Epitranscriptomic modifications in mesenchymal stem cell differentiation: advances, mechanistic insights, and beyond

Abstract

RNA modifications, known as the "epitranscriptome", represent a key layer of regulation that influences a wide array of biological processes in mesenchymal stem cells (MSCs). These modifications, catalyzed by specific enzymes, often termed "writers", "readers", and "erasers", can dynamically alter the MSCs' transcriptomic landscape, thereby modulating cell differentiation, proliferation, and responses to environmental cues. These enzymes include members of the classes METTL, IGF2BP, WTAP, YTHD, FTO, NAT, and others. Many of these RNA-modifying agents are active during MSC lineage differentiation. This review provides a comprehensive overview of the current understanding of different RNA modifications in MSCs, their roles in regulating stem cell behavior, and their implications in MSC-based therapies. It delves into how RNA modifications impact MSC biology, the functional significance of individual modifications, and the complex interplay among these modifications. We further discuss how these intricate regulatory mechanisms contribute to the functional diversity of MSCs, and how they might be harnessed for therapeutic applications. The review also highlights current challenges and potential future directions in the study of RNA modifications in MSCs, emphasizing the need for innovative tools to precisely map these modifications and decipher their context-specific effects. Collectively, this work paves the way for a deeper understanding of the role of the epitranscriptome in MSC biology, potentially advancing therapeutic strategies in regenerative medicine and MSC-based therapies.

Fig. 1. Origin, characteristics, therapeutic strategies, and applications of MSCs.

From diverse tissue origins such as bone marrow, blood, umbilical cord, and adipose tissue, MSCs exhibit a spectrum of unique attributes, including exceptional self-renewal capacity, robust proliferation, versatile differentiation potential and complex paracrine functionality, governed by intricate regulatory networks. Additionally, MSCs exhibit tropism towards injured tissues, contributing to the healing of wounds, repair of bone defects, and recovery from myocardial injuries. They also have a potent immunomodulatory capacity, demonstrated through dynamic interactions with a broad array of immune cells.

Fig. 2. Types of RNA modifications and their influential role in RNA metabolism.

A plethora of significant RNA modifications, including m⁶A, m⁷G, m⁵C, m¹A, ac⁴C, and Ψ, each with distinct chemical structures, have been identified. The intricate interplay among “writer”, “eraser”, and “reader” proteins orchestrates the dynamic nature of these modifications. “Writer” proteins add these modifications to target RNA molecules, “reader” proteins recognize and interpret these modified codes, and “eraser” proteins are responsible for the removal of these modifications. These processes collectively regulate various aspects of RNA processing, such as RNA splicing, translation, degradation, and the fine-tuning of RNA expression.

Fig. 3. Influences of RNA modification on osteogenic differentiation of MSCs.

The dynamic nature of RNA methylation, governed by METTL3, METTL14, and WTAP, plays a crucial role in the regulation of mRNA, lncRNA, and miRNA functions. Within this complex network, m⁶A modification initiates a cascade of signaling events, while m⁶A “reader” proteins, specifically YTHDF1 and IGFBP1-3, finely regulate gene expression, RNA stability, and translation efficiency of osteogenesis-associated transcripts. Furthermore, demethylases FTO and ALKBH5 contribute to MSC osteogenesis by orchestrating the demethylation of various RNA targets, which can result in alterations in the expression of critical osteogenic markers, thereby influencing MSC osteogenic fate. Importantly, the ac⁴C “writer” protein NAT10 modulates the trajectory of MSC osteogenesis by precisely controlling the abundance of ac⁴C modifications within mRNA transcripts. These findings highlight the significant role of RNA modification in directing osteogenic differentiation, opening up new possibilities for manipulating MSC fate.

Fig. 4. The pivotal role of RNA modifications in MSC adipogenic differentiation.

The precise regulation of RNA modifications is integral in maintaining the delicate balance between osteogenesis and adipogenesis in MSCs. Key regulators, namely the RNA methylases METTL3, WTAP and NSUN2, modulate this balance by altering m⁶A and m⁵C modifications of mRNA, respectively, hence governing associated RNA processes. In contrast, demethylases FTO and ALKBH5 serve as crucial mediators, directing MSC adipogenic differentiation via coordinated RNA demethylation processes. Moreover, the m⁶A “reader” proteins YTHDF2 and IGF2BP2, alongside the m⁵C modifying enzymes YBX1 and ALYREF, actively participate in refining the adipogenic differentiation of MSCs. By selectively binding to m⁶A or m⁵C sites within mRNA transcripts, these regulatory factors significantly influence transcript stability, translation efficiency, RNA splicing, and the complex regulation of associated RNA and gene expression networks. Overall, these dynamic RNA modifications contribute to the delicate regulation of adipogenic differentiation in MSCs.

Fig. 5. Role of RNA modifications in other lineage-specific differentiations of MSCs.

RNA modifications serve as pivotal regulators in the complex orchestration of chondrogenic, dentinogenic, and vascular smooth muscle lineages, guided by the key role players: m⁶A methylases METTL3, m⁵C methylases NSUN4, and the demethylase FTO, in conjunction with their respective “reader” proteins. METTL3, particularly significant in modulating genes integral to dentin formation such as GDF6 and STC1, is instrumental in preserving mRNA stability. Intriguingly, the enhancement of METTL3 expression leads to increased stability of these dentinogenesis-associated transcripts. Moreover, the upregulation of FTO escalates the demethylation of m⁶A modifications on Nanog mRNA, thereby significantly boosting the differentiation potential of MSCs. These modifying enzymes intricately regulate gene expression, RNA stability, transcriptional efficiency, and other essential RNA processes, providing deeper understanding of the roles these factors play in steering cellular fate.

Fig. 6. RNA modifications and their impact on MSCs proliferation, self-renewal, migration, and senescence.

RNA modifications play an integral role in dictating the biological behaviors of MSCs, including proliferation, self-renewal, migration, and senescence, governed by intricate regulatory machinery involving enzymes like methylases and demethylases. These enzymes precisely modulate m⁶A, ac⁴C, and m⁷G modifications in mRNA, thereby significantly impacting the stability, transcriptional efficiency, and consequent gene expression profiles. Notably, the METTL3/YTHDF2-mediated methylation of Grp78 fosters osteoblast proliferation and differentiation, and the METTL1/WDR4 complex, regulating m⁷G methylation, is essential for embryonic ESC self-renewal and differentiation. Moreover, METTL3 overexpression enhances BMMSC migration via increased m⁶A modification of HDAC5 mRNA and provides a safeguard against premature stem cell senescence by stabilizing MIS12 mRNA transcripts through m⁶A modification. This intricate regulation of RNA modifications, orchestrated by “writer” and “reader” enzymes, profoundly impacts MSC proliferation, self-renewal, migration, and senescence, demonstrating the multifaceted influence of the epitranscriptome on MSC biology.

Fig. 7. Role of RNA modifications in MSC paracrine signaling.

Exosomes derived from MSCs serve as crucial mediators of intercellular communication, leveraging the intricate machinery of “writer”, “eraser”, and “reader” proteins to manage RNA modification processes. These exosomes, filled with RNA-modifying enzymes, demonstrate a notable capacity to finely tune the abundance of RNA transcripts through precise m⁶A modifications. For example, MSC-derived exosomes carry maturely modified miR-34a-5p, a process finely orchestrated by the coordinated action of METTL3 and IGF2BP3, leading to significant alleviation of intestinal ischemia/reperfusion injury. Additionally, exosomes from BMMSCs, enriched with ALKBH5 shRNA, display an extraordinary ability to suppress the malignant behaviors of triple-negative breast cancer cells. Hence, these RNA-modifying enzymes, or their targeting agonists/inhibitors encapsulated within exosomes, emerge as essential orchestrators, effectively guiding immunomodulatory responses, promoting tissue repair, and offering a vast range of promising applications in the realm of cancer therapy.