Functions and mechanisms of circular RNAs in regulating stem cell differentiation

Authors: Zhengjun Lin, Xianzhe Tang, Jia Wan, Xianghong Zhang, Chunfeng Liu, Tang Liu

ABSTRACT

Stem cells are unspecialized cells that can copy themselves and turn into many different adult cell types. Their ability to differentiate is central to normal tissue maintenance, wound repair and regenerative medicine. Circular RNAs (circRNAs) are a recently recognized type of RNA that form closed loops rather than the usual linear chains. They are unusually stable in cells and can influence gene activity in several ways. Over the last decade many studies have found that circRNA levels change when stem cells shift from a stem-like state into specialized cells, and that some circRNAs actively control that process. This review summarizes recent findings on how circRNAs affect the differentiation of multiple stem cell types, what mechanisms are likely involved, and what this could mean for regenerative medicine and stem cell banking.

KEY POINTS (plain language)

• What are circRNAs? Small, stable looped RNAs made from pieces of regular genes. They usually do not make proteins but can bind and regulate other RNAs or proteins.
• How do they work? The best-supported mechanisms: "sponging" microRNAs (miRNAs) so those miRNAs can't block target genes; binding or sequestering RNA-binding proteins (RBPs); acting as scaffolds for protein interactions; and in some cases being translated into small proteins.
• Why this matters for stem cells: circRNA levels change during differentiation of embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs) and muscle stem cells. Some circRNAs help keep stem cells "stem-like," others push them to become bone, fat, nerve or muscle.
• Clinical relevance: circRNAs are promising biomarkers to measure the quality and differentiation potential of stem cells, and manipulating circRNAs may improve stem-cell therapies for bone repair, heart repair, muscle injury and more.

• Introduction — a simple view

Stem cells can renew themselves and become many tissue types. Controlling when they stay stem-like versus when they differentiate is essential for development, healing and preventing disease. Researchers have been mapping the networks that control differentiation for years. One relatively new layer of control involves circular RNAs (circRNAs). CircRNAs are formed when the normal RNA splicing machinery joins the piece at the end of an exon back to an earlier splice site, creating a closed loop. Because they lack the free ends of linear RNAs, circRNAs are more resistant to degradation and can accumulate in cells. They can act locally in the nucleus or in the cytoplasm and can affect gene expression through several mechanisms [7,9,14].

• How circRNAs act (plain language)

• MicroRNA "sponges": many circRNAs have binding sites for microRNAs (small RNAs that normally block gene expression). By binding those microRNAs, circRNAs reduce the microRNAs' ability to repress their target genes. A classic example is CDR1as, which binds many copies of miR-7 [19].
• Protein interactions: some circRNAs bind RNA-binding proteins and change what those proteins do. Others act as scaffolds that bring enzymes and substrates together, changing the activity of signalling pathways [22–24].
• Possible translation: a few circRNAs can be read by ribosomes and produce short proteins; this remains an active research area [25–27].

• circRNAs during stem cell differentiation — what the studies show

High-throughput sequencing and microarray studies have revealed thousands of circRNAs and showed that many change in abundance during stem cell differentiation. Key examples and findings follow, grouped by cell type. (Reference numbers indicate primary studies in the literature.)

3.1 Embryonic stem cells (ESCs)

• Several circRNAs are enriched in undifferentiated human ESCs and appear to help maintain pluripotency (the ability to form many cell types). For example, circBIRC6 and circCORO1C help keep human ESCs in a stem-like state by counteracting miRNAs (miR-34a and miR-145) that push cells to differentiate [51].

3.2 Mesenchymal stem cells (MSCs) — many clinical applications

MSCs (from bone marrow, fat, dental pulp, umbilical cord and other tissues) are widely used in regenerative medicine. Many circRNAs influence whether MSCs turn into bone (osteogenesis), cartilage (chondrogenesis) or fat (adipogenesis):

• Bone marrow MSCs (BMSCs): circDAB1 promotes BMSC proliferation and osteogenesis by sponging miR-1270/miR-944 and raising RBPJ/DAB1 expression [57]. hsacirc0074834 promotes bone formation and blood vessel growth (osteogenesis–angiogenesis coupling) via miR-942-5p/ZEB1-VEGF and helped bone regeneration in animal models [58]. By contrast, CDR1as (which binds miR-7) can shift BMSC fate toward fat and away from bone in steroid-induced osteonecrosis models, showing circRNAs can be involved in disease-related stem cell dysfunction [41]. Other circRNAs (circRNA0006393, hsacirc_0076906) have been linked to osteoporosis and glucocorticoid-induced bone loss, and their manipulation improved bone markers and bone mineral density in experimental models [59,60].

• Adipose-derived MSCs (ADSCs): circFOXP1 and circRNA-23525 (aka circRNA-23,525) support osteogenic differentiation of ADSCs and helped bone regeneration in preclinical studies — relevant for using fat-derived cells to repair bone in patients [66,33]. On the other hand, circH19 seems to block adipocyte differentiation in some settings [71].

• Dental pulp MSCs (DPSCs) and periodontal ligament stem cells (PDLSCs): several circRNAs (circSIPA1L1, circLPAR1, circRNA124534, CDR1as) promote tooth/dentin or periodontal tissue formation by regulating Smad, β‑catenin and RUNX signalling pathways; some of these acted via exosomes (small vesicles) that can transfer circRNAs between cells [74–77,80]. Exosomal circLPAR1 from DPSCs promoted osteogenesis in recipient DPSCs [75].

Clinical context: Many MSC-based therapies rely on robust osteogenesis (bone formation) or angiogenesis (new blood vessels). The circRNAs listed above influence those processes in cell and animal experiments, suggesting two practical applications:

• Biomarkers: circRNA patterns could help assess the quality and differentiation potential of MSCs before and after cryopreservation in a stem cell bank.
• Targets: modifying circRNA levels (for example, increasing circDAB1 or hsacirc0074834) may make MSCs more likely to form bone when used in therapies.

3.3 Muscle stem cells, satellite cells and myoblasts

• circRNAs such as CDR1as can promote muscle differentiation by freeing pro-myogenic genes from microRNA inhibition, and others (circHIPK3, circSVIL, circFGFR2, circSamd4, circTTN) were shown to support myoblast differentiation across species [88,93–99]. Some circRNAs act as inhibitors of myogenesis (circHUWE1, circFoxO3, circLMO7), giving researchers tools to fine-tune muscle regeneration.

3.4 Neural, epidermal and intestinal stem cells

• The brain is rich in circRNAs. Certain circRNAs increase during neural differentiation and affect neuron formation; for example, hsacirc0002468 promoted neuronal differentiation via a miR-561/E2F8 pathway [110].
• In epidermal stem cells, circZNF91 is upregulated during keratinocyte formation and likely influences skin renewal via miR-23b-3p and TGF-β/SMAD signalling [35,105].
• circPan3 in intestinal stem cells promotes self-renewal by stabilizing an interleukin receptor messenger and linking innate immune signals to stem cell maintenance [107].

3.5 Large-scale profiles

• Studies found thousands of circRNAs across different stem cell types. For example, 5,602 circRNAs were identified in hiPSCs and derived cardiomyocytes (320 differed between states) [36]. Hundreds to thousands of circRNAs change during differentiation of BMSCs, ADSCs, DPSCs, myoblasts and other stem cells (see referenced profiling studies) [37–39,42–49].

• Mechanistic themes (simplified)

• circRNA–miRNA–mRNA networks: many circRNAs act as competing endogenous RNAs (ceRNAs) — they reduce miRNA activity and thereby increase expression of important transcription factors (like RUNX2 for bone or MYOD for muscle).
• circRNA–protein interactions: circRNAs can bind RBPs and alter splicing, stability or translation of other RNAs.
• Exosomal delivery: circRNAs packaged in exosomes can move between cells and deliver pro-regenerative signals (e.g., circHIPK3 in exosomes reduced pyroptosis and improved ischemic muscle repair in mice) [113].
• Translation: a small subset of circRNAs can produce short proteins whose functions are still being explored [25–27,28].

• Challenges and limits (what this means for clinical translation)

• Detection and abundance: most circRNAs are much less abundant than the linear RNAs from the same gene, making them harder to detect reliably. Their unique back-spliced junction is the key signature but can be difficult to quantify precisely in low-input clinical samples [111,112].
• Mechanism uncertainty: the popular miRNA sponge explanation fits some circRNAs (like CDR1as) but not all. Many functional claims come from overexpression or knockdown in cell lines — more physiological and in vivo validation is needed.
• Choosing targets: many dysregulated circRNAs have been reported, but identifying which are robust, safe and effective to manipulate for therapy requires larger, well-controlled animal studies and ultimately clinical trials.
• Clinical readiness: only a few circRNAs have been tested in relevant animal repair models (for example, exosomal circHIPK3 in ischemic muscle) [113]. Moving from experiments to approved therapies will require standardized assays, delivery methods, dosing and safety studies.

• Relevance to stem cell banking, longevity and regenerative therapies (practical takeaways)

• Quality control for banked cells: circRNA profiles could become part of potency testing for banked stem cells (cord blood, adipose-derived MSCs, umbilical-cord MSCs). Because circRNAs are stable, they may be especially useful for assessing frozen samples and predicting how well cells will differentiate when used later.
• Personalized regenerative medicine: profiling circRNAs from a donor’s banked cells could help choose or pre-treat cells to favor bone, cartilage, nerve or muscle repair, improving outcomes for aging-related conditions (e.g., osteoporosis, muscle loss, heart disease).
• Extending healthy function: stem cell therapies guided by circRNA knowledge offer a route to restore tissue function during aging. For example, boosting circRNAs that encourage osteogenesis or vascularization could improve bone healing in older patients.
• Therapeutic development: circRNAs or their antagonists (antisense oligonucleotides, small molecules, or exosome-delivered circRNAs) may become tools to enhance stem-cell therapies or to treat diseases caused by faulty stem cell differentiation.

• Conclusions and next steps

Research over the past decade shows circRNAs are abundant, stable and often dynamically regulated during stem cell differentiation across tissues. Several circRNAs have clear effects on osteogenesis, myogenesis, neurogenesis and more, and some improve tissue repair in animals. For clinical translation and for making stem cell banking more effective, the field needs: rigorous, standardized methods to detect and quantify circRNAs in clinical samples; more in vivo validation of candidate circRNAs; and tests that link circRNA patterns in banked cells to therapeutic outcomes. Ultimately, circRNA-based assays or interventions could strengthen quality control for stored stem cells and improve regenerative treatments for aging and injury.

Selected study examples and helpful references (representative studies cited above):

• circBIRC6 maintains pluripotency in human ESCs [51]
• Large circRNA catalogues in hiPSCs and cardiomyocytes (thousands detected) [36]
• circDAB1 promotes BMSC osteogenesis via miR-1270/miR-944 → RBPJ/DAB1 [57]
• hsacirc0074834 promotes osteogenesis and angiogenesis and improved bone repair in vivo [58]
• CDR1as shifts BMSC fate toward adipogenesis in steroid-induced osteonecrosis [41]
• circFOXP1 supports ADSC osteogenesis and bone repair [66]
• circSIPA1L1, circLPAR1 and circRNA124534 drive dental pulp stem cell osteogenesis [74–76]
• Exosomal circHIPK3 protected muscle and aided ischemic repair in mice [113]

(For a full list of studies and review references see the open literature on circRNAs and stem cell differentiation.)

Acknowledgements: This simplified review draws on peer-reviewed research summarized in the literature on circRNAs and stem cell differentiation.

Notes for patients and stem cell bank clients

If you are considering stem cell banking (cord blood, cord tissue, adipose or other sources), this research matters because the molecular quality of stored cells affects future usefulness. In the near future, tests based on circRNA patterns may help labs report how likely banked cells are to become bone, cartilage, nerve or muscle — improving decisions about storage and future use. Current clinical applications of circRNA-based approaches are still at the research stage, but they represent a promising direction for improving regenerative medicine and healthy aging.