The Regulatory Role of m⁶A Modification in the Function and Signaling Pathways of Animal Stem Cells

Abstract

As a type of cell with self-renewal ability and multi-directional differentiation potential, stem cells are closely related to their functions, such as reprogramming transcription factors, histone modifications, and energy metabolism. m⁶A (N⁶-methyladenosine modification) is one of the most abundant modifications in RNA, and dynamic reversible m⁶A modification plays an important role in regulating stem cell function. This review moves beyond listing isolated functions and instead adopts an integrated perspective, viewing m⁶A as a temporal regulator of cellular state transitions. We discuss how m⁶A dynamically regulates stem cell pluripotency, coordinates epigenetic and metabolic reprogramming, and serves as a central hub integrating key signaling pathways (Wnt, PI3K-AKT, JAK-STAT, and Hippo). Finally, using somatic reprogramming as an example, we elucidate the stage-specific role of m⁶A in complex fate transitions. This comprehensive exposition not only clarifies the context-dependent logic of m⁶A regulation but also provides a precise framework for targeting the m⁶A axis in regenerative medicine and cancer therapy.

1. Introduction

Stem cells, as a type of cell with multi-directional differentiation potential and self-renewal ability, can differentiate into cells of a specific tissue type [1]. Stem cell function is closely related to reprogramming transcription factors [2], histone modifications [3], and energy metabolism [4]. With the continuous development of life science research, human understanding of stem cells is gradually deepening, and research achievements related to stem cells are playing an increasingly important role in disease treatment, regenerative medicine, and other fields [5].

In recent years, post-transcriptional RNA modifications have emerged as a form of epigenetic regulation extensively involved in gene expression control. Nucleotide modifications are present in various RNA transcripts, both coding and non-coding [6]. m⁶A modification, as an important RNA modification, participates in regulating mRNA processing and metabolism in various biological processes [7].

In 1974, m⁶A was first discovered to be the main form of mRNA methylation in mammals. As the most common and abundant post-transcriptional RNA modification in eukaryotic cells, it accounts for over 50% of all methylated ribonucleotides in total RNA in cells, with an average of 3–5 m⁶A sites per mRNA [8,9]. The m⁶A modification is distributed in the RRACH consensus sequence (where R = A/G and H = A/C/U), mainly enriched in the termination codon and 3′ untranslated regions (3′ UTRs) [10]. m⁶A modification is dynamically regulated by the interactions between various m⁶A-specific protein families, known as methyltransferases (writers), demethylases (erasers), and readers [11]. m⁶A modification affects almost every step of RNA metabolism, including processes such as alternative splicing (AS), stability regulation, degradation, translation, and nuclear export [12]. This in turn drives many biological processes, including circadian rhythms, T cell differentiation, stem cell renewal and differentiation, the epithelial–mesenchymal transition, etc. [13,14]. In stem cells, m⁶A controls the functional changes in stem cells by regulating processes such as RNA splicing, maturation, stability, translation, and localization [15]. Research has found that m⁶A plays an important role in regulating stem cell function, such as regulating reprogramming transcription factors [16], histone modifications [17], energy metabolism [18], and reprogramming efficiency of induced iPSCs (pluripotent stem cells) [19].

m⁶A modification plays a central role in the development of individual organisms on a cellular basis. This review introduces m⁶A modification and focuses on the regulatory role of m⁶A in stem cell pluripotency maintenance, histone modification, energy metabolism, and iPS cell induction. At the same time, the regulatory role of m⁶A in key signaling pathways of stem cells was elucidated. This review helps to better understand RNA modification and its mechanisms in the field of life sciences and provides prospects for future research on cell proliferation and human regenerative medicine.

2. m⁶A Modification

m⁶A modification, as a highly reversible process, regulates nearly every step of RNA metabolism [12]. It is dynamically installed, removed, and interpreted by writers, erasers, and readers, respectively. Extensive biochemical and structural studies have established that the METTL3-METTL14 heterodimer constitutes the catalytic core of the m⁶A methyltransferase complex (MTC), with METTL3 providing the catalytic site and METTL14 stabilizing RNA substrate binding [20,21]. This core complex invariably associates with adaptor proteins such as WTAP, which is essential for directing m⁶A deposition to proper genomic loci. The MTC modulates cellular functions in HeLa cells, leukemia cells, pancreatic cancer cells, and others, playing vital roles in related disease pathogenesis and treatment [22,23]. Demethylases, including ALKBH5 and FTO, remove m6A marks [24]. Readers, primarily the YTH domain family proteins (YTHDF and YTHDC classes), along with HNRNP and IGF2BP proteins, recognize and execute the functional outcomes of m6A [11,25].

m6A regulates mRNA alternative splicing (AS), stability, degradation, translation, and nuclear export [26] (Figure 1A). The methyltransferase localizes to nuclear speckles [27]. WTAP promotes METTL3/14 accumulation in speckles to influence AS, while FTO regulates AS by preventing SRSF2 recruitment [28,29]. YTHDC1 modulates AS by binding splicing factors SRSF3/10 within nuclear speckles. Upon export to the cytoplasm, m6A-modified transcripts are recognized by cytoplasmic readers, enhancing RNA export efficiency—a process relevant to stem cell disease therapeutics [30,31]. IGF2BPs stabilize mRNA by recruiting HuR, MATR3, and PABPC [32]. YTHDF2 recruits the CCR4-NOT deadenylase complex to promote mRNA decay, whereas YTHDF1 facilitates translation by recruiting eIF3 [33,34].

m6A also modulates non-coding RNAs. METTL3 and HNRNPA2B1 interact with DGCR8 and DROSHA to regulate pri-miRNA processing into mature miRNA [35,36]. circRNA, generated through backsplicing of pre-mRNA, lacks a 5′ cap and often relies on m6A modification within its 5′UTR for cap-independent translation, a process regulated by YTHDF3 [37,38] (Figure 1B). Furthermore, YTHDF proteins influence embryonic development, stem cell fate, adipogenesis, and tumorigenesis, highlighting their potential as predictive biomarkers and therapeutic targets [39].

The functions of m⁶A “read–write–erase” proteins may extend beyond their classical roles. A key concept is distinguishing between actions dependent on their enzymatic or binding activities versus those dependent on their scaffold/structural protein functions.

m⁶A-dependent mechanisms: Function directly relies on the protein’s catalytic activity (e.g., METTL3’s methyltransferase activity) or binding activity (e.g., YTHDF2 recognizing m⁶A marks) [40]. Examples include METTL3-mediated m⁶A deposition [41] and YTHDF2-mediated mRNA degradation following m⁶A recognition [42]. m⁶A-independent mechanisms: Function arises from their capacity to serve as protein interaction platforms or components of chromatin-regulating complexes, independent of enzymatic/binding activity. A classic example is METTL14: it regulates H3K27me3 levels by interacting with the PRC2 complex and recruiting KDM5B, a function independent of its methyltransferase activity [43,44]. This discovery critically expands the functional paradigm of the core “writer” proteins, revealing that they can serve as scaffold molecules to directly bridge RNA methylation with chromatin remodeling. It underscores a key caution in the field: the phenotypic consequences of depleting an m⁶A factor may not always be attributable to the loss of m⁶A marks per se but could arise from disrupting its non-catalytic, structural roles. Similarly, YTHDC2 recruits the histone methyltransferase MLL1 in cancer stem cells [45], a role that may also be partially independent of its reading function.

Figure 1. (A) Dynamic m⁶A modification of mRNA. The schematic depicts the reversible m⁶A modification cycle. Writers (e.g., the METTL3/14 complex) deposit m⁶A marks using S-adenosylmethionine (SAM) as the methyl donor [21]. Erasers (FTO and ALKBH5) remove these marks. Reader proteins (e.g., YTHDF family, YTHDC family, IGF2BPs, and HNRNPs) recognize m⁶A and determine the functional outcome for the modified mRNA, influencing its splicing, export, stability, or translation [11,24,25]. (B) m⁶A modification of non-coding RNAs. Beyond mRNA, m⁶A also regulates the biogenesis and function of non-coding RNAs. METTL3 facilitates the processing of primary microRNAs (pri-miRNAs) into mature miRNAs. Additionally, m⁶A modification on circular RNAs (circRNAs) can promote their cap-independent translation through reader protein (e.g., YTHDF3)-mediated recruitment of translation initiation factors [37,38].

Figure 1. (A) Dynamic m⁶A modification of mRNA. The schematic depicts the reversible m⁶A modification cycle. Writers (e.g., the METTL3/14 complex) deposit m⁶A marks using S-adenosylmethionine (SAM) as the methyl donor [21]. Erasers (FTO and ALKBH5) remove these marks. Reader proteins (e.g., YTHDF family, YTHDC family, IGF2BPs, and HNRNPs) recognize m⁶A and determine the functional outcome for the modified mRNA, influencing its splicing, export, stability, or translation [11,24,25]. (B) m⁶A modification of non-coding RNAs. Beyond mRNA, m⁶A also regulates the biogenesis and function of non-coding RNAs. METTL3 facilitates the processing of primary microRNAs (pri-miRNAs) into mature miRNAs. Additionally, m⁶A modification on circular RNAs (circRNAs) can promote their cap-independent translation through reader protein (e.g., YTHDF3)-mediated recruitment of translation initiation factors [37,38].

3. m⁶A Modification and Stem Cell Pluripotency

The maintenance and exit from pluripotency are fundamental to stem cell biology. This section addresses a pivotal question: How does m⁶A, often perceived as a stabilizer of pluripotency, also facilitate the timely transition to differentiation? We will first delineate the canonical paradigm where m⁶A safeguards pluripotency by stabilizing transcripts of core factors like NANOG. We will then contrast this with emerging evidence that m⁶A-mediated decay of a subset of pluripotency transcripts is essential for differentiation priming. This duality highlights m⁶A’s role as a dynamic modulator rather than a static lock, with non-coding RNAs (e.g., linc1281) serving as key intermediaries in this process.

The maintenance of pluripotency is crucial for stem cell function, governing their proliferation and differentiation [46]. This state requires high levels of transcriptional and ribosomal activity [47], processes that are centrally regulated by m⁶A RNA modification [15]. m⁶A safeguards pluripotency primarily by stabilizing the mRNAs of key pluripotent factors [48,49]. It should be noted that the role of m⁶A in pluripotency is context-dependent and not a single-mode phenomenon. Contrary to the “stability model,” Geula et al. found that m⁶A methylation is essential for the timely release of initial pluripotency and promotes differentiation by facilitating the turnover of certain initial pluripotency transcripts [49]. This apparent paradox suggests that m⁶A does not simply “lock in” a pluripotent state but rather functions as a dynamic modulator of RNA fate, where its outcome (stabilization vs. decay) is determined by specific cellular contexts, transcript identities, and the complement of bound reader proteins. ERK phosphorylates METTL3 at the S43/S50/S525 site, phosphorylates WTAP at the S306/S341 site, deubiquitinates through USP5, and finally produces stable m⁶A methyltransferase complexes. The lack of METTL3/WTAP phosphorylation reduces the decay of m⁶A-labeled pluripotent factor transcripts and maintains the pluripotency of mESC (mouse embryonic stem cells) [50]. This regulatory axis of METTL3 phosphorylation is also functional in other stem cell contexts, such as dental pulp and pancreatic cancer stem cells [51].

METTL3 is a key protein regulating stem cell pluripotency [52]. A pivotal yet context-sensitive finding is that depletion of METTL3 in mouse embryonic stem cells (mESCs) can, counterintuitively, lead to increased Nanog expression and impede differentiation [53]. This contrasts with its general role in stabilizing pluripotency transcripts, underscoring that METTL3’s function is not monolithic. It suggests that in naive pluripotency, METTL3 may preferentially target a subset of transcripts for degradation to prime cells for differentiation, a function that appears to be highly dependent on the specific cellular state. The m⁶A machinery intricately controls core pluripotency factors: the methyltransferase complex (MTC) interacts with SMAD2/3 [54], while the demethylase ALKBH5 partners with ZFP217 to fine-tune Nanog expression [55]. Furthermore, readers like IGF2BPs stabilize the transcripts of OCT4 and SOX2 by binding to their CDS and 3’UTRs, directly reinforcing the pluripotent state [32,56].

Beyond canonical mRNA targets, m⁶A modification regulates stem cell pluripotency through select long non-coding RNAs (lncRNAs) that function primarily in the cytoplasmic regulatory layer [57]. A key example is linc1281, whose m⁶A modification enhances its function as a competing endogenous RNA (ceRNA). By sponging pluripotency-inhibitory let-7 family miRNAs, m⁶A-modified linc1281 preserves the expression of pluripotency factors, thereby maintaining mESC identity [58]. Other lncRNAs like H19 are also m⁶A targets and may operate in similar ceRNA networks [59]. This regulatory axis intersects with the well-known let-7/Lin28 negative feedback loop [60], illustrating how m⁶A can modulate existing post-transcriptional circuits to reinforce the pluripotent state. It is important to distinguish this cytoplasmic, miRNA-mediated regulation from the nuclear, chromatin-directed mechanisms of m⁶A discussed in the following section.

Emerging Consensus: A foundational principle is that m⁶A functions as a bidirectional rheostat. It maintains the pluripotent ground state by stabilizing core factor mRNAs (e.g., via IGF2BPs [32,56]) but also primes cells for differentiation by promoting the turnover of specific naïve pluripotency transcripts [49]. This duality is essential for state transitions.

Model-Dependent Insights and Gaps: The current mechanistic map is predominantly drawn from mouse embryonic stem cells (mESCs). While the conservation of core writers (METTL3) and readers is evident, their precise targets and functional outcomes in human ESCs, adult stem cells, or organoid models remain less defined, representing a critical area for comparative studies.

Key Unresolved Controversy: The field is actively debating the functional logic of cytoplasmic readers, particularly the YTHDF family. Evidence points to both context-specific specialization (e.g., distinct roles for YTHDF1 and YTHDF2 in porcine iPSCs [16]) and functional redundancy (e.g., cooperative requirement of YTHDF2/3 in murine reprogramming [61]). Resolving this “redundancy vs. specificity” paradox is essential for predicting the consequences of m⁶A deposition and for designing targeted interventions.

4. m⁶A Modification and Histones

Moving beyond direct mRNA regulation, m⁶A exerts a profound influence on the cellular state by interfacing directly with the epigenome. This section focuses on m⁶A’s role as a critical bridge between RNA metabolism and chromatin dynamics. We will detail how m⁶A modification of chromatin-associated RNAs (carRNAs) and its interaction with histone-modifying complexes (e.g., PRC2 and SETDB1) actively sculpt the chromatin landscape. This mechanism is not separate from pluripotency control but is a key effector pathway by which m⁶A stabilizes or alters transcriptional programs, including those governing pluripotency and differentiation.

The opening or closing of specific chromatin regions during stem cell proliferation and differentiation is influenced by histone methylation and acetylation [62]. m⁶A modification participates in various histone modifications and regulates chromatin changes [38,63] (Figure 2).

m⁶A modification orchestrates chromatin dynamics in stem cells through multiple, interconnected mechanisms. A primary pathway involves the regulation of chromatin-associated regulatory RNAs (carRNAs). In mESCs, METTL3-mediated m⁶A modification of carRNAs (including promoter-/enhancer-associated and repeat RNAs) facilitates an open chromatin state by recruiting chromatin modifiers [64]. Specifically, METTL3 interacts with SETDB1 and TRIM28 to localize to retrotransposons like IAPs. This targeting, mediated by the methyltransferase complex (MTC) at IAP 5′UTRs, regulates heterochromatin marker deposition and is essential for chromatin integrity and homeostasis [13,66,67].

Beyond carRNAs, m⁶A directly influences histone modification landscapes. METTL14 interacts with the PRC2 complex and recruits the demethylase KDM5B to modulate H3K27me3 levels [17,43,44]. Conversely, the demethylase FTO erases m⁶A marks on LINE1 retrotransposon RNA, regulating its abundance and local chromatin condensation [68]. The m⁶A reader YTHDC1 recognizes m⁶A on LINE1 and ERVK transcripts, recruiting SETDB1 to enforce transcriptional silencing and maintain mESC homeostasis [69,70,71].

This chromatin-regulatory capacity of m⁶A extends to pathological stem cell contexts. In cancer stem cells, YTHDC2 recruits the histone methyltransferase MLL1 to enhance H3K4me3 and oncogene transcription [45]. In acute myeloid leukemia stem cells, RBFOX2 recognizes m⁶A on carRNAs and recruits RBM15 to promote repeat RNA methylation, an event that suppresses leukemogenesis and promotes differentiation [72].

The recruitment of m⁶A regulators to specific chromatin loci is not passive but is actively controlled. Extracellular signals can modulate their activity and localization via post-translational modifications. For instance, ERK-mediated phosphorylation of METTL3 and WTAP, stabilized by USP5-mediated deubiquitination, enhances the integrity and nuclear retention of the methyltransferase complex, potentially directing it to pluripotency gene loci in response to growth factors [50]. Furthermore, sequence-specific transcription factors serve as critical recruiters. The TGF-β pathway effectors SMAD2/3 physically interact with the MTC, thereby coupling extracellular morphogen signals to m⁶A deposition on key pluripotency transcripts like Nanog [54]. Conversely, the transcription factor ZFP217 can partner with the demethylase ALKBH5, forming a complex that is recruited to specific promoters to fine-tune local RNA methylation and histone modification landscapes [55]. These examples illustrate a paradigm where upstream signaling cascades and transcription factors act as the “directors”, guiding the “writers” and “erasers” of m⁶A to precise genomic addresses to execute state-specific epigenetic programs

Core Established Mechanism: It is now well-established that m⁶A on chromatin-associated RNAs (carRNAs) serves as a direct signal for recruiting histone modifiers (e.g., SETDB1 and PRC2) to specific loci, thereby influencing local chromatin states and transcription [64,66]. This provides a concrete molecular bridge between the epitranscriptome and epigenome.

Contextual Variability and Challenges: While the principle is clear, its functional impact is highly context-dependent. The same reader, YTHDC1, can mediate silencing of retrotransposons in mESCs [69] but may have opposite effects in cancer contexts. Furthermore, distinguishing the effects of m⁶A on carRNAs from its concurrent regulation of protein-coding mRNAs at the same locus presents a significant technical and interpretative challenge.

Major Unanswered Question: A central unresolved issue is the hierarchical relationship and potential feedback loops between m⁶A on chromatin and canonical histone marks. Does m⁶A deposition direct histone modification, or do existing chromatin states recruit the m⁶A machinery? Disentangling this causality is crucial for understanding the sequence of events during epigenetic reprogramming.

5. m⁶A Modification and Energy Metabolism

Cell fate is closely linked to metabolic status. This section explores how m⁶A, as a core regulator of stem cell metabolism, serves as a critical interface between gene expression and metabolic flux. We will analyze how m⁶A targets key enzymes in glycolysis and oxidative phosphorylation, thereby controlling the metabolic switch between self-renewal and differentiation. Crucially, we highlight that this metabolic regulation is not an isolated function; it directly fuels the epigenetic machinery (e.g., by providing acetyl-CoA for histone acetylation or α-KG for demethylases), thereby creating a cohesive “m⁶A–metabolism–epigenetics” axis that dictates cell fate.

Energy metabolism directly participates in the maintenance of stem cell pluripotency, stem cell proliferation and differentiation, and affects stem cell function [73,74]. Oxidative phosphorylation and glycolysis are the two main ways for stem cells to obtain ATP [75,76]. m⁶A modification regulates the transcription and translation of key genes involved in oxidative phosphorylation and glycolysis, affecting stem cell energy metabolism [18,77] (Figure 3).

Figure 3 not only illustrates the regulation of metabolic enzymes by m⁶A but also implies its role in reshaping cellular metabolic states to influence stem cell fate. In stem cell biology, the balance between glycolysis and oxidative phosphorylation (OXPHOS) carries distinct functional implications. Active glycolysis (the Warburg effect) typically supports self-renewal and pluripotency maintenance. It not only provides ATP but, more importantly, supplies biosynthetic precursors for cell proliferation. Metabolites such as acetyl-CoA produced from glycolytic flux serve as substrates for histone acetylation, thereby maintaining an open chromatin state and expression of pluripotency genes [78]. Conversely, a shift towards OXPHOS is often associated with differentiation. OXPHOS meets the higher energy demands of differentiated cells more efficiently. Moreover, metabolites from the TCA cycle (e.g., α-ketoglutarate) are essential cofactors for epigenetic modifiers (e.g., TET and KDM families), which activate differentiation-related gene programs [73,79]. Therefore, by precisely regulating the metabolic switches depicted in Figure 3, m⁶A exerts an indirect yet profound influence on stem cell fate decisions.

In the glycolysis of stem cells, the YTHDF1/eEF2 complex and IGFBP3 regulate the 5’UTR of PDK4 (pyruvate dehydrogenase kinase 4) [80], and IGFBPs interact with the 5’/3’UTR of HK2 (hexokinase 2) and the 3’UTR of GLUT1 (facilitative glucose transporter), promoting the expression of HK2 and GLUT1 [81]. YTHDF1 regulates the expression of HK2 and PKM2 (pyruvate kinase isozyme type M2) [18,82], while R-2-HG upregulates LDHB (lactate dehydrogenase B) expression by mediating FTO and YTHDF2, promoting glycolysis [83].

METTL3 and METTL14 jointly regulate APC (adenomatous polyposis coli) expression, which increases c-MYC (myelocytomatosis viral oncogene homolog) and PKM2 expression [84]. METTL3 directly interacts with ACLY (ATP citrate lyase) and SLC25A1 (solute carrier family 25 member 1) [85], affecting glycolysis. Activated glycolysis serves as a switch for controlling histone acetylation, regulating stem cell pluripotency [78]. In mitochondria, ALKBH5 regulates the TCA (tricarboxylic acid cycle) [86] (Figure 3). ALKBH5 reduces DRP1 (Dynamin-related protein 1) methylation levels, inhibits mitochondrial fission, and controls stem cell energy metabolism [87]. In gastric cancer stem cells, METTL3 modifies the 3’UTR of NDUFA4 (NADH dehydrogenase 1 alpha subcomplex 4) mRNA, recruits IGF2BP1 to increase NDUFA4 mRNA stability, and promotes oxidative metabolism [88]. NDUFA4 enhances stem cell glycolysis to promote proliferation and differentiation [18]. In iPSCs, OXPHOS and glycolysis are in a balanced state, regulating the proliferation and differentiation of iPSCs through the aminoglycation of OCT4 and SOX2. YTHDF2 directly regulates OCT4 and SOX2 [61,75,89]. The intermediate products produced in OXPHOS and glycolysis are not only important substrates for protein acetylation but also essential for maintaining the chromatin structural characteristics of stem cells [90]. m⁶A regulates energy metabolism in stem cells, which has significant implications and potential for cancer stem cell renewal and differentiation, tumor treatment resistance, tumor metabolism, and tumor therapy [18].

In summary, the profound significance of m⁶A’s regulation of stem cell energy metabolism lies in its direct shaping of the cellular epigenetic landscape. Glycolysis and the TCA cycle serve not only as energy-producing processes but also as supply stations for key metabolic precursors of histone modifications. By regulating the expression of metabolic enzymes, m⁶A precisely controls the flow of these precursors: enhanced glycolysis leads to the accumulation of pyruvate and lactate. Pyruvate can be converted into acetyl-CoA, providing substrates for histone acetylation (e.g., H3K27ac); lactic acid directly drives histone lactylation (e.g., H3K18la). Both modifications correlate positively with chromatin accessibility and pluripotency gene activation [91]. TCA cycle intermediates like α-ketoglutarate (α-KG) serve as essential cofactors for histone demethylases (e.g., KDM) and DNA demethylases TET. m⁶A modulates TCA via ALKBH5 [86] or promotes acetyl-CoA production by stabilizing PDHA1 mRNA through YTHDC2 [79], effectively controlling the availability of the “metabolic tool” used to erase repressive epigenetic marks.

Collectively, these findings support a model in which m⁶A modification functions as an upstream regulator within a cohesive “m⁶A–metabolism–epigenetics” axis. By directing metabolic reprogramming and modulating the availability of critical metabolites (such as acetyl-CoA and α-KG), m⁶A installation indirectly governs the dynamics of histone modifications and chromatin states, culminating in fate decisions in stem cells. This axis provides a mechanistic framework for understanding the profound impact of metabolism on cellular reprogramming and pluripotency.

Strong Correlative Evidence: A robust body of data demonstrates that m⁶A regulates key metabolic enzymes and that metabolic shifts (e.g., glycolytic flux) alter the availability of epigenetic cofactors (acetyl-CoA, α-KG) [78,91]. The temporal correlation of these events during fate changes is strongly supportive of an integrated axis.

Causality and Quantitative Gaps: Much of the evidence remains correlative. Precisely how the dynamic, often subtle, changes in m⁶A on individual metabolic enzyme mRNAs quantitatively translate into metabolite pool shifts sufficient to drive global epigenetic changes is not fully mapped. The field lacks dynamic, quantitative flux models that incorporate m⁶A kinetics.

Primary Controversy: The predominant focus has been on how m⁶A-driven metabolism fuels epigenetics. However, the reverse regulation—how epigenetic states or metabolic signals control the m⁶A machinery itself (e.g., via transcription or PTMs of writers/erasers)—is equally important but less explored. The axis is likely bidirectional, and its unidirectional portrayal is an oversimplification.

Figure 3. The illustration integrates two interconnected regulatory modules controlled by m⁶A. Left panel, glycolysis promotion: m⁶A reader proteins (e.g., YTHDF1 and IGFBPs) enhance glycolytic flux by upregulating the expression of key glycolytic enzymes (HK2 and PKM2) [18,81] and the transporter GLUT1. Right panel, mitochondrial function and OXPHOS modulation: m⁶A also finely tunes mitochondrial function. YTHDF1 influences mitochondrial dynamics by regulating HIF-1α and DRP1 [80]. The demethylase ALKBH5 modulates TCA cycle intermediates (e.g., L-2-HG) and acetyl-CoA levels, thereby affecting oxidative phosphorylation (OXPHOS) efficiency and ATP production [86,87]. Critical Integration Point: These two modules are not isolated. Pyruvate from glycolysis feeds into mitochondrial metabolism, while mitochondrial outputs (e.g., acetyl-CoA and α-ketoglutarate) serve as key precursors for epigenetic processes like histone modification. Thus, by coordinating these two metabolic arms, m⁶A shapes the metabolic state that determines stem cell fate, exemplifying the “m⁶A–metabolism–epigenetics” axis.

Figure 3. The illustration integrates two interconnected regulatory modules controlled by m⁶A. Left panel, glycolysis promotion: m⁶A reader proteins (e.g., YTHDF1 and IGFBPs) enhance glycolytic flux by upregulating the expression of key glycolytic enzymes (HK2 and PKM2) [18,81] and the transporter GLUT1. Right panel, mitochondrial function and OXPHOS modulation: m⁶A also finely tunes mitochondrial function. YTHDF1 influences mitochondrial dynamics by regulating HIF-1α and DRP1 [80]. The demethylase ALKBH5 modulates TCA cycle intermediates (e.g., L-2-HG) and acetyl-CoA levels, thereby affecting oxidative phosphorylation (OXPHOS) efficiency and ATP production [86,87]. Critical Integration Point: These two modules are not isolated. Pyruvate from glycolysis feeds into mitochondrial metabolism, while mitochondrial outputs (e.g., acetyl-CoA and α-ketoglutarate) serve as key precursors for epigenetic processes like histone modification. Thus, by coordinating these two metabolic arms, m⁶A shapes the metabolic state that determines stem cell fate, exemplifying the “m⁶A–metabolism–epigenetics” axis.

6. m⁶A Modification and Signaling Pathways

Stem cell fate determination represents a coordinated response to intracellular and extracellular signals. Building upon the established role of m⁶A in regulating fundamental cellular states (Section 3, Section 4 and Section 5), this section further explores its higher-order functions as a sophisticated regulator and integrator of key signaling pathways. We will analyze how m⁶A modification of key nodal components (e.g., APC in Wnt and PTEN in PI3K-AKT) dynamically modulates the activity of the Wnt, PI3K-AKT, JAK-STAT, and Hippo pathways. A particular focus will be on how m⁶A may orchestrate crosstalk between these pathways by simultaneously targeting shared nodes (e.g., GSK-3β), thereby amplifying fate-determining signals and ensuring coordinated cellular responses.

6.1. m⁶A Modification and Wnt Signaling Pathway

The Wnt pathway plays a crucial role in stem cell differentiation, proliferation, metabolism, and other processes [92,93]. Key components of this pathway, including APC, GSK-3β, AXIN, and β-catenin, are subject to m⁶A modification, which dynamically regulates their expression and function [94].

Studies primarily in colorectal and gastric cancer models have revealed a key oncogenic axis: METTL3 promotes Wnt/β-catenin signaling by recruiting YTHDF reader proteins to degrade APC mRNA [84]. The consequent accumulation of β-catenin upregulates downstream targets like c-MYC and PKM2, which in these cellular contexts drives a pro-proliferative glycolytic switch. This mechanism provides a clear example of how the m⁶A machinery can be co-opted to fuel pathological stemness, though its activity in normal stem cell homeostasis requires further investigation. Conversely, METTL3 can also inhibit cell motility by downregulating c-Met (cellular mesenchymal–epithelial transition factor), which in turn suppresses the membrane localization of β-catenin and its interaction with E-cadherin, thereby playing a role in controlling cancer cell proliferation [95].

The expression of FTO and ALKBH5 is reduced, directly increasing the methylation level of receptor protein FZD10 mRNA and the expression of β-catenin, activating the Wnt pathway, and thereby affecting the gene expression of epithelial ovarian cancer (EOC) [96]. ALKBH5 binds to AXIN2 mRNA of the Wnt pathway, causing demethylation [97]. The FTO promoter region LEF/TCF binds to β-catenin, modifies MYC, and maintains stem cell homeostasis [98]. FTO serves as a key regulatory factor for Wnt to trigger EMT, altering the 3′ end processing of key mRNA in the Wnt signaling cascade, leading to EMT [99]. YTHDF1 directly promotes the translation of the Wnt signal transduction effector factor TCF7L2/TCF4, enhances the activity of β-catenin, and affects the pluripotency of stem cells [100] (Figure 4). YTHDF2 recognizes and targets m⁶A-modified GSK-3β mRNA for degradation, enhances β-catenin activity, reduces YTHDF2 expression, inhibits Wnt/β-catenin/Cyclin D1 pathway-related protein expression, leads to G0/G1 phase arrest in cells, and ultimately inhibits CRC cell proliferation [101].

6.2. m⁶A Modification and PI3K-AKT Signaling Pathway

The PI3K-AKT signaling pathway is crucial for stem cell survival, proliferation, and apoptosis [102]. Notably, key proteins within this pathway are closely regulated by m6A modification [103]. For instance, METTL3-mediated m6A modification directly modulates the PI3K-AKT pathway. Knockdown of METTL3 inhibits the phosphorylation of AKT (also known as RAC-alpha serine/threonine protein kinase), thereby suppressing PI3K/AKT activation and subsequently inhibiting glycolysis in stem cells [104]. Furthermore, reduced expression of METTL3 and EPPK1 (epiplakin 1) [105] can upregulate PI3K (phosphatidylinositol 3-kinase) expression, collectively disrupting stem cell homeostasis [106].

Overexpression of METTL14 increases the m⁶A enrichment of PTEN (phosphatase and tensin homolog), promoting PTEN expression. The increased expression of PTEN significantly inhibits the activation of the PI3K/AKT pathway [107] (Figure 5). METTL14 affects the expression of transcription factor SOX4 (sex-determining region Y-box transcription factor 4), and the degradation of SOX4 mRNA depends on YTHDF2. Low levels of SOX4 increase the expression levels of PI3K and AKT proteins, activate glycolysis in stem cells, and have a certain inhibitory effect on the malignant process of CRC [108]. Lowering the expression of YTHDF1 significantly reduces key proteins in the PI3K/AKT signaling pathway, such as PI3K, AKT, and mTOR (mammalian target of rapamycin). YTHDF1 mainly regulates the translation of AKT2 and AKT3 rather than transcription [109,110]. Low levels of YTHDF2 significantly increase the expression of LHPP (phospholysine phosphohistidine inorganic pyrophosphate phosphatase) and growth inhibitor NKX3-1 (NK3 homeobox 1), inhibit AKT phosphorylation, and significantly suppress the proliferation and migration of PCa cells. At the same time, reduced AKT phosphorylation levels affect the expression of glycolytic genes and the maintenance of stem cell pluripotency [111]. FTO, as a downstream target of PI3K-AKT, activates PI3K-AKT through a transcription factor FOXO6 (forkhead box protein O 6), affecting the energy metabolism and pluripotency of stem cells [112].

6.3. m⁶A Modification and JAK-STAT Signaling Pathway

The JAK-STAT pathway regulates the proliferation and apoptosis of stem cells [113], and the expression of key proteins in the pathway is regulated by m⁶A modification at the transcriptional level [94]. METTL3 directly modifies the JAK1 (just another kinase) mRNA, while METTL3 and YTHDF2 maintain the stability of JAK1 mRNA [114]. After m⁶A modification, the JAK-STAT signal directly participates in the regulation of iPSCs [16]. METTL3 maintains the pluripotency of iPS cells by mediating the expression of JAK2-STAT3. KLF4 is a direct downstream target of JAK-STAT3, which is activated by phosphorylated STAT3 (signal transducer and activator of transcription 3) and indirectly activates SOX2. Overexpression of METTL3 directly enhances the phosphorylation level of STAT3 and increases the expression of SOX2 and KLF4 [16,115]. SOCS3 (suppressor of cytokine signaling 3) is a key negative regulator of the JAK2-STAT3 pathway. METTL3 modifies JAK2 and SOCS3, and JAK2 and SOCS3 are targets of YTHDF1 and YTHDF2, respectively. Decreasing the m⁶A modification of JAK2 and SOCS3 inhibits YTHDF1-mediated JAK2 translation, blocks YTHDF2-dependent SOCS3 mRNA decay, and impairs the pluripotency of iPSCs [16] (Figure 6).

6.4. m⁶A Modification and Hippo Signaling Pathway

The Hippo pathway directly participates in the complex biological processes of stem cells and plays an important role in their proliferation, self-renewal, and differentiation [117]. m⁶A modification regulates the Hippo signaling pathway through key regulatory factors such as LATS1, YAP, and TAZ [94]. m⁶A modification directly affects the translation of YAP1 mRNA, regulating the proliferation and differentiation of stem cells [118].

The increase in METTL3 expression directly activates the Hippo pathway and regulates YAP nuclear translocation, affecting the proliferation and differentiation of CRC cells through lncRNA [119,120]. LATS1 directly regulates the phosphorylation level of YAP/TAZ, increases METTL3 or decreases YTHDF2 expression, and activates YAP/TAZ in the Hippo signaling pathway [121] (Figure 7). The combination of YTHDF2 and YAP mRNA reduces the stability of mRNA, while YTHDF2 recruits AGO2 (argonaute RISC catalytic component 2) to promote the degradation of YAP mRNA, affecting the production of related tumor factors and inhibiting the occurrence of tumors in breast cancer [122,123]. YTHDF2/3 regulates somatic reprogramming by regulating the relationship between the Hippo pathway and EMT [61]. YTHDF2/3 recruits the disenergizing enzyme complex CCR4-NOT and PAN2/PAN3 to degrade TEAD2 mRNA, reduce TEAD2 expression, promote YAP/TAZ nuclear translocation and MET [124,125,126]. The increased expression of YTHDF2/3 promotes YAP nuclear translocation, promotes the expression of pluripotent factors, and accelerates somatic reprogramming [127].

In summary, key components of signaling pathways such as Wnt, PI3K-AKT, JAK-STAT, and Hippo, after being modified with m⁶A, play a series of regulatory roles in stem cells. These signaling pathways are directly involved in the regulation of somatic reprogramming (

Table 1. Regulatory effects of different signaling pathways on somatic reprogramming.

Signal Pathway	Mechanisms for Regulating Reprogramming	References
Wnt	After activation, β-catenin binds to the effector factor TCF to maintain the expression of pluripotent genes, such as SOX2. The efficiency of reprogramming is regulated by the binding of β-catenin and repressor protein TCF7L1.	[128,129]
PI3K-AKT	The pluripotent factor SOX2 is a direct target of phosphorylated AKT, and phosphorylated AKT directly regulates the expression of SOX2, regulating the induction efficiency of iPS cells. Activated AKT can replace bFGF and improve reprogramming efficiency.	[130,131]
JAK-STAT	Activated STAT3 interacts with Nanog or KLF4, respectively, to improve reprogramming efficiency. In the later stage of reprogramming, STAT3 activates the endogenous OCT4 gene, improving reprogramming efficiency.	[132,133]
Hippo	LATS2 inhibits reprogramming by antagonizing TAZ factors. Reducing the expression of LATS2 promotes the nuclear translocation of YAP. After translocation, YAP regulates reprogramming by interacting with OCT4 and SOX2 and combines with TEAD alone to regulate reprogramming.	[134,135,136]

).

6.5. m⁶A Modification: A Core Integrator of Stem Cell Signaling Network Crosstalk

The Wnt, PI3K-AKT, JAK-STAT, and Hippo pathways do not operate independently but form a dense regulatory network that collectively determines stem cell fate. m⁶A modification may play a central role in integrating signals and amplifying effects by simultaneously targeting key nodes across multiple pathways. Wnt-PI3K-AKT Synergy: These two pathways engage in well-known crosstalk. On the one hand, the PI3K-AKT pathway stabilizes β-catenin by phosphorylating and inhibiting GSK-3β, thereby positively reinforcing Wnt signaling. This review highlights that m⁶A degradation of GSK-3β mRNA via YTHDF2 [101] and downregulation of APC expression through the METTL3-YTHDF axis [84] both lead to β-catenin accumulation. This suggests m⁶A may simultaneously weaken two major negative regulatory mechanisms of β-catenin (APC and GSK-3β), synergistically amplifying Wnt pathway output. Transcriptional Cooperation Between Hippo and Wnt: Upon nuclear translocation, Hippo downstream effectors YAP/TAZ not only bind to TEAD family transcription factors but also interact with Wnt pathway effectors TCF/LEF, jointly regulating an overlapping set of target genes (e.g., CYR61 and CTGF). This review indicates that m⁶A influences YAP activity by degrading Tead2 mRNA via YTHDF2/3 [61,126]. This suggests m⁶A may indirectly regulate the cooperative efficiency between YAP/TAZ and Wnt pathway effectors by modulating the Hippo pathway, thereby broadly impacting gene programs controlling proliferation and pluripotency.

These interactions demonstrate that m⁶A’s regulation of individual signaling molecules (e.g., GSK-3β, APC, and Tead2) amplifies and integrates effects through networked crosstalk, ultimately coordinating global regulation of stem cell self-renewal, differentiation, or reprogramming. Future studies should increasingly employ systems biology approaches to map the coupling between the m⁶A modification landscape and the dynamics of stem cell signaling networks.

Established Node-Specific Regulation: There is strong evidence that m⁶A can modulate individual key nodal proteins across major pathways (e.g., APC in Wnt and PTEN in PI3K-AKT) [84,107]. This establishes m⁶A as a bona fide post-transcriptional regulator of signaling cascades.

The “Integration” Caveat: Most examples show m⁶A acting on parallel, independent nodes. True network integration would require evidence that a single m⁶A event simultaneously coordinates the activity of two or more pathways in a coordinated manner (e.g., via a shared regulator like GSK-3β). Such higher-order coordination is suggested but not definitively proven in stem cell fate decisions.

Critical Disconnect between Models: A profound gap exists between the detailed oncogenic signaling roles of m⁶A in cancer stem cells (CSCs) and the understanding of its function in physiological stem cell signaling during development or tissue repair. It is unclear whether the aggressive, pro-growth wiring seen in CSCs reflects a hijacking or an amplification of normal stem cell mechanisms. Bridging this gap is essential for therapeutic safety.

7. m⁶A Modification and Somatic Reprogramming

Somatic reprogramming is an integrative paradigm. The complete conversion of a somatic cell to a pluripotent state requires the synchronized rewiring of all regulatory layers discussed thus far. This section uses reprogramming as a model process to illustrate the stage-specific, coordinating function of m⁶A. We will delineate how, in a temporally ordered manner, m⁶A (1) initiates dedifferentiation and metabolic reprogramming (leveraging mechanisms from Section 3 and Section 5); (2) regulates the EMT/MET balance and consolidates the pluripotency network (integrating controls from Section 3, Section 4 and Section 6); and (3) finalizes mitochondrial and epigenetic remodeling. This analysis showcases m⁶A not as a collection of parts, but as a unified system driving complex state transitions.

Somatic reprogramming refers to the reprogramming of terminally differentiated cells into iPSCs [137]. This process is typically initiated by the co-expression of key transcription factors, notably OCT4, SOX2, KLF4, and c-MYC [138]. Successful reprogramming entails a coordinated cascade of events, including the silencing of somatic genes, a metabolic switch from oxidative phosphorylation to glycolysis (OGS), a mesenchymal-to-epithelial transition (MET), and the activation of the core pluripotency network [139]. Given the profound epigenetic remodeling required, the regulation of epigenetic modifications, such as m⁶A, is a critical determinant of reprogramming efficiency [140]. m⁶A modification, as a key layer of epigenetic transcriptional regulation, plays a core, temporally and stage-specific role at different phases of this process. By precisely regulating transcription factors, cellular state transitions, and energy metabolism, it synergistically drives cells toward reprogramming into a pluripotent state. Following initiation in the early stage, cells must first disrupt existing somatic programs and activate metabolic shifts supporting pluripotency acquisition. The core task of the intermediate stage involves activating the central pluripotency transcription factor network and completing metabolic reprogramming (MET) to establish stable epithelial-like iPSC characteristics. In the late stage of reprogramming, cells must consolidate the pluripotent epigenetic state and complete mitochondrial function remodeling to achieve full establishment and maintenance of pluripotency.

7.1. Early Phase: Initiation, Dedifferentiation, and Metabolic Reprogramming

The m⁶A machinery precisely orchestrates the expression of core pluripotency factors to facilitate reprogramming. Writers such as METTL3 and METTL14 create a permissive epigenetic landscape: METTL3 broadly promotes miRNA expression [141] and, via the mediator ZFP217, upregulates OCT4, SOX2, and NANOG [19]; meanwhile, METTL14 overexpression induces a senescence-associated secretory phenotype (SASP), whose component IL-6 enhances iPSC induction efficiency [142]. Subsequently, readers YTHDF2 and YTHDF3 execute the clearance of somatic program mRNAs (e.g., Tead2 and Tgfb1) by recruiting the CCR4-NOT and PAN2-PAN3 deadenylase complexes, respectively, thereby removing barriers to reprogramming [61,124,125]. Collectively, through these writer- and reader-mediated actions, m⁶A modification exerts central control over the pluripotency transcription factor network, including OCT4, SOX2, Nanog, and KLF4 (

Table 2. Regulation of m⁶A modification on reprogramming transcription factors and pluripotent factors.

m6A Modifier	Transcription Factors	Molecular Mechanism	Reference
METTL3	Nanog	METTL3-METTL14-WTAP complex binds with SMAD2/3 to regulate Nanog levels and cellular pluripotency	[54]
	SOX2	METTL3 deficiency interferes with the expression of JAK2 and SOSC3, inactivates the JAK2 pathway, blocks SOX2 transcription, and inhibits piPSC differentiation	[16]
	KLF4	METTL3 deficiency interferes with JAK2 and SOSC3 expression, blocks KLF4 transcription, and inhibits piPSC differentiation	[16]
METTL14	OCT4	Overexpression of METTL14 increases the expression of OCT4 and improves reprogramming efficiency	[125]
ALKBH5	Nanog	In the late stage of reprogramming, overexpression of ALKBH5 improves reprogramming efficiency by stabilizing the transcript of Nanog	[143]
YTHDF1	OCT4	RNA/DNA binding protein binds to OCT4 and YTHDF1 promoters to regulate hiPSC differentiation	[144]

).

7.2. The Role of m⁶A Modification in EMT

The dynamic balance between epithelial–mesenchymal transition (EMT) and its reverse process, mesenchymal–epithelial transition (MET), is crucial for somatic reprogramming [145,146] (Figure 4). Early reprogramming involves a transient EMT phase, which activates epigenetic modifiers like Bmi1 and Ezh2 [147]. Subsequently, the establishment of MET is essential; it is characterized by the downregulation of TGF-β, Snail, and Zeb family members [145].

The m⁶A modification system acts as a key regulator of this EMT-MET equilibrium. It directly targets core EMT regulators: for instance, METTL3 and YTHDF1 control the translation of Snail mRNA, thereby influencing EMT/MET dynamics [148]. Furthermore, the m⁶A reader proteins YTHDF2 and YTHDF3 promote the MET phase and enhance reprogramming efficiency by degrading Tead2 mRNA. This reduction in TEAD2 levels prevents nuclear accumulation of YAP/TAZ, a key event that otherwise hinders MET [61,126] (Figure 8). Thus, through coordinated actions on multiple nodes, m⁶A finely tunes the EMT-MET switch to facilitate reprogramming.

7.3. m⁶A Modification and Energy Metabolism in Somatic Reprogramming

m⁶A modification plays a pivotal role in reprogramming by orchestrating the essential metabolic switch and modulating mitochondrial function [18,48,77]. A core event is the oxidative phosphorylation to glycolysis (OGS) transition. This process is initiated by ERRα/γ-activated OXPHOS and subsequently driven by a shift towards glycolysis [149,150,151]. In addition, the expression of ERRα is regulated by HIF-1α (hypoxia inducible factor-1α) [152]. HIF-1α directly modifies glycolytic kinases such as PDK1, PKM2, and HK2 to promote OGS and improve the efficiency of somatic reprogramming [153,154]. HIF-1α is regulated by YTHDF1, which works together with ATG2A (autophagy-related 2A) and ATG14 (autophagy-related 14) to regulate HIF-1α [155]. Activated glycolysis enhances cellular acetyl-CoA and lactate levels, enhances H3K27ac (acetyl histone H3 Lys27) and lactylation of H3K18la (histone 3 on lysine residue 18) at pluripotent gene loci, and promotes somatic reprogramming [91].

Mitochondrial physiology, encompassing dynamics and the permeability transition pore (mPTP), is critically regulated by m⁶A and directly influences somatic reprogramming [156,157,158,159] (Figure 9A,B). m⁶A supports the metabolic reprogramming of cells by enhancing mitochondrial oxidative capacity. It promotes acetyl-CoA production, a key substrate for histone acetylation: YTHDC2 stabilizes PDHA1 mRNA to boost nuclear acetyl-CoA levels [79,160]. This aligns with the role of ATP synthase in coupling metabolic shifts to reprogramming [161], while METTL3 can regulate mitochondrial ATP synthase activity [162]. In parallel, m⁶A exerts precise control over the mPTP, a pivotal gateway in mitochondrial signaling. The opening of the mPTP elevates mitochondrial reactive oxygen species (mtROS), which in turn activates (via miR-101c) the histone demethylase PHF8. PHF8 erases repressive histone marks (H3K9me2/H3K27me3), thereby facilitating epigenetic reprogramming [76,157] (Figure 9B). METTL3 contributes to this regulatory layer by controlling the expression of the mPTP-associated transcription factor NRF1 [158]. Therefore, through coordinated regulation of mitochondrial metabolism and permeability-dependent signaling, m⁶A modification plays an essential role in establishing the mitochondrial conditions necessary for somatic reprogramming.

Well-Defined Stage-Specific Functions: The field has reached a consensus on the temporal sequence of m⁶A actions: early somatic gene silencing (via YTHDF2/3), mid-phase MET promotion, and late-stage metabolic/epigenetic consolidation [61,124,125]. This provides a refined view beyond simple “pro-” or “anti-” reprogramming effects.

The In Vitro Bottleneck: This exquisite mechanistic understanding is almost entirely confined to fibroblast reprogramming in culture. A major, unresolved question is whether this same playbook operates during in vivo reprogramming (e.g., in injury models) or in reprogramming clinically relevant human somatic cells (e.g., blood or hepatocytes), which may have distinct m⁶A landscapes and dependencies.

Therapeutic Translation Challenge: The biggest controversy lies in the application. While modulating m⁶A can enhance iPSC generation in vitro, the potential risks of altering such a fundamental regulatory layer in vivo (e.g., for in situ regeneration) are immense and unknown. The field must confront the trade-off between efficiency and safety, moving from proof-of-concept to clinically viable strategies.

8. Current Challenges and Future Perspectives

Based on the integrated perspective of this review, we arrive at a core conclusion: the strong context dependence of m⁶A modification not only reflects its complexity but also constitutes its functional essence as a precise regulator of stem cell fate. The varying, sometimes opposing, outcomes described for factors like METTL3 or YTHDF2 across different stem cell systems (Section 3, Section 6 and Section 7) are logical consequences of their roles within specific molecular contexts. This framework forces a shift from asking “What does protein X do?” to “What does protein X do, in which cell state, and in response to what signals?”

The methyltransferase METTL3 illustrates this principle. In mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs), METTL3 primarily supports pluripotency by stabilizing transcripts of core factors such as Nanog and facilitating the silencing of somatic genes during reprogramming [16,53]. In stark contrast, within cancer stem cells (CSCs) of various malignancies—including leukemia, gastric, and pancreatic cancer—METTL3 often functions as an oncogene. It promotes unlimited proliferation, metabolic reprogramming (e.g., enhanced glycolysis), and drug resistance by activating pro-survival pathways like Wnt/β-catenin and PI3K/AKT [84,104]. This functional duality likely originates from cell-type-specific RNA targeting and interactions with distinct upstream signals and epigenetic landscapes. A similar context-dependent pattern is observed for reader proteins. YTHDF2 can promote induced pluripotency by degrading differentiation-related mRNAs (e.g., Tead2) [61], yet it acts as a tumor suppressor in prostate cancer by destabilizing oncogenic transcripts [111]. Likewise, the demethylases ALKBH5 and FTO fine-tune pluripotency in ESCs but are frequently upregulated in glioblastoma or leukemia stem cells to drive self-renewal, block differentiation, and aid immune evasion [24,68]. This profound heterogeneity underscores a critical caveat: broad statements such as “METTL3 promotes stemness” or “YTHDF2 inhibits stemness” are oversimplified and potentially misleading. Consequently, therapeutic strategies targeting the m⁶A pathway must be designed to selectively disrupt pathological modifications in diseased cells while preserving the physiological homeostasis of normal stem cells.

The functional output of m⁶A is shaped by the intrinsic nature of the stem cell. Embryonic stem cells (ESCs) employ m⁶A as a molecular “rheostat” to enable state transitions, such as priming for differentiation by destabilizing naïve pluripotency transcripts [49]. Adult somatic stem cells, however, often utilize m⁶A to maintain homeostasis within their niche, precisely regulating fate decisions in response to local cues [74]. Cancer stem cells (CSCs) represent a pathological adaptation, hijacking the m⁶A machinery to enforce a plastic, pro-survival state that fuels tumorigenesis, therapy resistance, and metastasis [18]. Recognizing this spectrum of physiological versus pathological roles is fundamental for developing precise interventions.

Significant debate persists regarding the functions of cytoplasmic m⁶A readers, particularly the YTHDF family (YTHDF1/2/3). Early models assigned specialized roles—translation promotion (YTHDF1), decay mediation (YTHDF2), and cooperative function (YTHDF3) [33,125]. Recent evidence, however, suggests considerable redundancy. During somatic reprogramming, for instance, the combined knockdown of YTHDF2 and YTHDF3, but not single knockdowns, is necessary to block the degradation of specific mRNAs like Tead2 [61,124]. Conversely, other contexts reveal clear specialization; in porcine iPSCs, YTHDF1 specifically regulates JAK2 translation, while YTHDF2 uniquely controls SOCS3 mRNA decay [16]. Resolving this redundancy-specificity paradox will require integrated approaches, including simultaneous multi-gene knockout, spatial proteomics, and single-cell analyses, to define condition-specific reader complexes and their precise targets.

A major translational challenge lies in bridging the gap between robust in vitro mechanistic findings and their in vivo validation. Most current knowledge derives from cultured stem cells or cancer lines, which, while indispensable for mechanistic dissection, cannot fully capture the complexity of native niches, systemic metabolism, and immune interactions. For example, while the role of YTHDF2/3 in reprogramming is well-established in vitro [61], their functions in adult stem cell homeostasis in vivo remain less clear. Similarly, although METTL3 inhibitors show promise in suppressing CSC growth in culture and xenograft models [22], their impact on normal tissue regeneration and long-term safety is largely unknown. Future research must prioritize physiologically relevant models—such as genetically engineered animals and patient-derived organoids—to validate mechanisms and rigorously assess the potential on-target toxicities of perturbing this fundamental RNA regulatory pathway.

9. Conclusions

Stem cells possess the capacity for self-renewal and multipotent differentiation, with their functional changes regulated by multiple factors, including transcription factors, histone modifications, and energy metabolism.

As the most abundant RNA modification in eukaryotic cells, m⁶A plays a huge role in almost every aspect of biological regulation by regulating RNA modification in different ways. At the same time, we are gradually mastering the functions of m⁶A modification and the key regulatory factors of methylation and demethylation. The dynamic regulation of m⁶A modification is a new post-transcriptional regulation mechanism, which is closely related to the regulation of stem cell pluripotency maintenance, histone modification, energy metabolism, and reprogramming. Energy metabolism directly affects the pluripotency, proliferation, and differentiation of stem cells. Histone modification regulates the pluripotency and differentiation of stem cells by regulating the opening and closing of chromatin. Pluripotency maintenance, histone modification, and energy metabolism are all involved in regulating somatic reprogramming. m⁶A modification is involved in the regulation of signaling pathways such as Wnt, PI3K-AKT, JAK-STAT, and Hippo, which directly regulate stem cell function and somatic reprogramming. The evidence synthesized through this integrative framework reveals that m⁶A regulation is fundamentally context-dependent and multi-layered. The apparent contradictions, such as METTL3 promoting pluripotency in ESCs but oncogenesis in CSCs, are resolved when viewed through the lens of distinct cellular “contexts”—different target RNA repertoires, signaling environments, and epigenetic landscapes. This underscores that m⁶A machinery components are not simply “on/off” switches for stemness but versatile tools whose functional output is defined by the system in which they operate. Future research must therefore prioritize defining these contexts through integrated omics approaches and sophisticated in vivo models. By doing so, we can move from a phenomenological catalog of m⁶A functions towards a predictive understanding of its role in fate decisions, unlocking its precise therapeutic potential in regeneration and disease. These studies reveal a fascinating but unexplored regulation of the interaction between gene expression and protein translation and the need for coordination with other regulatory networks. To move forward, the field must rigorously address the translational gap between in vitro mechanisms and in vivo physiology, leveraging advanced models to unlock the true therapeutic potential of targeting m⁶A in regenerative medicine and oncology.

Although the m⁶A-targeted modification of stem cells has shown broad prospects in the medical field, there are still challenges in related research. These related studies are still in their infancy, and many potential mechanisms have not yet been discovered. More in vivo studies and clinical trials are urgently needed to confirm the potential clinical significance of m⁶A modification in stem cell therapy, which will significantly improve the effectiveness of stem cell therapy in the treatment of human diseases.

In different types of stem cells, m⁶A modification affects the biological behavior of stem cells by altering the stability of key mRNA involved in stem cell function regulation and by activating or inhibiting related signaling pathways. Therefore, further exploration of the regulatory mechanisms of m⁶A modification in stem cell function and somatic reprogramming will provide great prospects for research in regenerative medicine, disease treatment, and drug screening.