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AB0103 Establishment of a human induced pluripotent stem cell-line from patients with hand ostheoarthritis

2018; BMJ; Linguagem: Inglês

10.1136/annrheumdis-2018-eular.4585

1468-2060

Rocío Castro-Viñuelas, Clara Sanjurjo‐Rodríguez, María Piñeiro-Ramil, Tamara Hermida‐Gómez, Isaac Fuentes‐Boquete, Francisco Javier de Toro Santos, Francisco J. Blanco, Silvia Díaz‐Prado,

Biomedical Ethics and Regulation

Background To date, there is no drug able cure such a prevalent disorder as it is hand osteoarthritis (OA). Cell therapies using stem cells have emerged as a promising strategy to explore and develop new treatments. Specifically, induced pluripotent stem cells (iPSc) are considered ideal tools not only for this purpose, but also for modelling the disease. The advantages of using an established iPSc line are unlimited cell source with regeneration capacity and chondrogenic differentiation potential. However, there are not many studies published generating iPSc from patients with hand OA. Objectives The aim of this study has been to generate an iPSc-line from human fibroblasts obtained from patients with radiographic hand OA, which can be useful for drug discovery, disease modelling and regenerative medicine applications. Methods Patients with radiographic hand OA and a healthy control were selected for the study. Using the explant culture technique, fibroblasts from 3 mm skin biopsies of these patients were isolated. These cells were histologically characterised and positivity for fibroblast markers was quantified. These cells were also karyotyped in order to confirm that chromosomal abnormalities did not exist before reprogramming. Four transcriptional factors were used for the reprogramming process: Oct4, Sox2, Klf4 and c-Myc. These were delivered using a non-integrative methodology that involves the use of Sendai virus. Cell lines obtained were clonally expanded over 20 passages and characterised for pluripotency markers by immunohistochemistry and RT-PCR, before and after reprogramming (figure 1). The best two clones of each one of the patients were selected according to the expression of the pluripotency markers to establish the cell-line. Results Cells were isolated from skin biopsies of two patients with radiographic hand OA and one healthy donor. Histological and immunohistochemical analyses showed that the 85%–95% of cells in the culture were fibroblasts, which presented a normal karyotype: 46, XX. Three weeks after reprogramming, embryonic stem cell-like colonies emerged in culture. These cells were positivity stained for alkaline phosphatase activity and the pluripotency markers Tra1–81 and Nanog. Alkaline phosphatase staining shows in blue the colonies correctly reprogrammed. Molecular analyses showed high relative expression levels of the endogenous pluripotency associated genes OCT4, SOX2, KLF4, NANOG and CRIPTO in the iPSc, but not in the no-reprogrammed fibroblasts. No presence of Sendai Virus-related genes was detected. Conclusions Fibroblasts were successfully isolated from all the patients. The reprogramming process using Sendai virus enabled us to generate iPSc from two patients with radiographic hand OA and one healthy donor. Acknowledgements Fundación Española de Reumatología; CIBER-BBN; REDICENT; GPC (Xunta de Galicia); Diputación da Coruña; Xunta de Galicia y Fondo Social Europeo, Servicio de genética del Hospital Teresa Herrera; Servicio de Radiofísica del Centro Oncológico de Galicia, Universidade da Coruña. Disclosure of Interest None declared