ORIGINAL RESEARCH REPORT Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes Huan Ting Ong, 1,2 Sharon L. Redmond, 1,3 Robert J. Marano, 1,3 Marcus D. Atlas, 1,3 Magnus von Unge, 4,5 Peder Aabel, 4 and Rodney J. Dilley 1,3,6 Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratino- cytes (HaCaT). ADSC conditioned media (CM ADSC ) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CM ADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CM ADSC . Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-a). Several of these may contribute to the activity of conditioned media on the kerati- nocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell–derived approach to TM wound healing. Keywords: gene expression, growth factors, MSC, wound healing Introduction T he skin is a protective barrier against the external environment and is routinely subjected to abrasive or penetrating injuries. Wound healing commences immedi- ately after injury with an orchestrated activation of intracel- lular and intercellular pathways to reestablish tissue integrity and homeostasis [1]. The tympanic membrane is a special- ized skin-like structure, which is protected from casual trauma by its anatomical location deep within the bony ear canal, but it remains subject to injury from air pressure, iat- rogenic or accidental perforation by objects inserted into the ear canal, or most commonly as a consequence of otitis media. The tympanic membrane also has a robust injury re- sponse, but failure to heal occurs in up to 15% of patients, leaving a chronic perforation. The mechanisms of healing and failure-to-heal are of considerable interest, but relatively poorly defined. Unlike cutaneous wounds, which have a source of regenerative tissue at the base [2], TM perforations are full-thickness wounds and rely entirely on tissues at their lateral margin for regenerative responses. TM regeneration is very effective, with up to 90% of TM perforations healing spontaneously without intervention [3,4]. Keratinocytes lead the wound healing process by migrating across the perfora- tion and proliferating to restore the epidermal layer, followed by the underlying granulation tissue and mucosal epithelial tissues to restore the middle and inner layers, respectively [5,6]. Where healing fails, chronic wounds may attract sur- gical treatment, but with increasing knowledge of TM bi- ology it is envisaged that nonsurgical treatment with bioactive molecules may soon prove suitable in many cases [7], thus reducing cost and providing greater access to treatment outside of a surgical facility. A number of molec- ular entities have been tested as suitable treatments, but without a clear choice of factors emerging; research is on- going in this area. 1 Ear Science Institute Australia, Nedlands, Australia. 2 School of Veterinary and Life Sciences, Murdoch University, Murdoch, Australia. 3 Ear Sciences Centre, University of Western Australia, Perth, Australia. 4 Division of Surgery, Akershus University Hospital and University of Oslo, Oslo, Norway. 5 Centre for Clinical Research Va ¨stera ˚s, University of Uppsala, Uppsala, Sweden. 6 Centre for Cell Therapy and Regenerative Medicine, University of Western Australia, Perth, Australia. STEM CELLS AND DEVELOPMENT Volume 00, Number 00, 2017 Ó Mary Ann Liebert, Inc. DOI: 10.1089/scd.2016.0204 1