo. They have been intensively studied as a promising cell source for regenerative medicine. Recent studies have revealed that both human and mouse ESCs are deficient in expressing IFNs and have attenuated inflammatory responses. Apparently, the ability to express IFNs and respond to certain inflammatory cytokines is not “innate” to ESCs but rather is developmentally acquired by somatic cells during differentiation. Accumulating evidence supports a hypothesis that the attenuated innate immune response may serve as a protective mechanism allowing ESCs to avoid immunological cytotoxicity. This review describes our current understanding of the molecular basis that shapes the immune properties of ESCs. We highlight the recent findings on Dicer and dsRNA-activated protein kinase R as novel regulators of ESC fate and antiviral immunity and discuss how ESCs use alternative mechanisms to accommodate their stem cell properties.
Embryonic stem cells (ESCs) have attracted considerable attention for their potential applications in regenerative medicine. Along with the landmark discoveries in ESC self-renewal, pluripotency, and differentiation, we have also witnessed many surprising findings in the basic biology of these cells. The unique immunological property of ESCs is one such finding demonstrating that they are immunologically divergent from our conventional view of “innate immunity” established in differentiated somatic cells (1, 2). Innate immunity encompasses a variety of mechanisms as the first line of defense in an organism against a broad range of pathogens. At the cellular level, the IFN antiviral response and inflammatory response constitute the two major mechanisms of innate immunity presumably developed in most, if not all, types of somatic cells in vertebrates (3, 4). It is surprising to find that pluripotent cells, including human and mouse ESCs (hESCs and mESCs) as well as induced pluripotent stem cells (iPSCs), do not express type I IFNs in response to viral stimuli and have an attenuated or lack response to a wide range of pathogen-associated molecular patterns (PAMPs) and inflammatory cytokines (5–11). These findings raise interesting questions about how ESCs deal with immunological challenges in an early embryo and their biological implications, which have been discussed from the perspectives of stem cell–based regenerative medicine (12), antiviral innate immunity (2, 13), and embryogenesis (1). In this review, we briefly revisit these questions, update our recent progress in understanding the molecular basis that defines the innate immunity of ESCs, and highlight the novel roles of Dicer and protein kinase R (PKR) in the antiviral response and in ESC fate determination.
Attenuated innate immune response—a common feature of ESCs and its biological implications
ESCs are deficient in expressing type I IFNs and have an attenuated response to inflammatory cytokines
ESCs are pluripotent cells derived from the inner cell mass of the blastocyst, the preimplantation embryo. They can be kept at the undifferentiated state with unlimited capacity for proliferation (self-renewal) and can be induced to differentiate into different cell lineages (pluripotency). These two features constitute the cellular basis for their applications in regenerative medicine (14). Several early studies reported that cells differentiated from hESCs and mESCs show lower levels of response to certain inflammatory cytokines than do their in vivo–differentiated counterparts (15–20). mESCs are susceptible to bacterial infection, but they do not show typical immune responses as seen in differentiated cells (21). These findings inspired the investigation of the immunological properties of ESCs. Subsequent studies, including a series of studies from our laboratory, have demonstrated that mESCs, hESCs, and iPSCs lack a response to LPS, TNF-α, and IL-1 (9, 18, 22–24), and they also fail to express IFN-α and IFN-β (type I IFNs) in response to viral infection or synthetic viral RNA analogs (5, 6, 8, 10, 11, 25). Therefore, the ability to express IFNs and respond to inflammatory cytokines, which is considered to be innate to differentiated somatic cells, is not (or at least not completely) “innate” to ESCs but is rather acquired by somatic cells during differentiation. This is illustrated in mESC-differentiated fibroblasts (mESC-FBs), which progressively acquire the ability to express IFN-β and respond to TNF-α during in vitro differentiation (8). The acquisition and maturation of innate immune systems during development are also evident from mouse models and developmental studies in humans (26–28).
Although some notable species differences in immunological properties between mESCs and hESCs have been discussed in more detail in a previous review (1), it is apparent that the deficiency in expressing type I IFNs and the lack of responses to certain inflammatory cytokines are common features of all PSCs, which we refer to as an “attenuated innate immune response” for the convenience of discussion. Importantly, however, note that this term does not necessarily entail that ESCs have “attenuated innate immunity,” as cellular innate immunity is comprised of different forms, and ESCs may use alternative mechanisms to gain innate immunity as we discuss later. We also note that more studies have used mESCs and that the data from hESCs are relatively limited. This makes a direct comparison between the two species difficult, and some conclusions are primarily based on the studies of mESCs.
The attenuated innate immune response in ESCs may serve as a protective mechanism to avoid immunological cytotoxicity
The biological implications of the attenuated innate immune response in ESCs can be speculated on from different views. At the cellular and molecular levels, it appears that the mechanisms controlling innate immune responses and self-renewal/pluripotency are not compatible in ESCs (2). IFNs are well known to inhibit cell proliferation and induce differentiation (29, 30). The potential damage of the IFN response to the stemness of ESCs was illustrated in a study where forced activation of the IFN pathway caused dysregulation of many pluripotency- and lineage-specific genes in human iPSCs (hiPSCs) and mESCs, resulting in enhanced but aberrant cardiomyocyte differentiation (31). Therefore, it is rational for ESCs not to produce IFNs when they are dedicated to cell proliferation for embryogenesis. From the perspective of immunology, the immune response is a double-edged sword: it serves as a part of defense mechanisms, but it also causes collateral damage to the host cells, known as immunological cytotoxicity (32, 33). The immunological cytotoxicity can be tolerated in the tissue of a developed organism, but it could be detrimental to ESCs in an early embryo. In particular, the immune response is a prominent event during implantation when a blastocyst is exposed to inflammatory cytokines, which is necessary for implantation yet can negatively affect cell viability and proliferation (34, 35). It would be essential for ESCs to avoid this immunological cytotoxicity. We recently demonstrated that treatment with IFN-α, IFN-β, or IFN-γ (a type II IFN) alone caused low levels of the inhibitory effect on proliferation of fibroblasts, but this effect was significantly potentiated by TNF-α. In particular, the combination of TNF-α with IFN-γ (TNF-α/IFN-γ) effectively killed mESC-FBs and human fibroblasts. TNF-α and IFN-γ are known as “embryotoxic cytokines” for their detrimental effect on early embryos when dysregulated (35). Remarkably, mESCs, hESCs, and hiPSCs were insensitive to the cytotoxicity of TNF-α/IFN-γ or any of the above-mentioned cytokines alone (9, 22, 23). Therefore, we proposed that the attenuated innate immune response in ESCs could be an adaptive feature for ESCs to avoid immunological insults in a blastocyst (1).
The molecular basis for the lack of IFN expression and inflammatory response in ESCs
At the cellular level, immune responses are elicited by various PAMPs, such as viral DNA/RNA and bacterial endotoxins. TLRs are membrane-bound receptors that detect a wide variety of PAMPs (36). Cytoplasmic receptors, such as retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated protein (MDA5), play key roles in detecting viral RNA (37). Although PAMPs activate different pathways, the signal transduction commonly converges at NF-κB. Together with other transcription factors, such as IFN regulatory factor (IRF)3/7 and AP-1, NF-κB activates the transcription of IFNs, cytokines, and other immune mediators that participate in various aspects of innate and adaptive immune responses (36). Through autocrine and paracrine mechanisms, IFNs bind to their cell surface receptor complex and activate the JAK-STAT pathway, which induces numerous IFN-stimulated genes (ISGs) that promote the cell to enter an “antiviral state” (38). The IFN system, including the capacity to produce and respond to IFNs, has evolved as a major innate antiviral mechanism in vertebrates.
The molecular basis underlying the attenuated innate immune response in ESCs is not completely understood. Studies have shown that the major receptors for viral RNA (TLR3, RIG-I, and MDA5), LPS (TLR4), and TNF-α (TNFR1) are either expressed at low levels or not functional in ESCs (6, 9, 10, 23). More importantly, NF-κB, the master transcription factor in mediating many types of immune responses, is inactive in ESCs (8, 9, 39–41). These findings explain, at least partly, the attenuated innate immune responses in ESCs. Although the deficiency of ESCs in expressing IFNs has only been reported for IFN-α and IFN-β (type I IFNs), this is likely the case for IFN-γ (a type II IFN) as well because its expression also requires NF-κB transcription activity (42).
A compelling, yet-to-be-answered question, is how the immunological properties of ESCs are shaped by the stem cell state. Based on the dynamic changes of innate immune responses during in vitro ESC differentiation and iPSC reprogramming, there is an apparent reciprocal inhibition between the pluripotent state and the innate immune response (2). This is illustrated by the fact that differentiating ESCs lose pluripotency while gaining the ability to express IFNs and respond to inflammatory cytokines (7, 8, 11, 23). Conversely, fibroblasts lose the capacity to express IFNs after being reprogrammed into iPSCs (10, 11). The incompatibility between the pluripotent state and the IFN system was further demonstrated in studies where activation of the IFN pathway impaired the stem cell state of mESCs and hiPSCs (31, 43).
The stemness of ESCs and viral infectivity
hESCs and mESCs are susceptible to a variety of viruses with different levels of infectivity and cytopathogenicity (5, 6, 8, 25, 44, 45), including the Zika, rubella, and Sendai viruses, which are known to impact developing embryos (46–50) However, only a few studies have investigated viral replication cycles of specific types of viruses. It was reported that mESCs are partly permissive for HSV type 1 but restrict the influenza virus replication cycle. Viral replication analysis revealed that the ineffective replication of HSV type 1 and incomplete replication of influenza virus are associated with improper glycosylation of viral proteins in mESCs, which could be attributed to the different glycosylation profiles between mESCs and mESC-differentiated cells (25). hESCs are permissive to coxsackievirus B similarly to hESC-differentiated cells (44). However, hESCs are highly refractory to Japanese encephalitis virus whereas hESC-differentiated neuroepithelial precursor cells (NPCs) are highly permissive to this virus. The dramatic difference can be explained by the finding that vimentin is the receptor that mediates viral entry in NPCs, and it is expressed in NPCs but not in hESCs (51). Therefore, ESCs are permissive for some viruses but restrict others. Although the stem cell state could directly or indirectly restrict the infectivity and replication of certain viruses, we are not aware of studies that have specifically investigated whether pluripotency factors may directly affect viral infectivity and replication.
Utilization of alternative immune defense mechanisms by ESCs
Intrinsic antiviral pathways
The hypothesis that the attenuated innate immune response in ESCs serves as a mechanism to avoid immunological toxicity only makes sense if ESCs are protected by other means. A preimplantation blastocyst is enclosed in a zona pellucida, a thick extracellular coat layer that can provide a physical barrier to microbial pathogens. The zona pellucida could effectively prevent embryonic cells from infection by many but not all types of viruses (52, 53). ESCs could presumably serve as good hosts for viral replication if infected, as they are rapidly proliferating cells with deficiencies in IFN production. However, ESCs have been found to be less susceptible to the infection by many types of viruses, and therefore ESCs may use alternative immune defense mechanisms that differ from those used by differentiated somatic cells. It was recently reported that ESCs are more resistant to several types of flaviviruses and negative-strand RNA viruses than ESC-differentiated cells due to the high level expression of a set of pre-existing ISGs (intrinsic ISGs) in ESCs (45). Unlike canonical ISGs that must be induced by IFNs, intrinsic ISGs in ESCs are constitutively expressed in the absence of IFNs. The antiviral activity provided by pre-existing antiviral molecules has been known as intrinsic antiviral immunity (54, 55). PKR and RNase L are well characterized among this class of antiviral molecules (33, 56). They can be directly and immediately activated upon viral infection to inhibit viral replication by blocking viral protein synthesis and by degrading viral RNA, respectively (54, 55). However, the expression and activity of PKR and RNase L can be further upregulated by IFNs, so they can also be considered as part of the IFN response (54). Although there are limited data on RNase L in ESCs, PKR is expressed in both hESCs and mESCs (6,11), which could provide important intrinsic antiviral immunity to ESCs, as is discussed later.
ESCs can gain antiviral activity from IFNs via paracrine signaling
Although mESCs and hESCs do not produce IFNs, they show low-level responses to IFN-α and IFN-β and express ISGs (7, 44, 57, 58). Interestingly, hESCs, but not mESCs, can also respond to IFN-γ (23). This means that ESCs could gain antiviral activity from IFNs via a paracrine mechanism in the blastocyst. How ESCs can avoid potential negative effects of IFNs, as previously discussed, is a clear question yet to be answered. However, two scenarios can be envisioned from our current knowledge. First, IFN-induced cellular responses in ESCs are substantially lower than in differentiated cells, which could provide antiviral activity without reaching the level that causes cytotoxicity. Second, the cytotoxicity of IFNs is synergistically potentiated by TNF-α, a well-known phenomenon in differentiated cells (59). The fact that both mESCs and hESCs lack a response to TNF-α could allow them to avoid or diminish the cytotoxic effects of TNF-α and IFNs (23).
The RNA interference antiviral pathway
The RNA interference (RNAi) antiviral pathway is widely used by invertebrates and plants in which the IFN system is absent (60, 61). In this mechanism, dsRNA intermediates generated during virus replication are processed by Dicer into small interfering RNAs (siRNAs), which leads to the cleavage of viral RNA. It is generally believed that the RNAi antiviral mechanism is not used in mammalian cells (62), presumably because the powerful IFN-based innate immunity and adaptive immune system in mammals may render the RNAi antiviral activity unnecessary. Mechanistically, the molecular machinery for siRNA processing in mammalian cells is optimized for microRNA (miRNA) biogenesis (61). However, siRNA of viral origin has been detected in virus-infected mESCs and newborn mice, suggesting that the RNAi antiviral mechanism might be functional at the early stage of development (63, 64). Interestingly, it was recently reported that a novel isoform of Dicer that is more effective in generating viral siRNA may exist in mESCs and hiPSCs (65). These findings support the hypothesis that mammalian cells may have adapted distinct antiviral mechanisms: the IFN system is primarily used by differentiated somatic cells, whereas RNAi could be used in ESCs in which the IFN system is not fully developed (2). However, whether RNAi is used as a prevalent antiviral mechanism in ESCs is still a matter of debate (62, 66–69). The major antiviral pathways in differentiated mammalian cells and ESCs are summarized in (Fig. 1.
Dicer is a repressor of the IFN response and the PKR pathway in ESCs
Dicer-deficient mESCs acquire the ability to express type I IFNs and have constitutively activated PKR
Dicer is a key enzyme in miRNA and siRNA biogenesis in mammalian cells, and its deletion is embryonic lethal in mice (70). However, Dicer knockout mESCs (Dicer−/− mESCs) are viable. These cells are normal in self-renewal but have a slower proliferation rate and are differentiation deficient (71, 72). One would assume that Dicer−/− mESCs would be more susceptible to viral infection than wild-type mESCs if the RNAi mechanism contributes to antiviral activity. Surprisingly, it was found that Dicer−/− mESCs were in fact more resistant to infection of Theiler’s murine encephalomyelitis virus and influenza A, but this could be rationally explained by the finding that Dicer−/− mESCs have gained the ability to express IFN-β (73). Using synthetic dsRNA as viral RNA mimics, we further demonstrated that Dicer−/− mESCs have constitutively activated PKR, in addition to being able to express IFN-β, which may together contribute to their increased antiviral activity (43, 73).
We proposed that the deficiency in expressing IFNs is an intrinsic feature of pluripotent cells and that the IFN system is developmentally acquired during differentiation (1, 12). The finding that Dicer−/− mESCs at the undifferentiated state can gain some ability to express IFNs indicates that the lack of IFN expression in ESCs is not entirely restricted by the stem cell state but is also repressed by Dicer. The significance of these findings in Dicer−/− mESCs lies in the fact that they revealed a role of Dicer as a repressor of IFN expression and the PKR pathway, which provides not only a novel explanation for the attenuated antiviral response in ESCs, but also new insights into the roles of Dicer and PKR in antiviral innate immunity and in the regulation of ESC properties.
Dicer represses IFN expression in ESCs via miRNAs
Considering the key role of Dicer in miRNA biogenesis, it is rational to believe that miRNA deficiency is responsible for the increased antiviral responses in Dicer−/−mESCs. Both hESCs and mESCs express a set of miRNAs (ESC-miRNAs) important for the establishment and maintenance of stemness (74). While there is limited knowledge about the specific miRNAs that regulate ESC immunological properties, the miR-290 cluster of mESC-miRNAs was reported to directly target the mRNA of the RelA subunit of NF-κB (75), which could contribute to the lack of IFN expression. In agreement, NF-κB can indeed be activated in Dicer−/− mESCs but not in wild-type mESCs (73). miR-673 is another miRNA that may repress IFN expression by targeting mitochondrial antiviral-signaling protein (MAVS), a key signaling molecule essential for IFN expression that is upregulated in Dicer−/− mESCs due to the lack of miR-673. However, miR-673 is only conserved in rodents (73). The specific miRNAs that repress IFN expression in hESCs and additional miRNAs that may regulate immune properties of ESCs remain to be identified.
Dicer represses the PKR pathway in ESCs and cellular RNA processing
In addition to its key role in siRNA and miRNA biogenesis, Dicer also processes different types of cellular RNAs, including noncoding RNAs, RNA with dsRNA structures, and the transcripts of transposable elements (TEs) (76–78). Accumulation of these cellular RNAs could elicit antiviral responses in the absence of pathogens known as “sterile inflammation,” leading to cellular damage similar to that caused by viral RNA (79). TEs, including long interspersed elements, long terminal repeats, and short interspersed nuclear elements (SINEs), occupy a large portion of the human and mouse genome. They participate in different cellular processes and are particularly active in early embryos (80). Their activities are tightly controlled by multiple mechanisms and can pose a potential threat to genome stability and elicit antiviral response if dysregulated (81–83). The regulation of TEs by Dicer was first reported in Dicer‐deficient mouse preimplantation embryos (84). It was recently reported that long interspersed element 1 (LINE‐1) transcripts are accumulated in Dicer−/− mESCs, which could contribute the failure of Dicer−/− mESCs to exit from the pluripotency state and increased apoptosis (85). As another example of TE regulation, the transcripts of Alu (a SINE in the human genome) is processed by Dicer in hESCs (86). Accumulation of Alu is known to cause an antiviral response and apoptosis in human cells (76, 87, 88), and it has been shown that Alu RNA, either endogenously transcribed or ectopically expressed, can activate PKR (77, 89, 90). B2 SINE, the mouse counterpart of Alu (82) is abundantly expressed in mESCs and is further accumulated in Dicer−/−mESCs (43). B2 RNA has a secondary structure with hairpins and loops, resembling the dsRNA structure (82, 91). Synthetic B2 RNA transfected into mESCs can activate PKR and lead to effects that are remarkably similar to dsRNA-induced inhibition of cell proliferation (43). These findings indicate that B2 RNA accumulation in Dicer−/−mESCs could contribute to the constitutive activation of PKR, but the underlying mechanisms remain to be further investigated. Other mechanisms, directly or indirectly related to miRNA deficiency, may also contribute to the constitutive PKR activation in Dicer−/−mESCs.
Contributions of miRNA deficiency, IFN response, and PKR activation to the phenotype of Dicer−/− ESCs
The phenotypic features of Dicer−/− mESCs, that is, a slower cell proliferation rate, reduced cell viability, and differentiation deficiency (71, 72), are ultimately attributed to the lack of Dicer. However, it is challenging to pinpoint the underlying molecular mechanisms because Dicer can regulate cellular processes by miRNA biogenesis, cellular RNA processing, and by interactions with other RNA-binding proteins independent of its endoribonuclease activity, including its direct interaction with PKR (78, 92). This complexity is manifested in the case of ESC proliferation. mESCs have a unique cell cycle characterized by a shortened G1 phase and a rapid cell proliferation rate driven by a combination of high levels of cyclins A and E and low levels of cell cycle inhibitors (93). ESC-miRNAs directly target the mRNA of several cell cycle proteins, including p21 and p19, the two major cell cycle inhibitors (94, 95). Thus, in Dicer−/− mESCs, p21 and p19 proteins are expressed at much higher levels than in mESCs (43), correlating with the reduced proliferation of Dicer−/− mESCs. However, treatment of Dicer−/− mESCs with a PKR inhibitor or knockdown of PKR significantly increased cell proliferation without affecting the levels of p21 and p19, indicating that PKR may inhibit cell proliferation via mechanisms independent of miRNA action (43). It is conceivable that global translation inhibition, a key function of PKR, caused by constitutive PKR activation in Dicer−/− mESCs could be one of the mechanisms that negatively affects cell proliferation and viability (32).
It appears that Dicer is more critical for the viability of hESCs because Dicer knockout leads to apoptosis (96), whereas Dicer−/− mESCs only show reduced cell viability. However, Dicer−/− mESCs have increased sensitivity to the cytotoxicity resulting from the antiviral response due to their IFN-β expression and constitutive PKR activation similar to somatic cells (43). The findings from Dicer−/− mESCs provide another line of evidence to support the hypothesis that the underdeveloped IFN system in ESCs can limit the damage resulting from the antiviral response, elicited either by viral pathogens or dysregulated intracellular RNA, such as aberrant production of TE transcripts (83).
Adaptation of PKR as an antiviral molecule and novel regulator of ESC fate
PKR as an antiviral molecule compatible with stem cell state
In differentiated somatic cells, PKR responds to a broad spectrum of viruses and provides antiviral activity via different mechanisms (97, 98). PKR is abundantly expressed in both hESCs and mESCs (6, 11). The data regarding the functionality of PKR in hESCs is rather limited, but PKR is fully functional in mESCs because it is activated by cellular RNA during the normal cell cycle and by synthetic viral RNA analogs and viral infection (6, 43, 90). Therefore, PKR activation could mount a quick antiviral response and provide critical antiviral innate immunity to ESCs in which the IFN system is not fully developed. Because PKR-mediated translation inhibition of viral proteins is a major mechanism to inhibit viral replication, the low-level translation rate in ESCs mediated by activated PKR (as is discussed in the next section) may render ESCs to not be an ideal host for viral replication. Interestingly, note that in differentiated cells, PKR can modulate IFN responses in different ways. One mechanism is by regulating the integrity of newly synthesized IFN mRNA as reported in mouse fibroblasts (99). In macrophages, it was recently demonstrated that activation of RIPK3 (receptor interacting serine/threonine protein kinase 3) can limit IFN-β transcription via interaction with MAVS. Alternatively, RIPK3 can also stabilize IFN-β mRNA by activating PKR, leading to IFN-β protein accumulation (100). These two actions together constitute a mechanism that enhances antiviral immunity while protecting the host cells (98, 100). It is tempting to speculate that PKR may play a similar role in differentiating ESCs when they begin to acquire limited capacity to transcribe IFN mRNAs.
It is known that PKR in somatic cells can negatively affect many cellular processes and even cause cell death as seen in differentiated cells (32, 33). In principle, the cytotoxicity caused by PKR activation could be as harmful to ESCs as that caused by IFN response. Surprisingly, this activation only causes a rather mild cell proliferation inhibition and downregulation of mESC marker transcripts without apparent long-term effects on mESC stem cell state (6, 43). It is apparent that the cytotoxicity resulting from PKR activation only causes serious cellular damage when the IFN response is also activated as seen in Dicer−/−mESCs and mESC-FBs (8, 43). Therefore, an attenuated IFN response in ESCs, together with other features as discussed, make PKR an antiviral molecule well adapted for the stem cell state, especially when the unique roles of PKR in the regulatory network in ESCs are considered.
The potential roles of PKR as a novel regulator of ESC self-renewal and differentiation
The pluripotency and self-renewal of ESCs are primarily maintained by a set of pluripotency transcription factors (including Oct4, Nanog, Sox2, and c-Myc) and modulated by additional mechanisms such as epigenetics (101). The newly proposed “translational switch” hypothesis has added a new dimension to the stem cell regulatory network. Recent studies have demonstrated that ESCs as well as adult stem cells use translational control as a mechanism governing gene expression profiles between self-renewal and differentiation (102, 103). The global translation rate in stem cells is kept at a low level in favor of self-renewal, whereas a high translation rate is needed for differentiation. Multiple mechanisms are involved in translational control (102–104). Among several translation regulators involved, eukaryotic initiation factor-2α (eIF2α) is a key component of the translational switch. p-eIF2α represses the translation of general cellular proteins but allows effective translation of stem cell–related mRNA, such as the mRNA of Nanog and c-Myc in mESCs, using upstream open reading frames (uORFs) (105). The fact that PKR is a key enzyme that phosphorylates eIF2α warrants its role as a critical component of the translational switch.
A uORF is an ORF within the 5′-untranslated region of an mRNA (106). Translation of the uORF usually inhibits downstream expression of the primary ORF, but it may increase translation of the main ORF in some mRNA, such as for proteins involved in stress responses (107, 108). Using uORF to regulate gene expression is recognized as a key mechanism by which cells can rapidly change their gene expression patterns in response to internal or external stimuli. Increasing evidence suggests that this mechanism is adapted by stem cells to regulate self-renewal. The mRNAs of Nanog and c-Myc are the two best studied genes in ESCs that use a uORF to promote their translation via p-eIF2α (105). A high level of p-eIF2α is maintained by LIF and bone morphogenic protein 4, two known factors that maintain mESC pluripotency. In contrast, differentiation signals trigger dephosphorylation of p-eIF2α, promoting the switch of the gene expression profile from self-renewal to differentiation (105).
It is known that miRNAs participate in differentiation via different pathways, including suppression of pluripotency genes and acceleration of differentiation (109). The lack of miRNAs essential for the differentiation process is conceivably an important contributor to the failure of Dicer−/− mESCs to exit from the stem cell state (110), but noncanonical functions of Dicer may also be involved (111). Based on the translational switch hypothesis that ESC differentiation is a process that demands a high rate of translation (102, 103), one can envision that constitutively activated PKR in Dicer−/− mESCs could slow down translation, thereby contributing to the differentiation deficiency of Dicer−/− mESCs.
In summary, Dicer and PKR could act as critical regulators of ESC proliferation, self-renewal, and differentiation depending on the timing, duration, and magnitude of their activation. (Fig. 2 highlights our current understanding of the roles of Dicer and PKR as novel regulators of ESC fate and antiviral innate immunity.
Two studies dating back 40 years ago reported that pluripotent murine teratocarcinomas (embryonic carcinomas derived from germ cells) do not produce type I IFNs upon viral infection (112, 113). The same findings in ESCs and iPSCs confirm that this is a property common to all types of PSCs and have led to what we know about the immune properties of PSCs today. Along with our understanding of the underlying molecular and cellular mechanisms, we have now begun to appreciate the physiological significance of the attenuated innate immune response in ESCs. The hypothesis that it serves as a mechanism to prevent immunological cytotoxicity has gained increasing support. The incompatibility between the molecular machineries that control the IFN response and stemness provides another important insight from a different perspective. Both scenarios highlight the rationale for mammals to use alternative defense mechanisms at different developmental stages. The recognition of Dicer as a repressor of antiviral responses revealed the novel roles of PKR in the regulation of diverse facets of ESC activities, not only as an antiviral molecule compatible with pluripotency, but also as a critical component of the translational switch that controls the fate of ESCs. It is interesting to note that the mechanisms of PKR action we discuss in the present review are well recognized in differentiated somatic cells. However, their utilization by ESCs at different developmental stages demonstrates how an early embryo can adapt and modify the PKR pathway to serve as a multifunctional regulator of ESCs beyond antiviral response, which is an excellent example that should prompt us to rethink what we have learned from differentiated somatic cells when applied to stem cells.
This work was supported by National Institutes of Health/National Institute of General Medical Sciences Grants R15GM109299 and R15GM128196.
Abbreviations used in this article:
eukaryotic initiation factor-2α
embryonic stem cell
induced pluripotent stem cell
neuroepithelial precursor cell
pathogen-associated molecular pattern
protein kinase R
retinoic acid–inducible gene I
short interspersed nuclear element
small interfering RNA
upstream open reading frame
The authors have no financial conflicts of interest.