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Establishment of an induced pluripotent stem cell-line from hand osteoarthritic patients

2018; Elsevier BV; Volume: 26; Linguagem: Inglês

10.1016/j.joca.2018.02.598

1522-9653

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,

Pluripotent Stem Cells Research

Purpose: Hand osteoarthritis (OA) is a prevalent disorder that leads to substantial pain and physical disability. Due to de lack of self-healing capacity of articular cartilage, OA is a challenging disease and to date, there is no drug able to cure it. Induced pluripotent stem cells (iPSc) are considered ideal tools for exploring human cell therapies and they have emerged as promising tools for modelling diseases. The advantages of using an established iPSc line are unlimited cell source with regeneration capacity and chondrogenic differentiation potential. However, not many studies generating iPSc from patients with OA have been published yet. Therefore, 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 as well as 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 characterized 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 factors were delivered by using the non-integrating method Sendai virus. Cell lines obtained were clonally expanded over 20 passages and characterized for pluripotency markers by immunohistochemistry and RT-PCR, before and after reprogramming (Fig. 1). 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 showed positivity for alkaline phosphatase activity and pluripotency markers, such as Tra1-81 and Nanog. Molecular analyses showed high relative expression levels of the pluripotency-related genes OCT4, SOX2, NANOG and CRIPTO in the iPSc but not in the 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 hand OA patients and one healthy donor. Acknowledgements: Fundación Española de Reumatología; Instituto de Salud Carlos III (PI17/02197); 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.