ABSTRACT
ABSTRACT Purposes: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. Methods: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-integrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. Results: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. Conclusions: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.
RESUMO Objetivos: Desenvolver um protocolo padrão, eficiente e xeno-livre, para a reprogramação de células-tronco pluripotentes induzidas, que possa ser usado durante as terapias de células-tronco pluripotentes induzidas para o tratamento de doenças degenerativas da retina, e comparar a eficácia relativa de sistemas de cultivo de origem animal e de origem não animal na geração de células-tronco pluripotentes induzidas. Métodos: Cultivos primários de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos foram induzidos a células-tronco pluripotentes induzidas usando um vírus não integrado sob dois sistemas xeno-livres; um sistema tradicional de reprogramação não xeno-livre também foi avaliado como parte deste estudo. Os clones de células-tronco pluripotentes induzidas foram selecionados e contados por coloração de células vivas. As eficiências de reprogramação foram avaliadas entre os diferentes fibroblastos e entre os diferentes sistemas de cultivo. Uma série de experimentos, como o PCR e a coloração por imunofluorescência, foram conduzidos para caracterizar as células-tronco pluripotentes induzidas. Resultados: Células-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e fibroblastos da cápsula de Tenon humanos foram estabelecidas com sucesso sob diferentes sistemas de reprogramação e exibiram propriedades de células-tronco pluripotentes induzidas. As eficiências de reprogramação das células-tronco pluripotentes induzidas usando o sistema de terapia celular, o sistema tradicional e o sistema E6/E8 foram 0,014, 0,028% e 0,001%, respectivamente. Além disso, as eficiências de reprogramação de células-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos usando todos os sistemas acima foram de 0,018% e 0,017%, respectivamente. Conclusões: O vírus Sendai pode ser usado para facilitar a reprogramação de fibroblastos oculares pelas células-tronco pluripotentes induzidas. Tanto os fibroblastos de pterígio humano quanto os fibroblastos da cápsula de Tenon humanos são seguros e eficientes para a reprogramação de células-tronco pluripotentes induzidas. Embora as eficiências de reprogramação das células-tronco pluripotentes induzidas de origem ocular sob condições xeno-livres não tenham sido superiores às eficiências observadas para o sistema tradicional de reprogramação, o sistema de reprogramação sistema de terapia celular é uma boa opção para a indução de células-tronco pluripotentes induzidas sob condições xeno-livres.
Subject(s)
Humans , Pterygium/pathology , Cell Culture Techniques/methods , Eye/cytology , Cellular Reprogramming/physiology , Induced Pluripotent Stem Cells/cytology , Fibroblasts/cytology , Cell Differentiation/physiology , Cell TransdifferentiationABSTRACT
PURPOSES: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. METHODS: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-in-tegrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. RESULTS: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. CONCLUSIONS: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.