Generation of an Immortalized Porcine ADSC Line and Comparative Analysis of Its Adipogenic Potential = 불멸화된 돼지 지방유래 줄기세포주의 확립과 지방분화능 비교 분석

국문 초록 (Abstract)

현대의 육류 소비의 증가에 따라 환경 오염 등 여러 문제가 대두되고 있으며, 배양육 기술은 이러한 축산업 증가의 여러 문제를 해결할 대안으로 제시되고 있다. 본 연구에서는 돼지 지방 유래 줄기세포(ADSC)의 자발적 불멸화를 통해 불멸화된 돼지 ADSC를 형성하고, 돼지 ADSC와의 차이를 분석하였다. 돼지 ADSC는 장기간의 계대배양에 따른 노화로 인해 증식력과 지방 분화 능력이 감소한다. 반면 불멸화된 돼지 ADSC는 장기간 계대배양에도 증식력이 유지되었으며, 텔로미어 유지 마커의 발현 유지와 노화 관련 마커의 발현 감소, 세포 주기 및 DNA 합성 마커의 발현이 증가하였다. 또한 지방 분화의 양과 크기도 돼지 ADSC와 거의 동일하였으며, 지방 분화 관련 마커의 발현도 비슷하게 유지되었다. 이 연구는 불멸화된 돼지 ADSC가 기존 돼지 ADSC의 노화로 인한 문제점을 해결하여 안정적 세포 및 세포 배양 지방 수급의 잠재력을 가지고 있음을 보여주어 배양육 생산에서 불멸화된 돼지 ADSC가 유망한 시스템으로서의 잠재적 응용 능력이 있음을 보여준다.

다국어 초록 (Multilingual Abstract)

The rapid increase in global meat consumption has contributed to severe environmental challenges associated with livestock production. Cultured meat technology has been introduced as a sustainable alternative to mitigate the ecological burden of traditional meat production systems. In this study, spontaneous immortalized porcine adipose-derived stem cells (ADSCs) were generated, and their biological characteristics were systematically compared with those of primary porcine ADSCs. Primary ADSCs exhibited a decline in proliferation and adipogenic capacity during long-term culture, consistent with cellular aging. In contrast, immortalized porcine ADSCs maintained high proliferative potential over extended passages, accompanied by continuous expression of telomere maintenance genes, reduced expression of senescence- associated markers, and enhanced activation of cell cycle and DNA replication regulators. Additionally, the amount and size of lipid droplets formed during adipogenic differentiation were similar to those observed in primary ADSCs, and the expression levels of adipogenic markers were similarly expressed. These results indicate that spontaneous immortalized porcine ADSCs supress the aging-related constraints of primary ADSCs, offering a consistent and sustainable source of cells and adipose tissue for applications in cultured meat research and production.

목차 (Table of Contents)

• List of Tables ⅲ
• List of Figures ⅳ
• ABSTRACT ⅴ
• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 3
• 2.1. Isolation and culture of porcine adipose derived stem cell (ADSC) ·· 3
• 2.2. Generate and culture of spontaneous Immortalized ADSC 3
• 2.3. Cell Proliferation analysis 4
• 2.4. Cell Counting Kit-8 (CCK-8) assay 4
• 2.5. Immunostaining 5
• 2.6. RNA extraction and qPCR analysis 5
• 2.7. RNA-sequencing 5
• 2.8. Bioinformatics 6
• 2.9. Adipogenic differentiation 7
• 2.10. BODIPY Staining 7
• 2.11. Oil Red O Staining and Quantification 7
• 2.12. Statistical Analysis 8
• Ⅲ. Results 11
• 3.1. Comparison of the proliferation capacity of B-ADSCs and Immortalized
• ADSCs 11
• 3.2. Comparative analysis of gene expression between B-ADSC and B-
• IM8 for qPCR 14
• 3.3. Transcriptional Signatures Underlying the Immortalized State of B-IM8
• Cells 16
• 3.4 Upregulation of Cell Cycle and DNA Replication Pathways Accompanied by Downregulation of Extracellular Signaling in B-IM8 19
• 3.5. Maintenance or Enhancement of Adipogenic Differentiation Capacity in Spontaneous Immortalized ADSCs 21
• Ⅳ. Discussion 24
• References 28
• Abstract (in Korean) 32