Regulation and intervention of stem cell differentiation by long non-coding RNAs: Mechanisms and therapeutic potential

Abstract

Long non-coding RNAs (lncRNAs) play pivotal roles in the regulation of gene expression, particularly in maintaining pluripotency and directing stem cell. By orchestrating stem cell fate decisions and lineage commitment through epigenetic, transcriptional, and post-transcriptional mechanisms, lncRNAs have emerged as key modulators in developmental biology. Their therapeutic potential has garnered increasing interest, especially in the contexts of regenerative medicine, disease modeling, targeted delivery systems, and precision therapeutics. This review presents a comprehensive overview of the mechanisms by which lncRNAs govern stem cell differentiation and examines emerging lncRNA-based therapeutic strategies, emphasizing major challenges and prospective research directions in this rapidly advancing field.

Key Words: Long non-coding RNAs; Stem cell differentiation; MicroRNA sponges; Epigenetic regulation; Regenerative medicine; Delivery and precise targeting; Stem cell therapy

Core Tip: As key regulators of stem cell differentiation, long non-coding RNAs (lncRNAs) orchestrate multilayered gene regulation through compartment-specific mechanisms. This review introduces a spatiotemporal framework linking lncRNA localization to epigenetic, transcriptional, and post-transcriptional control, and highlights emerging lncRNA-targeted therapies. By integrating mechanistic insights with therapeutic strategies, it redefines lncRNAs as dynamic spatial organizers of stem cell fate, offering new opportunities for regenerative medicine and precision therapy.

INTRODUCTION

Stem cell differentiation is a fundamental process in developmental biology and regenerative medicine, underpinning tissue homeostasis, repair, and therapeutic regeneration. This tightly regulated process is orchestrated by a complex network of transcription factors [e.g., octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), NANOG], signaling pathways (e.g., Wnt, Notch, Hippo), and epigenetic modifications. Embryonic stem cells (ESCs), derived from the inner cell mass of the mammalian blastocyst, undergo gastrulation to form the ectoderm, mesoderm, and endoderm - each giving rise to specialized tissues.

Recent studies have revealed that long non-coding RNAs (lncRNAs) - transcripts longer than 200 nucleotides with no protein-coding capacity - play crucial roles in stem cell maintenance and differentiation. Their functional diversity is shaped by unique structural conformations, biochemical properties, and subcellular localization[1]. LncRNAs participate in diverse biological processes, including embryogenesis, spermatogenesis, cell cycle regulation, and tumorigenesis[2], with increasing experimental evidence supporting their regulatory functions. Distributed across nuclear, cytoplasmic, and extracellular compartments, lncRNAs exert context-dependent regulatory effects through diverse gene regulatory modalities[3-5]. Compared to mRNAs, they display stronger nuclear enrichment[6], where they regulate stem cell differentiation via chromatin remodeling, transcriptional modulation, and alternative splicing. In the cytoplasm, they influence mRNA stability, translation, and post-translational modifications (PTMs).

This minireview provides a comprehensive overview of the multilayered mechanisms - epigenetic, transcriptional, and post-transcriptional - by which lncRNAs regulate stem cell fate. To better capture their functional complexity, we propose a compartmental framework based on subcellular localization - nucleus, cytoplasm, and membrane-less organelles - offering a spatiotemporal lens for understanding lncRNA-mediated regulation (Figure 1). We further summarize representative lncRNAs and their roles across distinct stem cell types and developmental lineages (Table 1). Finally, we discuss emerging lncRNA-targeted strategies - including CRISPR-based editing, antisense oligonucleotides (ASOs), and nanoparticle-based delivery - that, although not yet widely adopted in clinical settings, hold significant promise for regenerative medicine. By bridging mechanistic insight with therapeutic innovation, this review aims to provide a conceptual framework for advancing lncRNA-based interventions in stem cell biology.

Figure 1 Functions of long non-coding RNAs in different cellular compartments. Long non-coding RNAs participate in diverse cellular processes and play specific functions in different cellular compartments. Long non-coding RNAs (LncRNAs) A: interact with transcription factors to affect gene activation and transcription. For example, during endoderm differentiation, definitive endoderm-associated lncRNA1 promotes forkhead box transcription factor A2 expression and guides polycomb repressive complexes 2 to specific chromatin sites through cis-regulation and mediates transcriptional silencing by catalyzing H3K27me3; B: Collaborate with chromatin modification factors to alter chromatin status; C: Participate in the regulation of alternative splicing by interacting with splicing factors. For instance, in central nervous system differentiation, Pnky cooperates with polypyrimidine tract-binding protein 1 to control alternative splicing events; D: Serve as structural scaffolds that recruit proteins to assemble membrane-less organelles. A notable example is nuclear enriched abundant transcript 1, which facilitates the formation of paraspeckles by recruiting paraspeckle-associated proteins, resulting in the assembly of shell-like nuclear bodies; E: Are involved in mRNA turnover: They may function as molecular sponges, modulating microRNA (miRNA) stability to influence the repression of miRNA target mRNAs (a); they can also directly affect mRNA stability and degradation. For example, H19 acts as a competitive endogenous RNA by binding miR-130b-3p to regulate keratinocyte differentiation and interacts with polypyrimidine tract-binding protein 1 to stabilize sterol regulatory element-binding protein 1c mRNA (b); lncRNAs regulate mRNA translation (e.g., LncMyoD participates in the translation control of myogenesis via the MyoD-LncMyoD-insulin-like growth factor 2 mRNA-binding protein axis) (c); F: LncRNAs can function as scaffolds for the regulation of post-translational modifications, such as phosphorylation. For example, during blood differentiation, lncRNA in dendritic cells binds cytoplasmic signal transducer and activator of transcription 3, preventing its interaction with the tyrosine phosphatase Src homology region 2 domain containing phosphatase-1 and thereby inhibiting signal transducer and activator of transcription 3 dephosphorylation. Created in BioRender. Available from: https://BioRender.com/5f0vac6. DEANR1: Definitive endoderm-associated lncRNA1; CARMEN: (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA; NEAT1: Nuclear enriched abundant transcript 1; ESCs: Embryonic stem cells; miRNA: MicroRNA; ANCR: Anti-differentiation noncoding RNA; PTMs: Post-translational modifications; Lnc-DC: Long non-coding RNA in dendritic cell; lncRNA: Long non-coding RNA.

Table 1 Summary of long non-coding RNAs and their functions in stem cell differentiation.

LncRNAs	Germ layer or tissue	Localization (N, C)	Specific mechanism(s)	Ref.
DEANR1	Endoderm; pancreas	N	Interaction with transcriptional factors	[7]
GATA6-AS1	Endoderm	N	Interaction with transcriptional factors	[8]
RP11-380D23.2	Lung	N	Interaction with transcriptional factors	[9]
Linc-YY1	Muscle	N	Interaction with transcriptional factors	[10]
XIST	ESCs	N	Interaction with chromatin modifiers	[11-13]
T-UCstem1	ESCs	N, C	Interaction with chromatin modifiers; mRNA turnover: MiRNA sponge	[14]
MEG3	Central nervous system; enteric nervous system	N, C	Interaction with chromatin modifiers; mRNA turnover: MiRNA sponge	[15-17,48,49]
Fendrr	Heart; body wall	N	Interaction with chromatin modifiers	[20]
CARMEN	Heart	N	Interaction with chromatin modifiers	[21]
PRESS1	ESCs	N	Interaction with chromatin modifiers	[22]
CAT7/cat7l	Central nervous system	N	Interaction with chromatin modifiers	[23,24]
UC.291	Epidermis	N	Interaction with chromatin modifiers	[25]
yylncT	Mesoderm	N	Interaction with chromatin modifiers	[26]
MALAT1	Liver	N, C	Interaction with splicing factors; functioning in PTMs	[27-31]
Pnky	Central nervous system	N	Interaction with splicing factors	[32-34]
NEAT1	ESCs	N	Scaffold membrane-less organelles	[35-38]
HBL1	Heart	C	mRNA turnover: MiRNA sponge	[43]
Linc-ROR	ESCs	C	mRNA turnover: MiRNA sponge	[44,45]
H19	Epidermis	C	mRNA turnover: MiRNA sponge; modulate mRNA stability	[46,47]
TINCR	Epidermis	C	mRNA turnover: Modulate mRNA stability	[50]
ANCR	Endoderm	C	mRNA turnover: Modulate mRNA stability	[51]
UCA1	Blood	C	mRNA turnover: Modulate mRNA stability	[52]
LncMyoD	Muscle	C	mRNA turnover: Regulate mRNA translation	[53]
Lnc-DC	Blood	C	Functioning in PTMs	[54]

LncRNAs: Long non-coding RNAs; DEANR1: Definitive endoderm-associated lncRNA1; GATA6-AS1: GATA binding protein 6 antisense RNA 1; YY1: Yin Yang 1; XIST: X inactive-specific transcript; ESCs: Embryonic stem cells; MEG3: Maternally expressed gene 3; CARMEN: (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA; CAT7: Combat Application Tourniquet Generation 7; MALAT1: Metastasis-associated lung adenocarcinoma transcript-1; NEAT1: Nuclear enriched abundant transcript 1; HBL1: Heart brake long non-coding RNA 1; Linc-ROR: Long intergenic non-protein coding RNA-regulator of reprogramming; TINCR: Terminal differentiation induced noncoding RNA; ANCR: Anti-differentiation noncoding RNA; UCA1: Urothelial carcinoma associated 1; Lnc-DC: Long non-coding RNA in dendritic cell; miRNA: MicroRNA; PTMs: Post-translational modifications.

NUCLEAR LNCRNAS IN STEM CELL DIFFERENTIATION

LncRNAs participate in a wide range of cellular processes and exert their regulatory functions through diverse molecular mechanisms. Within the nucleus, lncRNAs primarily function by interacting with various protein partners to modify their activity and subsequently modulate gene expression (Figure 1). These interactions influence stem cell fate by shaping transcriptional programs and regulating alternative splicing, thereby playing critical roles in lineage specification and differentiation.

Interactions with transcription factors

The precise initiation of gene expression programs is central to stem cell differentiation. Transcription factors regulate target gene transcription through DNA binding, while recent studies demonstrate that lncRNAs serve as dynamic modulators that coordinate or antagonize transcription factors via spatial conformations or sequence complementarity, thereby reprogramming gene regulatory networks. For example, definitive endoderm-associated lncRNA1 (DEANR1) promotes forkhead box transcription factor A2 (FOXA2) expression through cis-regulation during differentiation of ESCs to endodermal and pancreatic cell lines, with FOXA2 overexpression rescuing differentiation defects caused by DEANR1 depletion (Figure 1A). Mechanistically, DEANR1 activation induces chromatin looping, bringing its locus closer to the FOXA2 promoter, where it facilitates SMAD2/3 recruitment, enhancing FOXA2 activation[7]. Similarly, GATA binding protein 6 (GATA6) antisense RNA 1 regulates GATA6 expression through interaction with SMAD2/3, mediating their binding to the GATA6 promoter region. GATA6 antisense RNA 1 depletion impairs human endoderm differentiation, while GATA6 overexpression rescues this differentiation defect, highlighting its role in embryonic development and lineage commitment[8].

LncRNAs also regulate transcription by competitively binding transcription factor-associated proteins. For instance, RP11-380D23.2 functions as a cis-regulator of paired-like homeodomain transcription factor 2 by competitively binding its potential repressor poly (ADP-ribose) polymerase 1 during distal lung differentiation[9]. Similarly, Linc-Yin Yang 1 (YY1) regulates ectodermal myogenic differentiation by disrupting YY1-histone deacetylase 3-polycomb repressive complex 2 (PRC2) interactions via steric hindrance, redirecting YY1 to myogenic gene promoters, where it cooperates with p300 to activate key differentiation regulators such as myogenin[10].

Epigenetic regulation by lncRNAs through interaction with chromatin modifiers

Beyond directly regulating transcription factor activity, lncRNAs also function as molecular scaffolds to recruit chromatin-modifying complexes to specific genomic loci. A well-characterized example is X inactive-specific transcript, which guides PRC2 to specific chromatin sites, mediating transcriptional silencing by catalyzing H3K27me3[11,12]. This process plays a crucial role in X chromosome inactivation during differentiation. As a key regulator of chromatin architecture, PRC2 modulates gene expression through histone modifications[13]. In addition to X inactive-specific transcript, lncRNAs such as T-UCstem1 and maternally expressed gene 3 (MEG3) exert regulatory functions via PRC2 in pluripotent stem cells (PSCs). T-UCstem1 exhibits dual functionality in ESCs, with its role dependent on subcellular localization. In the nucleus, T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing PRC2 at bivalent domains, whereas in the cytoplasm, it operates through distinct mechanisms that will be discussed later[14]. Similarly, MEG3 exhibits stage-specific regulatory roles during differentiation. As a maternally expressed lncRNA, MEG3 acts as a molecular scaffold, bridging the PRC2-Jumonji AT rich interacting domain 2 (JARID2) complex to epigenetically silence progenitor genes such as paired box protein 6 and iroquois-class homeobox protein 3, thereby guiding motor neuron maturation[15]. In human induced PSCs (iPSCs), MEG3 depletion disrupts the chromatin localization of PRC2, JARID2, and H3K27me3, emphasizing its essential role in the pluripotency-to-differentiation transition[16,17]. In addition to its role in central nervous system differentiation, the lncRNA MEG3 plays a role in the enteric nervous system, which will be discussed later.

LncRNAs can also guide PRC2 or Trithorax group/mixed lineage leukemia complexes to specific genomic loci, modulating histone modification and target gene transcription[18,19]. For instance, during cardiogenesis and body wall development, the lncRNA Fendrr binds both PRC2 and Trithorax group/mixed lineage leukemia complexes, dynamically reshaping chromatin landscapes involved in lateral mesoderm specification and differentiation[20]. Similarly, the human super enhancer-associated lncRNA (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA (CARMEN) governs cardiac specification, differentiation, and homeostasis by directly interacting with PRC2 components enhancer of zeste homolog 2 and suppressor of zeste 12, thereby guiding cardiac lineage commitment (Figure 1B)[21].

Not all lncRNAs function by guiding regulatory complexes to specific loci. Some act as molecular decoys, sequestering chromatin modifiers to regulate gene expression. For instance, PRESS1, a p53-targeted lncRNA highly expressed in human ESCs, binds to the histone deacetylase sirtuin 6, blocking its activity and thereby preserving H3K56ac/H3K9ac levels, which are crucial for maintaining open chromatin states and activating pluripotency-associated genes[22]. Another example is yylncT, a Yin-Yang lncRNA family member. It regulates mesodermal differentiation by interacting with DNMT3B at the BRACHYURY (T) locus, promoting its epigenetic activation and directing human PSCs toward mesodermal commitment[26].

LncRNAs can also fine-tune chromatin modifiers during differentiation, as exemplified by Combat Application Tourniquet Generation 7 (CAT7)[23]. In human ESCs (hESCs), CAT7 plays a pivotal role in dynamically regulating the chromatin-modifying activity of PRC1. CAT7 knockdown disrupts PRC1-mediated gene regulation, leading to aberrant suppression of genes such as motor neuron and pancreas homeobox 1, which are essential for motor neuron differentiation. Notably, this regulatory mechanism is evolutionarily conserved, as the zebrafish cat7L homolog recapitulates CAT7 function[23]. These findings reinforce the notion that lncRNAs modulate PcG binding and function at specific loci[16,24].

LncRNAs also modulate other chromatin-associated epigenetic regulators. The ultraconserved element UC.291 plays a crucial role in cutaneous differentiation by competitively binding to actin-like 6A, a core subunit of the BRG1/BRM-associated factor complex. This interaction impairs BRG1/BRM-associated factor recruitment to chromatin, thereby relieving epigenetic silencing of differentiation-associated genes such as Grainyhead like transcription factor 3 and Kruppel-like factor 4 and further promoting cellular differentiation[25]. There are many other lncRNAs involved in epidermal differentiation, such as H19, and these will be described later.

Regulation of alternative splicing by lncRNAs through splicing factor interactions

In eukaryotic cells, polycistronic transcription and alternative splicing contributes to mRNA and protein diversity, with transcript processing influencing gene function. LncRNAs play a crucial role in this process by regulating transcript maturation and alternative splicing (Figure 1C), thereby shaping gene expression programs and impacting cell differentiation.

Metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) is a ubiquitously expressed nuclear-restricted lncRNAs that has been extensively studied[27]. Its high abundance is attributed to strong promoter activity and enhanced stability of its transcribed RNA[28]. A key function of MALAT1 is in alternative splicing, primarily through interactions with serine/arginine-rich (SR) proteins. It influences the distribution of SR proteins and other splicing factors within nuclear speckle domains, while also modulating SR protein phosphorylation, thereby regulating the alternative splicing of multiple pre-mRNAs[29]. Recent studies have found that MALAT1 also functions as a molecular scaffold, interacting with key proteins to regulate signaling pathways. In multiple myeloma mesenchymal stem cells, it facilitates transforming growth factor-β activation and secretion by recruiting Sp1 to the promoter of latent transforming growth factor-beta-binding protein 3[30]. Additionally, in liver differentiation, MALAT1 forms a lncRNA-protein complex with Smad proteins, SETD2, and protein phosphatase magnesium-dependent 1A, promoting the dephosphorylation of p-Smad 2/3, and thereby terminating transforming growth factor-β/Smad signaling[31]. This highlights MALAT1’s multifunctionality, extending beyond splicing regulation to include epigenetic and signaling pathway modulation.

Pnky is a key regulator of neuronal differentiation and neural stem cell (NSC) migration, operating through a dual-mode mechanism. It interacts with the NSC maintenance factor polypyrimidine tract-binding protein 1 (PTBP1) to modulate the splicing of neurogenesis-associated mRNAs, thereby inhibiting neuronal differentiation[32,33]. Additionally, Pnky interacts with U2AF1, SARNP, Aly/Ref, and THOC7 to couple splicing with mRNA export, which influences NSC migration[34]. This suggests that Pnky actively participates in spliceosome assembly during migration, while modulating splicing factor recruitment via epigenetic regulation during differentiation.

Scaffolding of membrane-less organelles

While many lncRNAs possess protein-binding capabilities, others function as structural RNA molecules, recruiting multiple proteins to form membrane-less organelles that execute specialized nuclear functions. A notable example is nuclear enriched abundant transcript 1 (NEAT1), which assembles paraspeckles by recruiting various paraspeckle-associated proteins, forming a core-shell structure within the nucleus (Figure 1D). Paraspeckles regulate gene expression by restricting the nuclear export of specific RNAs and modulating protein distribution within the nucleus. NEAT1 and paraspeckles are widely present in the nuclei of cultured mammalian cells, but notably absent in human ESCs. Their expression progressively increases during differentiation, and once assembled, paraspeckles influence cell fate by sequestering specific RNAs and proteins.

One key mechanism by which paraspeckles regulate differentiation involves sequestering mRNAs that contain inverted repeated elements in their 3’-untranslated regions[35]. In mouse cells, these elements primarily consist of short interspersed nuclear elements[35], whereas in human cells, they are predominantly Alu retrotransposon elements[36-38]. Given the differential expression of NEAT1 before and after differentiation, mRNAs containing inverted repeats, such as the pluripotency factor Lin28, can be efficiently exported and translated in hESCs, thereby promoting differentiation. Furthermore, the quantitative regulation of NEAT1 transcripts and paraspeckle assembly, along with the sequestration or re-association of paraspeckle components such as CARM1 and TDP-43, are essential regulatory mechanisms in hESC differentiation. CARM1 and TDP-43 are essential regulators of pluripotency-associated genes, including Sox2 and Oct4, highlighting the critical role of NEAT1 and paraspeckles in stem cell fate determination.

CYTOPLASMIC LNCRNAS IN THE REGULATION OF STEM CELL DIFFERENTIATION

LncRNAs exhibit distinct subcellular localization patterns, with a substantial proportion transported to the cytoplasm where they regulate post-transcriptional gene expression. Within this compartment, lncRNAs contribute to the control of stem cell fate by modulating mRNA stability, influencing translational efficiency, and participating in the regulation of PTMs, as illustrated in Figure 1E and F.

LncRNA regulates mRNA turnover

Although lncRNAs do not encode proteins, they can regulate mRNA turnover by interacting with microRNAs (miRNAs) or by directly modulating mRNA translation and stability, thereby influencing stem cell fate and function. MiRNAs comprise a class of small non-coding RNAs with essential roles in the post-transcriptional regulation of gene expression, controlling hESC self-renewal, pluripotency, and differentiation[39,40]. As competitive endogenous RNAs (ceRNAs), lncRNAs sequester miRNAs to modulate their activity and influence gene expression[41,42]. A notable example is the human-specific cardiac regulatory lncRNA heart brake lncRNA 1 (HBL1), a negative regulator of cardiomyocyte differentiation. The transcription factor SOX2 directly binds to the HBL1 promoter and activates its transcription, resulting in high HBL1 expression in undifferentiated human PSCs. Within the RNA-induced silencing complex, HBL1 interacts with AGO2 and miR-1, effectively suppressing miR-1 activity during early cardiomyocyte development. Through this mechanism, HBL1 contributes to the maintenance of pluripotency and prevents premature differentiation[43]. Several other lncRNAs regulate stem cell pluripotency and differentiation through miRNA sponging. For example, the long intergenic non-protein coding RNA regulator of reprogramming (LINC-ROR) maintains ESC pluripotency by sequestering miR-145, thereby relieving its suppression on core pluripotency factors such as Oct4, Nanog, and Sox2[44,45]. Similarly, H19 functions as a ceRNA, binding miR-130b-3p to regulate keratinocyte differentiation, while also interacting with PTBP1 to stabilize sterol regulatory element-binding protein 1c mRNA[46,47]. Other lncRNAs exhibit stage-specific ceRNA activity. T-UCstem1 controls ESC proliferation by modulating the miR-9/Lin28b axis[14], while MEG3 sponges multiple miRNAs, including miR-211-5p and miR-140-5p, to influence neuronal development[48], osteogenesis, and adipogenesis[49]. These findings highlight the widespread role of lncRNAs as ceRNAs, fine-tuning stem cell fate and differentiation through dynamic miRNA sequestration.

In addition, lncRNAs can modulate mRNA stability by recruiting RNA-binding proteins that either promote degradation or enhance stabilization of target transcripts. For example, in the process of epidermal differentiation, terminal differentiation induced noncoding RNA forms a tripartite noncoding RNA-staufen-1-mRNA complex with differentiation-associated transcripts (e.g., keratin 80, ALOX12B), promoting their degradation via the staufen-1-mediated mRNA decay pathway, leading to epidermal terminal differentiation[50]. Moreover, several lncRNAs regulate mRNA stability through PTBP1 recruitment. Anti-differentiation noncoding RNA can act as an RNA scaffold that recruits PTBP1 to ID2 mRNA, enhancing its stability and inhibiting human amniotic mesenchymal stem cell differentiation into definitive endoderm[51]. Similarly, urothelial carcinoma associated 1 regulates heme biosynthesis and erythropoiesis by recruiting PTBP1 to ALAS2 mRNA[52].

There are also some lncRNAs that directly regulate mRNA translation. During myogenesis, the transcription factor MyoD activates downstream myogenic factors to drive muscle differentiation. LncMyoD, a key target of MyoD, competitively binds proliferative gene transcripts (e.g., c-MYC, N-RAS) through insulin-like growth factor 2 mRNA-binding protein. This modulates mRNA translation efficiency, thereby suppressing myoblast differentiation[53]. This MyoD-LncMyoD-insulin-like growth factor 2 mRNA-binding protein axis highlights a translation-based regulatory mechanism in myogenesis. Notably, LncMyoD is evolutionary conserved, exhibiting analogous functions in both humans and mice.

Modulation of PTMs

Beyond mRNA turnover, several lncRNAs can act as molecular scaffolds to recruit PTM enzymes (e.g., methyltransferases, deacetylases, ubiquitin ligases) to target proteins or chromatin regions, dynamically modulating protein modification states to steer stem cell differentiation trajectories (Figure 1F). The lncRNA in dendritic cell (DC) (lnc-DC) is exclusively expressed in human conventional DCs, where it regulates the expression of genes essential for antigen uptake and T cell activation. Mechanistically, during hematopoietic cell differentiation, lnc-DC binds to cytoplasmic signal transducer and activator of transcription 3 (STAT3), preventing its association with the tyrosine phosphatase Src homology region 2 domain containing phosphatase-1 and thereby inhibiting STAT3 dephosphorylation. This interaction enhances STAT3 phosphorylation at tyrosine 705, maintaining its transcriptional activity and downstream signaling. As a result, lnc-DC is critical for proper DC differentiation and immune function[54].

LncRNA-based therapeutic potential

The precise control of stem cell fate remains a central challenge in regenerative medicine. Owing to their multilayered regulatory capacity, lncRNAs have attracted attention as potential therapeutic targets. Although there are currently no clinical or preclinical examples of lncRNA-based therapies specifically applied to stem cell regulation, various strategies have been developed to modulate lncRNA expression or function in cellular systems, animal models, and other disease contexts. These include RNA interference, ASOs, CRISPR-based genome editing, and small molecules that modulate RNA stability or molecular interactions. Collectively, these approaches provide a conceptual and technical foundation for future lncRNA-directed regulation of stem cell differentiation.

Targeted intervention and delivery strategies for lncRNAs

Several effective approaches have been developed to interfere with lncRNA expression at the transcriptional and post-transcriptional levels. CRISPR interference and CRISPR-Cas9 genome editing are widely used in basic research to silence or delete lncRNA loci. However, such interventions may inadvertently disrupt local enhancer elements or chromatin architecture, introducing unpredictable effects at the DNA level. As an alternative, CRISPR-based RNA tethering strategies enable locus-specific recruitment of regulatory RNAs or proteins, promoting enhancer-promoter looping and transcriptional activation. This has been demonstrated with the divergent lncRNA Evx1as, which enhances mesendodermal differentiation through chromatin remodeling[55].

RNA interference-based methods, including short interfering RNAs (siRNAs) and short hairpin RNAs, effectively suppress cytoplasmic lncRNAs via RNA-induced silencing complex-mediated degradation, although their clinical utility is limited by poor in vivo stability and delivery efficiency[56]. In contrast, peptide nucleic acids and ASOs offer improved RNA stability and resistance to nucleases[57,58]. ASOs are widely used and have been clinically validated in targeting disease-associated mRNAs[59]. In murine models, ASO-mediated silencing of MALAT1 effectively suppressed tumor metastasis[60]. GapmeR, a next-generation ASO technology incorporating LNA modifications, further improves binding affinity and stability[61,62]. Notably, GapmeR-mediated knockdown of lincRNA-p21 - a KAP1-associated lncRNA involved in cardiac hypertrophy - attenuated pathological remodeling and showed synergy when combined with siRNAs[63].

In addition to RNA-targeting oligonucleotides, small molecules have emerged as promising tools for modulating lncRNA stability and interactions. For instance, NP-C86 inhibits the interaction between growth arrest-specific transcript 5 lncRNA and up-frameshift-1, repressing up-frameshift-1-mediating degradation of growth arrest-specific transcript 5, causing increased insulin receptor signaling and enhanced glucose uptake in adipocytes[64]. Similarly, high-throughput screening has identified two compounds targeting the MALAT1 triple helix, which significantly reduced MALAT1 levels, downregulated downstream gene expression, and suppressed mammary gland organoid branching[65]. These findings suggest that lncRNA stability and structural integrity can be selectively targeted to modulate biological outcomes.

Effective and tissue-specific delivery remains one of the major obstacles to translating lncRNA-targeted therapies into clinical applications. Conventional lipid-based nanoparticles and polymeric carriers have been widely investigated for siRNA and ASO delivery. For example, lipid-based delivery of receptor-tyrosine-kinase-like orphan receptor-1 antisense 1 siRNA in colorectal cancer suppressed its downstream targets dual-specificity phosphatase 5 and cyclin-dependent kinase-inhibitor 1[66]. However, challenges remain regarding nanoparticle size, surface chemistry, and biocompatibility. Adenoviral vectors and engineered extracellular vesicles, including exosomes, have gained attention for their ability to package and deliver RNA molecules with low immunogenicity. Notably, exosomal delivery of NEAT1 has been shown to promote bone regeneration via immune modulation, while PTENP1-loaded exosomes inhibited bladder cancer progression[67,68]. Although these studies have primarily focused on oncology and immunology, the underlying targeting and delivery strategies may be readily adapted for future therapeutic applications in regenerative medicine.

CONCLUSION

With their spatiotemporally regulated expression and broad functional repertoire, lncRNAs play critical roles in stem cell differentiation through epigenetic, transcriptional, and post-transcriptional mechanisms. While therapeutic strategies targeting lncRNAs - such as ASOs, siRNAs, CRISPR-based tools, and small molecules - have shown promise in cancer and other disease contexts, their application in stem cell-based treatments remains unexplored. This gap reflects both the inherent complexity of lncRNA biology and the technical limitations in selectively and precisely manipulating their function.

This review presents a compartment-specific framework for understanding lncRNA-mediated regulation of stem cell fate, organized by subcellular localization - including nuclear, cytoplasmic, and membrane-less compartments. By mapping these spatially distinct regulatory mechanisms, we provide an integrated view of how lncRNAs influence lineage specification, pluripotency maintenance, and developmental transitions. Moreover, by extending the scope beyond ESCs to include NSCs, mesenchymal stem cells, and lineage-specific progenitors, we highlight the conserved yet context-dependent nature of lncRNA function across diverse stem cell types. This perspective offers a broader, mechanistic basis for future lncRNA-guided interventions in regenerative biology.

Although lncRNA-targeted therapies for stem cell modulation are still in their infancy, a growing arsenal of tools, including CRISPR-based editing, RNA-targeting oligonucleotides, and small molecules, combined with advances in delivery technologies, lays the groundwork for future translation. Several key directions will be essential for progress. First, the challenge of low evolutionary conservation may be addressed through synthetic biology and modular engineering. Second, the development of safe, precise, and efficient delivery systems, such as ligand-targeted nanoparticles and engineered exosomes, will be critical for therapeutic application. Third, the integration of multi-omics technologies, including single-cell RNA sequencing, spatial transcriptomics, and RNA-binding protein interactome mapping, will help decode the dynamic lncRNA networks that shape stem cell differentiation in vivo. Despite these challenges, the ability of lncRNAs to fine-tune gene regulatory networks across multiple layers of control positions them as compelling candidates for next-generation cell fate engineering. With continued advances in RNA biology, delivery systems, and precision editing technologies, lncRNA-based therapeutics may ultimately unlock new frontiers in regenerative medicine.