OP0073 ESTABLISHMENT OF HUMAN INDUCED PLURIPOTENT STEM CELL-LINES (IPSC) FOR IN VITRO MODELLING HAND OSTHEOARTHRITIS

Background Research results in the field of hand OA are currently limited due to the unavailability of tissue samples and lack of animal models replicating the features of the disease in humans. Cellular in vitro models are important tools to elucidate molecular mechanisms and pathways that are involved in hand OA. Specifically, induced pluripotent stem cells (iPSc) are considered ideal tools for this purpose since they allow the use of unlimited cells with chondrogenic differentiation potential. However, there are not studies published generating iPSc from patients with hand OA. Objectives To generate and characterize iPSc-lines from patients with radiographic hand OA and healthy donors, which can be used as cellular models of the disease, for studying the pathogenesis of the disease in vitro and for testing new drugs. Methods Patients with hand OA (non erosive hand OA with right thumb OA) and a healthy control were selected for the study. Using the explant culture technique, fibroblasts from skin biopsies of these patients were isolated. Transcriptional factors Oct4, Sox2, Klf4 and c-Myc were used for the reprogramming process; which was performed by using a non-integrating method, the Sendai virus. Cell lines obtained were morphologically, phenotypically and functionally characterized. To evaluate weather these iPSc lines could be used as cellular model of hand OA, presence of single nucleotide polymorphisms (SNPs) within the genes ALDH1A2 and SMAD3 were studied by Sanger sequencing, before and after reprogramming. Variants rs3204689 and rs12901499 respectively have been associated with severe OA of the hand (Styrkarsdottir et al., 2014; Shu-Thao Gao et al., 2018). Finally, chondrogenic differentiation capacity of the “healthy” and “ill” iPSc-lines was studied by means of histological techniques. Results Fibroblasts were isolated from one patient with radiographic hand OA and one healthy donor. Three weeks after reprogramming, embryonic stem cell-like colonies emerged in culture. These cells showed positivity for alkaline phosphatase activity (fig. 1A) and the pluripotency markers Tra1-81 and Nanog (fig. 1B). Molecular analyses showed high relative expression levels of the pluripotency-related genes OCT4, SOX2, NANOG and CRIPTO in the iPSc. These cells were also able to give rise to cells from the three germ layers (fig. 1C). Indeed, during mesodermal differentiation, spontaneously beating cardiomyocytes were seen in culture. Regarding SNPs studies, cells from the patient with hand OA were homozygous for the at-risk allele in both genes studied, both before and after reprogramming (fig. 1D). The “ill” iPSc-line (MOAFiPS 15/645#7) showed worse chondrogenic differentiation than the “healthy” iPSc-line (NFiPS 15/637#7), as shown by the micromasses collagen and proteoglycan content (fig. 1E). Conclusion The generation of one iPSc-line form patients with hand OA is reported for the first time. The presence of the at-risk alleles within the ALDH1A2 and SMAD3 genes were maintained after fibroblast reprogramming. The iPSC lines obtained shown differences in their chondrogenic differentiation capacity, showing their usefulness to model hand OA in vitro, and to deeper study the role of these genetic variants in the pathogenesis of hand OA. References [1] Styrkarsdottir U, et al. (2014). Nat Genet. 46(5):498-502. [2] Shu-Tao Gao, et al. (2018) Ther Clin Risk Manag. 14: 929–936. Acknowledgement FER; ISCIII (PI17/02197); CIBER-BBN; REDICENT; GPC (Xunta de Galicia); Diputacion Coruna; Xunta de Galicia y Fondo Social Europeo, Servicio de Genetica Hospital Teresa Herrera; Servicio Radiofisica Centro Oncologico de Galicia, Universidade da Coruna. Disclosure of Interests Rocio Castro-Vinuelas: None declared, Clara Sanjurjo-Rodriguez: None declared, Maria Pineiro-Ramil: None declared, Tamara Hermida Gomez: None declared, Isaac Fuentes-Boquete: None declared, Javier de Toro-Santos: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, SILVIA MARIA DIAZ-PRADO: None declared