(F1117) FUNCTIONAL CHARACTERIZATION OF HUMAN TYMPANIC MEMBRANE-DERIVED PROGENITORS AND THEIR EXTRACELLULAR VESICLES UNDER SERUM-FREE CULTURE CONDITIONS
Professor NITTE (DEEMED TO BE UNIVERSITY) MANGALURU, Karnataka, India
Abstract: The transplantation of stem or progenitor cells derived from human tympanic membrane and their extracellular vesicles (EVs) into damaged sites can stimulate numerous regenerative functions through the secretion of various bioactive molecules. However, currently there is limited data on the cellular and phenotypic properties of progenitors and EVs responsible for regeneration of tympanic membrane. Therefore, the present study investigated the potency features along with markers expression in human tympanic membrane progenitors and their EVs for clinical applications using serum-free media. Tympanic membrane samples were used for establishing the cells in vitro. A plastic adherent progenitor cells displayed a small spindle-shape with enhanced cell number due to increased proliferation, and the growth was higher during culture expansion. The FACS assay determined the varied proportion of cells expressing integrin beta 1, cytokeratin 19 (epidermal/epithelium), nestin, beta III tubulin (neural), and vimentin (fibrous/dermal) and the positively stained cells were thus considered to be potential progenitors in the tympanic membrane. Further, an enriched round and discoid vesicles from the progenitors of tympanic membrane were successfully separated and the results of transmission electron microscopy (TEM) have shown the circular or elliptical vesicles with disc and cup-shaped structures, possessing a diameter about 50-150 nm. Nanoparticle tracking analysis (NTA) suggested the main peaks of EVs around 100 nm. The results of Western blotting showed that three positive protein markers (CD9, CD63, and CD81) were highly expressed and negative protein (Albumin) was very low or absent in EVs. Moreover, progenitors isolated EVs displayed anti-inflammatory and regenerative ability, and promoted angiogenesis and extracellular matrix remodeling, in vitro. In conclusion, tympanic membrane derived clinical-grade progenitors showed their potency features and the growth was relatively higher. Further, the expression of phenotypic markers indicated their plasticity and stemness properties. Morphology, particle size, and labeled protein marker detection confirmed EVs separation from progenitors of tympanic membrane, and provided a basis for investigating their regenerative functions in vivo.
Funding Source: Funded by Nitte (Deemed to be University)
