Attenuated innate immunity in embryonic stem cells: mechanisms and therapeutic applications

Abstract

Embryonic stem cells (ESCs) possess remarkable pluripotency and self-renewal capacity, making them a cornerstone of regenerative medicine. A defining yet underappreciated feature of ESCs is their attenuated innate immune response, characterized by suppressed expression of pattern recognition receptors, cytokines, and interferons. While this immune hyporesponsiveness helps preserve pluripotency and rapid proliferation during early embryogenesis, it also presents both challenges and opportunities for clinical application. This review summarizes recent findings on the molecular mechanisms underlying the innate immune deficiency of ESCs, including underdeveloped pathogen–sensing machinery and deficiencies in downstream signaling. We further explore the biological significance of this immune state in embryonic development and its implications for stem cell-based therapies. Specifically, we discuss how immune silence can mitigate inflammation during transplantation and be harnessed to improve mRNA-based differentiation and cancer vaccine strategies. Lastly, we highlight the challenges in balancing immune activation with stemness and outline future directions for optimizing ESCs-based therapeutics. A better understanding of ESC immunobiology will enable safer and more effective applications in regenerative medicine, oncology, and immunotherapy.

Introduction

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of pre-gastrulation embryos or from primordial germ cells in early gonads [1, 2]. These cells are defined by their capacity for unlimited self-renewal and the potential to differentiate into derivatives of all three embryonic germ layers under defined culture conditions using specific growth factors or cytokines [3]. The successful derivation of various cell types from ESCs has now demonstrated the principle and feasibility of their therapeutic application [4, 5]. Notably, both human ESCs (hESCs) and mouse ESCs (mESCs) exhibit a profoundly attenuated innate immune response compared to differentiated somatic counterparts [6, 7]. This innate immune hyporesponsiveness appears to be a conserved hallmark of ESCs, yet its biological implications remain poorly understood.

Regenerative medicine has harnessed the unique properties of ESCs to develop strategies for repairing tissues and organs with irreversible damage, offering great promise for clinical applications [8]. However, the inherently blunted innate immunity of ESC-derived cells represents a significant hurdle: following transplantation into injured sites, these cells may be more susceptible to microbial infection, potentially undermining their therapeutic efficacy [9]. Consequently, elucidating the mechanisms that govern innate immune competence in ESCs—and developing approaches to modulate their immunological profile—are critical for enhancing the safety and success of stem cell–based therapies.

In this review, we will provide a comprehensive overview of innate immune characteristics in ESCs, dissect the molecular underpinnings of their immune deficiencies, and trace the activation of innate immune pathways during in vitro differentiation. Finally, we will discuss recent advances and future prospects for leveraging the distinctive immunological features of ESCs in stem cell biology, immunology, and regenerative medicine.

The innate immune system of ESCs

Innate immunity serves as the organism’s indispensable first line of defense, comprising a complex network of cellular and molecular components that respond swiftly to pathogenic challenges [10]. The cellular arm is governed by specialized sentinel cells, primarily macrophages, natural killer (NK) cells, and dendritic cells (DCs), each possessing significant functional plasticity to detect threat, coordinate defense mechanisms, and influence subsequent adaptive immunity [11–13]. Macrophages, as versatile tissue-resident phagocytes, can polarized into distinct functional phenotypes: the pro-inflammatory M1 phenotype, typically activated by IFN-γ and TLR ligands, is highly effective at eliminating pathogens and secreting cytokines like TNF-α and IL-12; in contrast, the anti-inflammatory M2 phenotype, stimulated by IL-4 or IL-13, promotes tissue repair, immunoregulation, and inflammation resolution via the release of TGF-β and IL-10 [11]. NK cells provide critical defense against virally infected and malignant cells by directly inducing cytotoxicity through the release of perforin and granzymes, and producing IFN-γ. Their function is precisely regulated by a balance of activating and inhibitory receptors, the latter often engaging with Major Histocompatibility Complex (MHC) class I molecules on target cells [12]. DCs are the premier professional antigen-presenting cells. In their immature state, they surveil tissues for pathogens; following activation via PRRs, they undergo maturation,, upregulate MHC and co-stimulatory molecules, migrate to lymph nodes, and present processed antigens to naïve T cells, thereby initiating antigen-specific adaptive immunity [13]. At the molecular level, these cells, along with non-immune somatic cells, recognize conserved pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) [14]. Cell-surface Toll-like receptors (TLRs) and cytosolic sensors—such as RIG-I-like receptors (RLRs) for RNA and the cGAS-STING pathway for DNA—detect extracellular and intracellular PAMPs, respectively [15–17]. Engagement of these receptors triggers adaptor proteins (e.g., MyD88, TRIF for TLRs; MAVS for RLRs) and activates transcription factors such as interferon regulatory factors (IRFs), nuclear factor-κB (NF-κB), and activator protein-1 (AP-1). These factors drive expression of type I interferons (IFN-α/β) and pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-8), which are essential for direct antiviral and antibacterial defenses and for instructing and recruiting the cellular actors of the innate immune system [18, 19].

It is widely recognized that the diverse array of antiviral responses initiated by the IFN system can result in various detrimental outcomes in infected cells, such as impeding cell cycle progression or inducing cellular demise [20]. While the adverse impacts of these effects on infected cells in tissues may not cause significant harm to the developing organism, if infected, the consequences may be harmful to ESCs because they are the progenitors of all subsequent tissues of the developing organism. On the other hand, if ESCs lack effective antiviral mechanisms, viral infection will also be disastrous, as their progeny cells will also be infected [21].

Although developed in various differentiated somatic cells, innate immunity is not, or at least not fully, developed in ESCs. Recent studies of both mESCs and hESCs have revealed that they have attenuated innate immune responses to bacterial and viral pathogens and inflammatory cytokines [22, 23]. For example, the IFN system in ESCs is underdeveloped. In particular, both mESCs and hESCs do not express IFNα and IFNβ in response to viral infection or synthetic viral RNA analogs [23]. However, the IFN response mechanism in ESCs from the two species differ to a certain degree. mESCs retain a diminished, yet measurable, sensitivity to IFNα and IFNβ [24, 25]. Previous studies demonstrated that exogenous IFNα and IFNβ can induce the expression of several ISGs, protect mESCs from viral infection, and suppress replication of several types of viruses, but the magnitude of these responses remains significantly weaker than that observed in differentiated mouse fibroblasts [26, 27]. In contrast, hESCs barely respond to IFNβ [28]. However, IFNβ slightly inhibited infection of hESCs by Coxsackievirus, indicating that the IFN response pathway is not entirely inactive in these cells [29].

Furthermore, both mESCs and hESCs do not respond to lipopolysaccharide (LPS) [30, 31], a bacterial endotoxin that mimics bacterial infection in eliciting the inflammatory molecule expression [32]. Similarly, these cells fail to mount a reaction to polyinosinic: polycytidylic acid (polyIC) [30, 31], a synthetic dsRNA widely commonly employed as a viral dsRNA mimic [33]. Although different results have been reported regarding their responsiveness to LPS and polyIC [34–36], mESCs consistently lack the characteristic immune reactions seen in differentiated cells upon infection with live bacteria or viruses [37, 38].

These studies suggest that the innate immunity deficiency seems to be a common feature of hESCs and mESCs. The consistent absence of a fully developed viral defense mechanism in ESCs provides novel insights into the evolution of innate immunity during embryogenesis.

Activation of innate immune functions during in vitro differentiation

While recent studies indicate that the acquisition of innate immune functions during in vitro differentiation is generally a dynamic and stepwise process, this progression is not universal. Although undifferentiated ESCs exhibit severely blunted innate immunity, some ESC-derived cell (ESC-DCs) types continue to display sustained hyporesponsiveness even after differentiation, highlighting the heterogeneous development of immune competence across lineages [22, 27, 34, 39, 40].

As ESCs commit to specific lineages, they acquire increasingly mature immune responses, reflecting a gradual transition towards the immunocompetent state of somatic cells. For instance, during in vitro differentiation, ESC-DCs progressively upregulate innate immune genes. mESC-differentiated fibroblasts (mESC-FBs) show a modest but significant increase in IFN-β expression upon polyIC or viral stimulation compared to undifferentiated mESCs, although their response remains lower than that of fully differentiated fibroblasts [39, 41]. This increased responsiveness is accompanied by a transition in NF-κB activation—from an inactive state in ESCs to an active, nuclear-translocating state in differentiated cells—allowing the cells to respond to cytokines such as IL-1β and TNFα [41, 42]. In addition, NF-κB p65 protein levels and DNA-binding activity—which are comparably low in undifferentiated hESCs—rise upon retinoic acid–induced differentiation, and differentiated hESCs exhibit TNF-α-driven NF-κB nuclear translocation that fails to occur in undifferentiated hESCs [43]. It is noted that although mESC-EBs have gained the ability to respond to viral stimuli and inflammatory cytokines, their levels of responsiveness are substantially lower than their naturally differentiated counterparts but can be further developed along with their continued in vitro propagation [39, 41, 42]. For example, hESC-differentiated endothelial cells express typical cell-specific markers and display basic properties similar to human aortic endothelial cells, but they showed attenuated response to a wide range of pathogens and inflammatory cytokines [22].

Interestingly, the antiviral features of ESCs and ESC-DCs seem to maintain some evolutionary balance [44] (Fig. 1). Recent studies suggest that RNAi may operate in mESCs and tissue cells at an early developmental stage, which has led to the hypothesis that mammals may have adapted distinct antiviral mechanisms at different stages of organismal development: the IFN system is utilized by differentiated somatic cells, whereas RNAi may be used in ESCs as an alternative antiviral mechanism to the IFN system [45–48]. RNAi is a major antiviral mechanism in plants and invertebrates that lack IFN-based innate antiviral immunity, where a well-developed IFN system can mount multiple forms of antiviral responses [49, 50]. Using mouse models, it has been recently demonstrated that the RNAi mechanism is functional in mESCs, but its efficiency is significantly diminished in differentiated cells [48, 49]. ESCs compensate for attenuated protein-based immunity by leveraging RNAi to process viral dsRNA into siRNAs, while differentiated cells mainly rely on the IFN pathway, that is, the induction of ISGs to inhibit the virus. However, RNAi antiviral function in differentiated cells was impaired, as indicated by low viral siRNA abundance and suppression of Ago2 upon antiviral signal activation. In contrast, the IFN pathway in pluripotent cells is defective but retains a strong RNAi antiviral function, that is, RNAi can be directly used to directly cleave viral RNA to combat viral infection [52]. This RNAi-IFN dichotomy reflects an evolutionary trade-off. By deploying virus-specific and short-lived siRNA derived from invading viruses, the early embryo may prevent viral infection in ESCs and avoid potential negative effects of a full-scale IFN response. In contrast, somatic cells employ the powerful, multi-faceted antiviral activities conferred by the IFN system [51, 52]. Although compelling, this hypothesis remains actively debated. Furthermore, recent studies have revealed that a subset of ISGs (IFN-induced transmembrane proteins, IFITM) are constitutively expressed not only in hESCs but also in other pluripotent states, including mouse primordial germ cells and human naïve pluripotent stem cells [53–55]. Crucially, unlike typical ISGs in differentiated cells, IFITM proteins in ESCs are preexisting and may provide immediate viral protection, further underscoring the existence of alternative antiviral mechanisms in pluripotent cells.

Fig. 1.

Distinct antiviral mechanisms in differentiated versus undifferentiated cells. Differentiated somatic cells primarily rely on a IFN-based innate immune response to combat viral infections. Upon recognition of PAMPs such as LPS by TLRs, downstream signaling cascades are activated via TRAF6 and the IKK complex, leading to nuclear translocation of NF-κB and IRF5. These transcription factors induce the expression of pro-inflammatory cytokines and type I IFNs, which in turn activate ISGs to establish an antiviral state. In contrast, undifferentiated ESCs exhibit attenuated TLR signaling and IFN production. Instead, they utilize an RNAi-based mechanism as a primary antiviral strategy. Viral dsRNA is processed by Dicer into siRNAs, which are incorporated into the RNA-induced silencing complex (RISC) to mediate sequence-specific cleavage of viral RNA. This IFN-independent antiviral pathway enables ESCs to limit viral replication while avoiding the cytotoxic effects associated with IFN signaling

In summary, the acquisition of innate immune function during ESC differentiation is a heterogeneous and context-dependent process. While some lineages gradually mature towards immunocompetence, others retain features of immune hyporesponsiveness. This developmental transition is accompanied by a shift in antiviral strategies, from an RNAi-dominant mechanism in pluripotent cells to an IFN-dependent response in somatic cells.

Molecular mechanisms of innate immune deficiency in ESCs

The molecular basis for the underdeveloped innate immunity in ESCs is not completely understood, but the current data suggest that the deficiencies are at multiple levels. ESCs fail to mount the robust innate immune responses characteristic of differentiated somatic cells because of at least two interrelated factors: underdeveloped pathogen–sensing machinery and deficiencies in downstream signaling (Fig. 2).

Fig. 2.

Underdeveloped innate immunity in ESCs. ESCs display widespread downregulation of innate immune sensors and deficiencies in downstream signaling. Key pathogen-sensing receptors (e.g. MDA5, TLR3, TLR4) are expressed at very low levels or not functional in ESCs. Downstream signaling is also suppressed, with NF-κB remaining inactive and no IFNβ induction. Pluripotency factors (e.g. Oct4/Nanog) and miRNA (e.g. miR-290) inhibited NF-κB transcriptional activity

Underdeveloped pathogen–sensing machinery

Studies have shown that the major receptors for viral RNA (TLR3, RIG-I, and MDA5) are either expressed at low levels or not functional in ESCs [23, 31, 41, 56, 57]. The protein expression of LPS receptor (TLR4) and co-receptor (CD14) was absent in mESCs [23, 41], but LPS at a very high concentration (10 µg/ml) may elicit response in mESCs through TLR2 [58]. The expression of TNFR1 was up-regulated in mESC-FBs induced by Retinoic Acid [59]. TNFα could activate NFκB and induce the expression of ICAM1/IL6, but the intensity was lower than that of natural fibroblasts [22, 60]. However, TLR4 protein was still not expressed in mESC-FBs, and LPS was unable to activate NFκB or induce inflammatory genes [41]. Similar to mESCs, Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals [22, 60]. Neither TNFα nor LPS were able to activate NFκB or induce inflammatory genes in hESCs [22, 60]. These results suggest that the loss of protein expression of major receptor is critical for the failure of mESCs cells to respond to inflammation.

It has been reported that hESCs do not induce interferon production in response to cytoplasmic dsRNA [31]. Two major cytoplasmic dsRNA sensors, TLR3 and MDA5, are not expressed in hESCs [31, 61]. PKR is expressed in hESCs, but is not activated by transfected dsRNA [31]. In addition, RIG-I is expressed, but fails to respond to dsRNA because its signaling adapter, MITA/STING, is not expressed [31]. Finally, the interferon-inducible RNAse and oligoadenylate synthetase enzymes are also expressed at very low levels [31]. Upon differentiation of hESCs into trophoblasts, cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF [31].

Moreover, recent studies suggest that epigenetic modifications contribute significantly to the gradual activation of innate immunity during differentiation. The methylation status of PRR gene promoters, such as that of TLR4, appears to decrease as cells differentiate, resulting in increased expression of these receptors [34, 39]. In the reporter assay, the in vitro methylation assay suppressed TLR4 promoter activity. ChIP analysis revealed that in this region, histones H3 and H4 are hypoacetylated in ESC [34]. Moreover, treatment with trichostatin A led to a pronounced increase in TNFRp55 mRNA and promoter activity [62].

Taken together, These results reveal that the lack of an interferon response may be a general characteristic of ESCs and that this results from the systematic downregulation of a number of genes involved in pathogen–sensing.

Deficiencies in downstream signaling

The attenuated innate immune response in ESCs is not solely due to deficient pathogen sensing at the cell surface or cytosol. A profound functional deficiency exists in the core downstream signaling pathways that execute inflammatory and antiviral responses in ESCs and ESC-DCs.

The NF-κB signaling pathways, which are central to innate immunity, remain largely inactive in ESCs and ESC-DCs. Although there is an indication that NFκB might not be completely inactive in hESCs or hiPSCs, the findings from most studies demonstrated that NFκB is kept in an inactive state that cannot be turned on by immunological and inflammatory stimuli. In both mESCs and hESCs, RelA (p65) and p50, the subunits of the NFκB transcription factor, are expressed at relatively low levels but are upregulated upon differentiation [41, 60]. In mESCs, prototypical inflammatory stimuli—LPS, TNF-α, IL-1β—and even live virus infection cannot trigger NF-κB nuclear translocation or induce expression of ICAM-1 and IL-6, indicating a non-functional NF-κB module in their naïve state [41, 60]. Protein levels of NF-κB p65 and RelB were clearly enhanced during retinoic acid-induced differentiation. Furthermore, increased DNA binding activity of NF-κB in response to TNF-α, an agonist of NF-κB signaling, was seen in differentiated but not undifferentiated mESCs [41, 60]. Similarly, in equine ESCs, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls [63]. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the equine ESC-tenocytes [63].

Nanog, one of the key pluripotency markers, can directly bind to NF-κB proteins and inhibit NFκB transcriptional activity in mESCs [64]. Moreover, overexpression of NF-κB proteins promoted differentiation, whereas inhibition of NF-κB signalling, either by genetic ablation of the Ikκb gene or overexpression of the IκB super-repressor, increased expression of pluripotency markers [64]. Beyond Nanog, Oct4, another key pluripotency markers, also actively contributes to suppressing innate immune responses in ESCs, a critical aspect of their immunoprivileged state [65]. Oct4 functions as a master transcriptional regulator that directly binds to regulatory regions of key innate immune genes, including ISGs and components of the MHC class I antigen presentation pathway, actively repressing their expression [65, 66]. This repression is mechanistically enforced through Oct4’s recruitment of chromatin-modifying complexes that establish a repressive chromatin state [65]. Consequently, Oct4 orchestrates a multi-faceted suppression of pro-inflammatory pathways and immunostimulatory elements, actively maintaining the low immunogenicity essential for ESCs function and pluripotency. These results suggested that this quiescent state of key signaling hubs is actively maintained by pluripotency factors, which function as potent repressors of innate immune activation.

Furthermore, miRNA also plays a significant role in the immune regulation of ESCs [67]. For example, the expression of the IRF2 transcription factor is strongly upregulated in the absence of mi-290 and that the nuclear concentration of the RelA component of the NF-κB pathway upon stimulation with TNF-α is also increased [68]. Moreover, miR-294 targets the Irf2 transcription factor and modulates ‘IRF1,2,7’ motif and ‘NF-κB_Rel_RelA’ activities [68]. In addition, autophagy-related protein ATG5 is essential for both attenuated inflammatory response and differentiation of mouse ESCs and that attenuation of inflammatory signaling is required for mouse ESC differentiation [69]. Mechanistically, ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of β-TrCP1, resulting in the inhibition of nuclear factor κB (NF-κB) signaling and inflammatory response [69].

Beyond NF-κB, the phosphorylation and nuclear translocation of STAT1 is diminished upon IFN-β treatment, and the expression of key signaling molecules (IFNAR1, JNK1, STAT1) is still lower in ESCs than that of ESC-DCs [70, 71]. This attenuated response correlates with a high expression of suppressor of cytokine signaling 1 (SOCS1) [7]. In differentiated cells, SOCS1 is expressed at a low basal level in resting state, but it is rapidly upregulated and acts as a negative regulator of JAK/STAT signaling pathway, thereby limiting excessive actions of IFNs. However, SOCS1 is constitutively expressed at a high level in hESCs, and may repress IFNβ action in those cells [7]. Upon differentiation of hESCs into trophoblasts, cells acquire the ability to respond to IFN-β, and this is accompanied by a significant induction of STAT1 phosphorylation as well as a decrease in SOCS1 expression [7].

In summary, although the complete mechanism of ESC innate immune deficiency remains unclear, current studies have shown that insufficient expression of PRRs and defects in downstream signaling are two very important factors. Understanding these complex interactions is crucial for improving differentiation protocols and ensuring that ESC-derived cells acquire sufficient immune competence for therapeutic applications.

Biological significance of attenuated innate immunity in ESCs

The underdeveloped innate immune program in ESCs appears to be an adaptive mechanism to protect the pluripotent state from the cytotoxic and antiproliferative consequences of robust inflammatory signaling during early embryogenesis. In addition, prevention of harmful immune activation by endogenous retroelements may be also an important function.

Protection of proliferation and maintenance of pluripotency

While the question of why ESCs choose not to have the conventional IFN-based antiviral system and antibacterial mechanisms that are so well adapted by differentiated somatic cells could be speculated from different views, it could be better understood from the perspectives of reproductive immunology and embryogenesis. The attenuated innate immunity observed in ESCs represents a finely tuned evolutionary adaptation critical for balancing rapid embryonic development with protection against endogenous and exogenous threats. During early embryogenesis, ESCs prioritize proliferation and pluripotency maintenance over pathogen defense, as the sterile intrauterine environment minimizes exposure to external pathogens [72, 73]. Together with the protection by maternal immunity and the potential alternative antiviral/bacterial mechanisms yet to be further investigated in ESCs (such as RNAi and IFITM) [47, 53], pathogenic infection may not pose a major threat to ESCs in a pre-implantation blastocyst. However, immunological and inflammatory responses, the dynamic events that take place during and shortly after implantation, could have significant impacts on the embryo [74]. Immune and inflammatory responses have been viewed as a double-edged sword. On one hand, they defend the organism against pathogens; on the other hand, they can cause collateral damage to tissue cells through cell cycle inhibition and even cell death [74]. In most somatic cells, activation of innate immune sensors and downstream cytokines—particularly TNF-α and IFN-γ—triggers NF-κB and JAK-STAT pathways that lead to cell cycle arrest, apoptosis, or differentiation [73, 74]. These negative effects could be tolerated in a tissue of a developed organism, but the consequences could be detrimental to ESCs in an early embryo where rapid cell proliferation is their dedicated task essential for embryogenesis. Indeed, TNF-α and IFN-γ have long been recognized as “embryotoxic cytokines”, whose elevated levels impair blastocyst formation and pregnancy maintenance in both animal models and clinical observations [75, 76]. From this perspective, it would be beneficial for ESCs not to produce and respond to inflammatory cytokines when the blastocyst is exposed to high concentrations of inflammatory cytokines and increased populations of immune cells resulting from the implantation process or from maternal systemic or intrauterine infection.

Consistent with this evolutionary pressure, both mouse and human ESCs are remarkably insensitive to the cytotoxic effects of TNF-α, IFN-α/β, or IFN-γ—alone or in combination—whereas differentiated fibroblasts undergo marked cell-cycle inhibition and reduced viability under identical treatments [75, 76]. Conversely, forced engagement of the type I IFN system in pluripotent cells has been shown to misdirect their lineage commitment: ectopic IFN-β expression disrupts ectoderm and endoderm formation and compromises mesodermal sublineages, demonstrating a fundamental incompatibility between robust IFN signaling and the maintenance of pluripotency [69, 77].

The attenuated inflammatory cytokine production and response could serve as an adaptive mechanism that not only allows ESCs to avoid the cytotoxicity of inflammatory cytokines, but also to prevent further aggravation of immune and inflammation reactions associated with the implantation process. ESCs avoid cytokine-induced proliferation block and apoptosis, preserving their “stemness” during a critical window of development; and by leveraging RNAi, they retain a sequence-specific antiviral safeguard compatible with rapid self-renewal. Therefore, the diminished innate immune responses could help ESCs maintain the rapid rate of cell proliferation essential for early embryo development.

Prevention of harmful immune activation by endogenous retroelements

A compelling biological rationale for the attenuated innate immunity in ESCs lies in the need to manage endogenous retroelements (ERVs). The mammalian genome is replete with ERVs, which are remnants of ancient viral infections [78]. Their activities contribute to the shaping of host gene expression and the organization of host genome structure [79]. The majority of ERVs are silenced and maintain neutrality toward the host genome. During early embryonic development, particularly during epigenetic reprogramming, these ERVs can become transiently active [80, 81]. For example, Oct4 and Sox2 are frequently co-localized with ERV group members in mESCs, the recognition of ERVs by transcription factors suggests that ERVs can serve as promoters or enhancers [82]. An ORR1D2 locus that is bound by Oct4 and Sox2 can serve as a promoter to drive the expression of a retrotransposon-derived lncRNA, Lx8-SINE B2, which is critical for ESC metabolism [83, 84]. These results underscore the role of ERVs as enhancers and promoters to regulate gene expression during early development and in ESCs.

However, the transcripts of ERVs can form dsRNA structures or reverse-transcribed DNA, which are potent ligands for cytosolic RLRs and the cGAS-STING pathway, respectively [85]. If ESCs possessed robust innate immune sensing, this burst of endogenous nucleic acids would trigger a catastrophic interferon and inflammatory response, leading to cell death, developmental arrest, or genomic instability [80]. Therefore, the suppression of innate immune pathways is an essential adaptation to tolerate this period of inherent genomic “noise”. Pluripotency factors like Oct4 play a direct role in this tolerance by binding to ERV regulatory sequences and recruiting silencing machinery like Kap1/TRIM28, thereby keeping their expression in check and preventing the generation of immunostimulatory ligands [80]. In this light, the attenuated innate immunity is not merely a passive deficiency but an active, coordinated strategy to shield the precious, pluripotent progenitor of the entire organism from the potential self-inflicted harm of its own genome. This ensures the faithful and uninterrupted progression of early embryogenesis.

In summary, the attenuated innate immunity in ESCs serves as a sophisticated adaptive strategy with dual purposes: it safeguards rapid proliferation and pluripotency by avoiding the detrimental effects of inflammatory signaling during critical developmental windows, while simultaneously preventing aberrant immune activation by tolerating transient expression of endogenous retroelements. This coordinated suppression of immune pathways ensures genomic stability and uninterrupted embryogenesis, highlighting its essential role in early development. Harnessing these mechanisms—while circumventing their limitations—will be critical to advancing safe and effective stem cell therapies for inflammatory and degenerative diseases.

Application of attenuated innate immunity in ESCs

ESCs display an intrinsically dampened IFN response, a feature that—while a liability for antiviral defense—can be harnessed to improve safety and efficacy in regenerative medicine (Fig. 3).

Fig. 3.

Application of Attenuated Innate Immunity in ESCs. The figure summarizes three application of attenuated innate immunity in ESCs: (1) mitigation of inflammatory damage in stem cell therapy, (2) developing mRNA-directed differentiation strategies based on the attenuated antiviral response in ESCs, and (3) ESCs-based stem cell vaccines

Mitigation of inflammatory damage in stem cell therapy

ESCs are characterized by their potential to differentiate into a variety of cell lineages (pluripotency) and unlimited capacity for proliferation (self-renewal). These properties make them a promising cell source for regenerative medicine [86]. The intrinsically blunted innate immune response of ESCs, while a vulnerability in pathogen defense, transforms into a significant therapeutic advantage during cell transplantation. When introduced into injured or diseased tissues, ESC-derived grafts are inevitably exposed to a hostile microenvironment rich in DAMPs released from necrotic host cells. In conventional somatic cells, these DAMPs are potent agonists for PRRs, triggering robust NF-κB and IRF signaling cascades that drive the production of pro-inflammatory cytokines and chemokines [14]. This response initiates and amplifies local inflammation, recruiting innate immune cells like macrophages, which can ultimately lead to the destruction of the transplanted graft [87].

ESCs, however, are uniquely equipped to evade this detrimental cycle due to their underdeveloped innate immune machinery. First, a key mechanism is their failure to express functional PRRs. As detailed above, due to their failure to express functional PRRs, ESCs are unable to recognize DAMPs, thereby preventing the initiation of inflammatory cascades [31, 57]. This inability to sense “danger signals” prevents the initiation of the inflammatory cascade at its very origin. Second, even if upstream signals were present, the critical downstream signaling hub, NF-κB, remains inactive in naive ESCs [41, 60]. Without a functional NF-κB pathway, ESCs cannot transcribe and secrete the array of cytokines and chemokines that would otherwise alert and recruit the host’s innate immune system to the transplant site.

This collective innate immune hyporesponsiveness creates a state of “inflammatory silence” around the ESC graft. By not contributing to the pro-inflammatory milieu, ESCs avoid self-inflicted damage and do not amplify the destructive inflammatory loop. This is particularly advantageous in treating chronic inflammatory diseases, where sustained innate immune activation is a primary driver of tissue destruction [88, 89]. The ESC graft essentially behaves as a passive, non-inflammatory entity, thereby enhancing its own chances of survival and integration.

However, on the other hand, the immune and inflammatory responses play key roles in an organism’s defense against infectious agents [10, 14]. Conceivably, the fate and functionality of transplanted cells could be compromised if they do not have such vital mechanisms. Many important questions remain to be answered, such as whether the host innate and adaptive immune function may confer immune protection to the grafted cells that lack innate immunity, and to what extent do the signals from the host environment promote their maturation. However, the in vivo studies that could help to answer these questions are currently lacking. To fully understand the importance of the innate immune response in ESC-based cell therapy, it will be essential to have a complete characterization of ESC-DCs by in vitro and in vivo studies.

Developing mRNA-directed differentiation strategies based on the attenuated antiviral response in ESCs

In cell reprogramming, mRNA-mediated gene delivery has gained prominence as a replacement for lentiviral vectors, owing to the non-integrating and transient nature of mRNA [90]. This technique involves the direct introduction of synthetic mRNA into host cells to express reprogramming factors, thereby circumventing the need for viral or DNA-based vectors. A major biological challenge, however, is that a synthetic mRNA is detected as a viral RNA by host cells and induces strong antiviral responses, resulting in IFN induction and reduced viability of host cells [91, 92]. Recent studies have demonstrated that the attenuated innate immunity of ESCs makes them exceptionally tolerant to repeated transfection with synthetic mRNA [93]. The research reported that synthetic mRNA encoding ETV2, a transcription factor that promotes vascular differentiation, can be effectively expressed in mESCs with expected transcription activity, therefore, demonstrating the feasibility of using synthetic mRNA as an alternative to viral vectors in directed ESC differentiation [93].

ESCs-Based stem cell vaccines

Another innovative application leverages the unique immune properties of ESCs in developing whole-cell vaccines. ESCs naturally express a spectrum of oncofetal antigens (e.g., OCT4, SSEA‑3/4, CEA, AFP) shared with many tumors. Stem cell vaccines are effective in promoting the pre-existing anti-cancer immune responses. Whole‑cell vaccination with irradiated ESCs can therefore prime host immunity against a constellation of tumor‑associated antigens (TAAs) without genetic modification [94–96]. Therefore, Marek’s disease [97], hepatic tumors [98] and lung cancer [99] are the essential candidate diseases for applying ESC-derived vaccines. More recently, an ESCs-derived lung cancer vaccine has demonstrated its effectiveness in preventing lung tumor development, and the tumor-preventing efficacy of ESC-based vaccine is reliant on the differentiation properties of these stem cells [100]. ESC-based vaccines also effectively suppress ovarian tumor development by generating complementary and cytotoxic T-cell responses that selectively target cancer stem cells in mice [101]. In addition, ESCs-Based Stem Cell Vaccines have also demonstrated significant anti-cancer potential in bladder cancer [102].

Challenges and future directions

In summary, while the attenuated innate immune response in ESCs provides a unique advantage by protecting these cells from immunological cytotoxicity during early development, it also poses potential risks in clinical applications. It raises several fundamental questions, such as whether or not transplanted ESC-DCs could be at risk if they lack a competent innate immunity against infections, whether and to what degree the host immunity can protect the grafted cells, and whether the attenuated immune response in ESC-DCs is beneficial or harmful to the interaction between the host tissue and grafted cells. Future research may be possible to design strategies that generate “customized” ESC-DCs with different levels of innate immunity depending on whether or not the innate immune response is desired in tissue regeneration. For example, the innate immunity development could be accelerated by the inclusion of “immunostimulants” (such as the cytokines used for the differentiation of innate immune cells) or other epigenetic modifiers during differentiation of structural tissue cells. To this end, it is imperative to understand the molecular mechanisms that control immunity development and they must be sought at the level of ESCs. Strategies such as mRNA-based directed differentiation, immune priming, and the development of ESC-based vaccines offer promising avenues. Advances in stem cell banking and culture optimization will further enhance the therapeutic potential of ESC-derived cells, ultimately leading to safer and more effective regenerative medicine therapies.

Abbreviations

AP-1

Activator protein-1

DCs

Dendritic cells

dsRNA

Double-stranded RNA

EBs

Embryoid bodies

ESCs

Embryonic stem cells

ESC-DCs

Embryonic stem cell-derived cells

hESCs

Human embryonic stem cells

IFN

Interferon

IFITM

Interferon-induced transmembrane proteins

IRFs

Interferon regulatory factors

ISGs

Interferon-stimulated genes

LPS

Lipopolysaccharide

mESCs

Mouse embryonic stem cells

MHC

Major histocompatibility complex

NF-κB

Nuclear factor kappa B

NK cells

Natural killer cells

PAMPs

Pathogen-associated molecular patterns

poly(I:C)

Polyinosinic: polycytidylic acid

PRRs

Pattern recognition receptors

RLRs

RIG-I-like receptors

RNAi

RNA interference

siRNAs

Small interfering RNAs

SOCS1

Suppressor of cytokine signaling 1

TAAs

Tumor-associated antigens

TLRs

Toll-like receptors

Author contributions

S.C. contributed to conceptualization, supervision, and writing—review and editing. R.W. contributed to project administration, and writing—review and editing. J.Y. contributed to conceptualization, writing—original draft preparation, and visualization. X.Z. contributed to writing—original draft preparation. H.L. contributed to writing—original draft preparation. X.Z. contributed to conceptualization and writing—original draft preparation. W.C. contributed to writing—original draft preparation. Q.X. contributed to conceptualization, funding acquisition and writing—original draft preparation. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Biological Breeding-National Science and Technology Major Project grant 2023ZD0405301, National Natural Science Foundation of China grant 32503009, the Special Project of National Modern Agricultural Industrial Technology System (CARS-41) and the China Agriculture Research System of MOF and MARA (CARS-42-13).

Data availability

The dataset supporting the conclusions of this article is included within the article.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The authors give consent for publication. The authors declare that they have not use AI-generated work in this manuscript.

Competing interests

The authors declare that they have no competing interests.