곰팡이 유래 다당류 및 p38 MAPK 억제제를 활용한 소 및 염소 근육줄기세포의 배양 효율 증진 및 최적화 = Optimization and Enhancement of Culture Efficiency in Bovine and Goat Satellite Cells Using Fungal-Derived Polysaccharides and p38 MAPK Inhibitors

다국어 초록 (Multilingual Abstract)

To address the decline in proliferative capacity and stemness of muscle stem cells in cultured meat production, this study investigated two optimization strategies: fungal-derived polysaccharides for bovine satellite cells (BSCs) and p38 MAPK inhibition for goat muscle stem cells (GSCs). First, we evaluated the effects of MP15R-polysaccharides (MP15R-P) on BSCs. Treatment with 5 µg/mL MP15R-P significantly enhanced cell viability, migration, and proliferation by promoting S-phase entry. Furthermore, MP15R-P upregulated PAX7 expression, preserving stemness, and improved myogenic differentiation efficiency. Second, for GSCs, we optimized expansion conditions using bFGF and the p38 MAPK inhibitor, SB203580. Long-term culture with 1 µM SB203580 maximized cell yield and maintained PAX7 expression up to passage 9. While high-concentration (10 µM) inhibition reversibly suppressed differentiation, lower concentrations allowed normal myogenesis. These findings demonstrate that utilizing bioactive polysaccharides and modulating p38 signaling are effective strategies to enhance stem cell expansion and differentiation for sustainable cultured meat bioprocessing.

목차 (Table of Contents)

• Ⅰ. BACKGROUND AND LITERATURE REVIEW 1
• Ⅱ. CHAPTER 1: Application of Polysaccharides fromMetarhizium pinghaense for the Expansion of Bovine Satellite Cells in Cultured Meat Production 10
• 2.1 INTRODUCTION 10
• 2.2 MATERIALS AND METHODS 13
• 2.2.1 Animal care 13
• 2.2.2 Bovine muscle stem cells isolation 13
• 2.2.3 Bovine satellite cell culture and differentiation 14
• 2.2.4 Cell proliferation analysis 15
• 2.2.5 Cell migration effect (wound healing assay) 15
• 2.2.6 Cell cycle analysis 15
• 2.2.7 Immunofluorescent analysis of cultured cells 16
• 2.2.8 PAX7+ cells population, fusion index percentage measurement 17
• 2.2.9 Gene expression analysis by qRT-PCR 17
• 2.2.10 Statistical analysis 18
• 2.3 RESULTS 20
• 2.3.1 Evaluation of BSCs viability following polysaccharidetreatment 20
• 2.3.2 Effect of MP15R on long term proliferation and migration of BSCs 22
• 2.3.3 Changes in cell cycle distribution of BSCs in response to MP15R during early passages 26
• 2.3.4 Expression of PAX7 and MYOD1 gene in BSCs cultured with MP15R-P 28
• 2.3.5 Effect of MP15R on the differentiation of BSCs 32
• 2.4 DISCUSSION 36
• Ⅲ. CHAPTER 2: INHIBITION OF P38 MAPK PATHWAY PRESERVES LONG TERM PROLIFERATION AND DOSE DEPENDENT SUPPRESSES DIFFERENTIATION IN GOAT SATELLITE CELLS 39
• 3.1 INTRODUCTION 39
• 3.2 MATERIAL AND METHODS 41
• 3.2.1 Goat muscle stem cells isolation 41
• 3.2.2 Goat satellite cell culture and differentiation 42
• 3.2.3 Immunofluorescence staining goat satellite cells 43
• 3.2.4 Quantification of myogenic markers and fusion index 43
• 3.2.5 Total RNA extraction and qPCR 44
• 3.2.6 Assessment of long term proliferation kinetics 45
• 3.2.7 Statistical analysis 45
• 3.3 RESULTS 47
• 3.3.1 Effects of SB203580 on the proliferation and maintenance of goat satellite cells 47
• 3.3.2 SB203580 promotes the long term proliferative potential of goat satellite cells (GSCs) in bFGF supplemented medium 52
• 3.3.3 Long term treatment with SB203580 preserves the PAX7+ stem cell pool during extended subculture 57
• 3.3.4 SB203580 Modulates Myogenic Differentiation of Goat Satellite Cells in a Concentration Dependent Manner 61
• 3.4 DISCUSSION 68
• Ⅳ. GENERAL CONCLUSION 71
• Ⅴ. REFERENCES 72
• Ⅵ. ABSTRACT(KOREAN) 84
• Ⅶ. ACKNOWLEDGMENTS 85