Optimizing culture conditions for myogenic differentiation of bovine mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are a promising alternative cell source for cultured meat production, but no robust protocols exist for farm animal MSCs to efficiently induce myogenic differentiation. Differentiation efficiency was evaluated under different conditions: normoxia versus hypoxia, with or without 5-Aza-2′-deoxycytidine (5-Aza) for 24h versus 21 days, horse serum versus fetal bovine serum (FBS), and supplementation with galectin-1 (Gal-1) versus the combination dexamethasone + hydrocortisone. A protocol was established by supplementing 10 µM 5-Aza, 100 nM Gal-1, and 10% FBS in normoxia, as confirmed by increased mRNA levels of MYF5, MYOD1, MRF4 and MYOG , decreased PAX3 , increased desmin, tropomyosin, and MyHC protein levels, and formation of myotube-like structures, as indicated by the presence of multinucleated cells. To study breed effects, bovine MSCs from Holstein Friesian (HF) and Belgian Blue (BB) were myogenically differentiated. Differences were observed with MYOG expression and MyHC expression being higher in BB-MSCs, although fusion into multinucleated myotubes was limited. Additionally, immunophenotypic profiles of HF-MSCs and BB-MSCs were assessed before and after myogenic differentiation. Following myogenic differentiation, significant reduction of MSC markers (CD29, CD44, CD90, and CD34) was observed. Our findings lay the foundation for further studies aimed at optimizing myogenic differentiation of MSCs.