Abstract
IntroductionSince the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.MethodsA 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.ResultsAfter 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.ConclusionFLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.
Highlights
Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs
IPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and stemness characteristics, which resemble those of ESCs [2,3]. iPSCs may have important potential clinical applications as drug screening platforms, in pathophysiological studies in dishes, and as candidate cell sources for regenerative medicine [4,5,6,7]
OA fibroblast-like synoviocyte (FLS) and rheumatoid arthritis (RA) FLS already expressed Klf4 before reprogramming process started. (B) Immunofluorescence staining against Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 in RA iPSCs
Summary
Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. Namely Oct, Klf, Sox, and c-Myc, were transduced into somatic cells to reprogram and generate iPSCs. Subsequently, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and stemness characteristics, which resemble those of ESCs [2,3]. The iPSCs used in pathophysiological studies in dishes were generated from the primary cells that originated from patients with neurological, hematological, metabolic, cardiovascular, primary immunodeficiency diseases, and so on [5,8,9,10]. These pioneering studies have detected many novel pathophysiological mechanisms, which were impossible to study previously because of the inaccessibility of disease tissues and cells. Patient-specific iPSCs are useful for studying diseases with complex mechanisms, which are affected by several factors that range from the genetic background to environmental modifications
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