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Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage
Published on: August 31, 2022
Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow
Lucas Hidenori Okamura1,2, Paloma Cordero2, Jaime Palomino2
1a Departamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária , Universidade Estadual Paulista "Júlio de Mesquita Filho" , Araçatuba , São Paulo , Brasil.
Abstract:
The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.
Insights
Bovine fetal mesenchymal stem cells (bfMSC) show potential for muscle development. Different protocols using 5-Aza, Galectin-1, or SkGM-2 induced varying degrees of myogenic differentiation, confirmed by muscle regulatory factor gene expression.
Area of Science:
- Stem cell biology
- Developmental biology
- Livestock science
Background:
- The myogenic potential of bovine fetal mesenchymal stem cells (bfMSC) from bone marrow (BM) is largely unexplored.
- Understanding this potential is crucial for myogenesis research and livestock production improvements.
Purpose of the Study:
- To investigate in vitro myogenic differentiation of bfMSC using three distinct protocols.
- To evaluate the effects of 5-Aza-2'-deoxycytidine (5-Aza), Galectin-1 (Gal-1), and SkGM-2 BulletKit on bfMSC differentiation.
Main Methods:
- Isolation of plastic-adherent bfMSC from fetal bovine BM.
- Assessment of post-thaw viability (>85% viability).
- Treatment with varying concentrations of 5-Aza, Gal-1, or SkGM-2, followed by analysis of muscle regulatory factor (MRF) gene expression (MYF5, MYF6, MYOD, DES) and protein detection (DES, MYF5).
Main Results:
- 100 µM 5-Aza significantly increased MRF mRNA levels compared to lower concentrations.
- Specific 5-Aza concentrations modulated MYOD, MYF6, MYF5, and DES mRNA expression over time.
- Gal-1 and SkGM-2 induced sequential MRF gene expression changes, indicative of myogenic progression.
- Immunodetection confirmed the presence of DES and MYF5 proteins in differentiated bfMSC.
Conclusions:
- The evaluated protocols promote myogenic differentiation in bfMSC to a certain extent.
- Changes in MRF gene expression and protein presence confirm partial myogenesis.
- bfMSC possess myogenic potential applicable to regenerative medicine and livestock studies.
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