PROTEOMIC CHARACTERIZATION OF HUMAN PLATELET LYSATE BY LABEL-FREE NLC-MS/MS: TOWARDS THE EFFECTORS OF ITS BENEFICIAL EFFECT IN REGENERATIVE MEDICINE

Introduction Hydrogel-forming polymeric scaffolds (HyPS) provides a favourable 3D biomimetic and biodegradable environment for Human Mesenchymal Stromal Cells (hMSCs) [1]. Recently, in HyPS engineered with hMSCs, human platelet lysate (hPL) has been shown as highly effective to stimulate the hMSCs cell-growth and osteo-chondral differentiation [2-4]. The proteins responsible for its beneficial effect have been not yet clarified and will be herein investigated by MS. Methods Quali-quantitative evaluation of hPL (n=4) compared to PlateletsPoor Plasma (PPP)(n=4) was achieved by label-free shotgun proteomics. IgG and albumin depleted and not depleted samples were trypsinized [5] and analysed by nLC-UHRTOF. Data was submitted to PEAKS studio and Mascot. Proteomic images of chondrogenic differentiated hMSCs from bone marrow (BM) and adipose tissue (AT), grown in HyPS for 21 days with hPL, were obtained by RapifleX MALDI Tissue Typer™ [6]. Results About 450 and 570 protein IDs were identified in not depleted and depleted hPL and PPP, respectively. 59 of them resulted specific of hPL and not present in PPP independently from depletion, representing a possible panel of factors involved in cell stimulating effect of this supplement. Relative quantification highlighted 7 and 43 proteins significantly altered in their abundances comparing hPL vs PPP in not depleted and in depleted samples (fold change>1.5; p<0.05; ≥2 unique peptides). 3 of these differences were in common, including a critical proliferation regulator. The functions and networks of the proteins of interest were investigated and compared. In situ proteome evaluation of AT-hMSCs and BM-hMSCs grown in HyPSin vitro models for 21 days in presence of hPL and chodrogenic medium was also provided. Histology evaluation was also performed with H&E. The molecular images of trypsinized samples were co registered with the stained counterparties and co-localization of specific peptide signals with differentiated cells were enlightened. Conclusions Shotgun analysis and label-free quantification of hPL and PPP allowed to ‘proteomically’ characterize the supplement and to highlight possible candidates responsible for the beneficial effect onto the proliferation and differentiation of in vitro models. MS imaging analysis of seeded scaffold permitted also to recognize proteomic profiles specific of chondrogenic differentiated cells for in situ monitoring cell expansion and maturation. Novel Aspect Proteomic characterization of hPL and MS imaging analysis give important information to clarify and monitor the hMSCs chondrogenic differentiation in in vitro HyPS3D model. References 1. Dey K., Agnelli S., et al. Int. J. Polym. Mater. Polym. Biomat. (2018). 2. Orlandi C, et al. Exp. Dermatol. (2018) Mar 31. 3. F. Re et al., Abstract, International Translational and Regenerative Medicine Conference, 25-27 April (2018), Rome 4. F. Re et al., Abstract number: B338, 44th Annual meeting of the European Society for Blood and Marrow Transplantation, 18-21 March (2018), Lisbon. 255 5. Raimondo F, et al. Mol. Biosyst. (2015) Jun;11(6):1708-16. 6. Galli M, et al. Biochim. Biophys. Acta. Jul;1865(7):817-827 (2017) Fundings The research leading to these results has received funding from FAR 2014–2017, Fondazione Gigi & Pupa Ferrari Onlus, UniBs Health & Wealth Project and Fondazione Comunità Bresciana.