To the Editor: Adipose-derived stem cells (ASCs) are promising multipotent stem cells for regeneration of skeletal muscle (SM) tissues.[1] However, the low myogenic differentiation ability limits the application.[2] Therefore, it is necessary to summarize the current differentiation strategy to improve the differentiation efficiency. Traditional methods (biochemical induction) and non-traditional strategies (co-culture, biomechanical approaches, and biomaterial utilization) are commonly used for the differentiation of ASCs into myogenic lineages. Although both strategies can effectively induce differentiation of myogenic cells, cellular unhealthy or immature myogenic differentiation status is still the greatest challenge for researcher. Theoretically, the combination of non-traditional methods and traditional methods has the potential to significantly enhance myogenesis of ASCs with remarkable efficacy, however, evidence is currrently insufficient. This review discusses the traditional and non-traditional strategies, as well as several combination motheds of above strategies, for inducing ASCs differentiation into myogenic cells [Figure 1].

Figure 1:

Overview of strategies on myogenic differentiation of ASCs. The figure was created with BioRender.com. ASCs: Adipose-derived stem cells; ECM: Extracellular matrix; FGF: Fibroblast growth factor; HUVEC: Human umbilical vein endothelial cell; IL: Interleukin; VEGF: Vascular endothelial growth factor.

Traditional strategies for inducing ASCs myogenesis: ASCs can present biochemical features that are consistent with skeletal myogenic cells when exposed to various induction factors. The DNA methylation inhibitor 5-azacytidine is commonly used to induce the differentiation of ASCs into multi-nucleated myogenic cells.[3] The addition of the chemokine interleukin (IL) 6 to culture media promotes the differentiation of ASCs towards the myogenic cell lineage.[4] Fibroblast growth factors (FGFs), such as FGF-8, can significantly enhance differentiation of ASCs into the myogenic lineage.[5] Additionally, H2 was shown to enhance MYOD1 gene expression of ASCs.[6] Moreover, polyhydroxylated fullerenes, such as fullerenol C60, have demonstrated excellent enhancing effects on the expression of myogenic transcription factors (TFs) in human ASCs.[7]

Non-traditional strategies for inducing ASCs myogenesis: ASCs myogenic differentiation is dependent on coupling and precise regulation of various types of SM cells. Several studies have confirmed that ASCs participate in the generation of myotubes in the presence of differentiating myoblasts (C2C12 cells). Milner et al[8] analyzed the expression profiles of muscle-specific genes (myogenin [MyoG] and myosin heavy chain [MHC]) of ASCs co-cultured with C2C12 cells, and detected single, unfused ASCs expressing MHC, indicating that ASCs fuse with myoblasts to form myotubes. Co-culturing ASCs with human umbilical vein endothelial cells (HUVECs) was shown to induce myotube formation and significantly increase MHC mRNA expression of ASCs.[9]

Biophysical stimuli alone can effectively enhance expression of early-stage myogenesis-related genes.[10] In addition, the combination of biochemical factors with mechanical signals is reported to promote myogenic differentiation of ASCs more effectively than biochemical factors or mechanical stimulation alone. For example, ASCs subjected to a dynamic culture with cyclic uniaxial strain (10% at 1 Hz) produced more MHC-positive cells with enhanced gene expression of MYOD1 and MyoG as compared to culture conditions with growth factor supplementation alone.[11] Therefore, cooperative stimulation may be required to induce differentiation of ASCs into myogenic cells and form myotubes.

Studies have showed that the decorin, a component of the extracellular matrix (ECM), can inhibit the activities of myostatin to promote myogenic differentiation of ASCs in vitro.[12] Therefore, study have focused on biomaterials derived from the decellularized extracellular matrix (dECM) for engineering of SM tissues.[2] Abbas et al[9] reported that the physical combination of dECM, vascular endothelial growth factor (VEGF), and HUVECs significantly increased myogenic differentiation of ASCs, indicating that such combinations of biomaterials have potential for engineering of SM tissues.

The material stiffness plays a critical role in regulating myogenic differentiation-related pathways of muscle precursor cells. Similarly, ASCs can also sense the stiffness of the microenvironment and differentiate into functional myotubes on substrates that mimic the ECM of SM.[13] Moreover, mounting biological evidence indicates that alignment of ASCs may promote greater terminal differentiation. Ergene et al[14] found that the acrylamide substrate in an aligned pattern, as opposed to an unpatterned matrix, induced the expression of fusion markers in ASCs and promoted myotube formation.

ASCs are well equipped to sense micro- and nanoscale geometric parameters. Previous study have found that while both aligned nanoscale and microscale topographic features can induce the expression of cytoskeletal proteins and myotube formation, nanoscale fibers are more efficient in inducing myotube formation than microscale fibers as the myotubes formed in nanoscale fibers are much longer.[15] Furthermore, coating the substrates with conductive gold nanoparticles upregulated the expression of myogenic markers, which was attributed to changes in inherent conductivity and subsequent upregulation of myogenesis.[16]

The chemical characteristics of synthetic materials can also affect myogenesis of ASCs. A few specific biomaterials have been shown to induce the maturation phenotype of myogenic cells derived from ASCs. Among the different types of synthetic materials suitable for constructing cell-culture scaffolds, those with biodegradable properties appear particularly attractive. In addition, biodegradable materials can be tailored according to the topographic, porosity, and other parameters to effectively control the cellular responses.[17]

Myogenic cells differentiated from ASCs are important for regeneration of functional SM tissues in the field of muscle tissue engineering. In the micro-environment, cells respond to biochemical stimulation, biomechanical conditions, and biomaterial features. Hence, clarification of the differentiation strategies for ASCs is crucial to gaining a greater appreciation of the differentiation processes and to modify current strategies to control the myogenic differentiation of ASCs.

Funding

This project was supported by the National Natural Science Funder of China (No. 81873939), the Peking University Medicine Sailing Program for Young Scholars’ Scientific & Technological Innovation (NO. BMU2024YFJHPY030), and the Postdoctoral Fellowship Program of CPSF (No. GZC20230151).

Conflicts of interest

None.

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