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Osteogenic differentiation is crucial in normal bone formation and pathological calcification, such as calcific aortic valve disease (CAVD). Understanding the proteomic and transcriptomic landscapes underlying this differentiation can unveil potential therapeutic targets for CAVD. In this study, we employed RNA sequencing transcriptomics and proteomics on a timsTOF Pro platform to explore the multiomics profiles of valve interstitial cells (VICs) and osteoblasts during osteogenic differentiation. For proteomics, we utilized 3 data acquisition/analysis techniques: data-dependent acquisition (DDA)-parallel accumulation serial fragmentation (PASEF) and data-independent acquisition (DIA)-PASEF with a classic library-based (DIA) and machine learning-based library-free search (DIA-ML). Using RNA sequencing data as a biological reference, we compared these 3 analytical techniques in the context of actual biological experiments. We use this comprehensive dataset to reveal distinct proteomic and transcriptomic profiles between VICs and osteoblasts, highlighting specific biological processes in their osteogenic differentiation pathways. The study identified potential therapeutic targets specific for VICs osteogenic differentiation in CAVD, including the MAOA and ERK1/2 pathway. From a technical perspective, we found that DIA-based methods demonstrate even higher superiority against DDA for more sophisticated human primary cell cultures than it was shown before on HeLa samples. While the classic library-based DIA approach has proved to be a gold standard for shotgun proteomics research, the DIA-ML offers significant advantages with a relatively minor compromise in data reliability, making it the method of choice for routine proteomics., (© The Author(s) 2025. Published by Oxford University Press GigaScience.) more...

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo. [ABSTRACT FROM AUTHOR] more...

Implementation of small-diameter tissue-engineered vascular grafts (TEVGs) into clinical practice is still delayed due to the frequent complications, including thrombosis, aneurysms, neointimal hyperplasia, calcification, atherosclerosis, and infection. Here, we conjugated a vasodilator/platelet inhibitor, iloprost, and an antimicrobial cationic amphiphilic drug, 1,5-bis-(4-tetradecyl-1,4-diazoniabicyclo [2.2.2]octan-1-yl) pentane tetrabromide, to the luminal surface of electrospun poly(ε-caprolactone) (PCL) TEVGs for preventing thrombosis and infection, additionally enveloped such TEVGs into the PCL sheath to preclude aneurysms, and implanted PCLIlo/CAD TEVGs into the ovine carotid artery (n = 12) for 6 months. The primary patency was 50% (6/12 animals). TEVGs were completely replaced with the vascular tissue, free from aneurysms, calcification, atherosclerosis and infection, completely endothelialised, and had clearly distinguishable medial and adventitial layers. Comparative proteomic profiling of TEVGs and contralateral carotid arteries found that TEVGs lacked contractile vascular smooth muscle cell markers, basement membrane components, and proteins mediating antioxidant defense, concurrently showing the protein signatures of upregulated protein synthesis, folding and assembly, enhanced energy metabolism, and macrophage-driven inflammation. Collectively, these results suggested a synchronised replacement of PCL with a newly formed vascular tissue but insufficient compliance of PCLIlo/CAD TEVGs, demanding their testing in the muscular artery position or stimulation of vascular smooth muscle cell specification after the implantation. [ABSTRACT FROM AUTHOR] more...