부산대학교 도서관

Background Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRS.sup.L274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation. Methods We used CRISPR/Cas9 homology-directed repair to stably integrate MetRS.sup.L274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRS.sup.L274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRS.sup.L274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays. Results Our results showed successful integration of MetRS.sup.L274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRS.sup.L274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells. Conclusions The MetRS.sup.L274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes. Keywords: Mesenchymal stromal cells (MSCs), Secretome, Cell-type specific proteomics, Mixed-culture disease models, Mutant methionyl-tRNA synthetase (MetRS.sup.L274G), Bioorthogonal non-canonical amino acid tagging (BONCAT), Azidonorleucine (ANL), CRISPR/Cas9, Induced-MSCs (iMSCs)
Author(s): Jeremy D. Burgess[sup.1,2,3], Danilyn Amerna[sup.3], Emily S. Norton[sup.1,2,4], Tammee M. Parsons[sup.3], Ralph B. Perkerson[sup.3], Ayman H. Faroqi[sup.1,3], Zbigniew K. Wszolek[sup.5], Hugo Guerrero Cazares[sup.4], Takahisa Kanekiyo[sup.3], Marion Delenclos[sup.3] and Pamela [...]