METHODS Three different protocols were tested: ▪Horse constitutes an important veterinary patient and a relevant animal model owed to their physiopathological analogies with human. ▪MSCs are promising therapy for several diseases, including musculoskeletal disorders. ▪However, MSCs present several drawbacks → senescence in culture and require repeated tissue harvesting: more heterogeneity and invasive procedures. ▪To increase MSC availability and reduce invasive procedures, it is possible to derive MSCs from iPSCs → iMSCs. ▪While human iMSCs are being intensively studied, only two equine studies have reported the generation of eiMSCs to date (Lepage et al., 2016 and Chung et al., 2019). INTRODUCTION Derivation of equine induced Mesenchymal Stem Cells (eiMSCs) from equine induced Pluripotent Stem Cells (eiPSCs): a promising step for veterinary and translational regenerative medicine María Belén Serrano1, Elvira Bernad1, Alina Cequier1,2, Francisco José Vázquez1,2, Antonio Romero1,2, Arantza Vitoria1,2, Pilar Zaragoza1, Clementina Rodellar1, Laura Barrachina1,2 (1) Laboratorio de Genética Bioquímica LAGENBIO – Instituto Agroalimentario de Aragón – IA2 (Universidad de Zaragoza – CITA) – Instituto de Investigación Sanitaria de Aragón (IIS), Zaragoza, Spain (2) Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, Zaragoza, Spain Contact: lbarrach@unizar.es RESULTS ➢Deriving eiMSCs from eiPSCs is feasible and these could be established as an interesting alternative to eMSCs. ➢We have detected some differences in eiMSCs among lines and compared to eMSCs, such as immunophenotype and their differentiation potential, mainly for chondrogenesis. However, variability of eMSCs has also been noted among tissue sources and between individuals. ➢Further studies are needed to analyse eiMSCs properties and immune function to improve cell therapies under One Medicine perspective. CONCLUSIONS Cell culture Spontaneous differentiation Directed differentiation ABBREVIATIONS: DMEM LG = Dulbecco`s Modified Eagle`s Medium Low Glucose; FBS = Fetal Bovine Serum; L-glut = L-glutamine; S = Streptomycin; P = Penicillin; VTN = Vitronectin; SB431542 = TGF-β receptor kinase inhibitor; bFGF = basic Fibroblast Growth Factor; eiNCCs = equine induced Neuro Crestal Cells; CDM = Chemically Defined Medium; CHIR99021 = GSK-3α/β inhibitor; AF = Attachment Factor; MEFs = Mouse Embryonic Fibroblasts; EGF = Epidermal Growth Factor. To obtain eiMSCs and compare their main characterization criteria over primary equine MSCs (eMSCs). OBJECTIVE PROTOCOL 3. Differentiation via neural crest pathway →3 stages PROTOCOL 1. Switch to MSC media Chung et al., 2019 PROTOCOL 2. Switch to specific differentiation media Lepage et al., 2016 Immunophenotype (flow cytometry) Trilineage differentiation Gene expression (qPCR) PID2020-116352GB-I00 The first 2 passages →VTN; next passages →tissue-treated culture plate DMEM LG 10% FBS 2mM L-glut 0.1 mg/mL S 100 U/mL P 10 µM SB431542 5 ng/mL bFGF The first 2 passages →VTN; next passages →tissue-treated culture plate The first 10 days of culture DMEM LG 10% FBS 2mM L-glut 0.1 mg/mL S 100 U/mL P 20 ng/mL bFGF The first 2 passages →VTN; next passages →tissue-treated culture plate CDM 10 µM SB431542 1 µM CHIR99021 VTN; 4 passages AF + MEFs; 10 days CDM 10 µM SB431542 20 ng/mL bFGF 20 ng/mL EGF 1. eiNCCs induction 2. eiNCCs expansion 3. eiMSCs induction Ctrl C A O Ctrl = control culture A = Adipogenic differentiation C = Chondrogenic differentiation O = Osteogenic differentiation Ctrl C A O eiMSCs line 1 eiMSCs line 3 eiMSCs line 2 Ctrl C A O Reference sample →each eiPSCs line PROTOCOL 1 eiPSCs eiPSCs PROTOCOL 2 1. eiNCCs induction 2. eiNCCs expansion 3. eiMSCs induction PROTOCOL 3 eiPSCs line 1 eiPSCs line 2 eiPSCs line 3 eiMSCs line 1 eiMSCs line 2 eiMSCs line 3 Fukuta et al., 2014; Chijimatsu et al., 2017; Nakamura et al., 2021; Kamiya et al., 2022 Bone marrow eMSCs DMEM LG 10% FBS 2mM L-glut 0.1 mg/mL S 100 U/mL P C