Induction of pluripotent stem cells by reprogramming human ocular fibroblasts under xeno-free conditions

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.

摘要
研究目的：开发一种高效且无异种成分（xeno-free）的标准眼源性诱导多能干细胞（induced pluripotent stem cell）重编程方案，用于基于诱导多能干细胞的细胞疗法治疗视网膜退行性疾病，并比较动物来源与非动物来源培养体系在诱导多能干细胞生成过程中的相对有效性。
研究方法：采用非整合型病毒，在两种无异种成分培养体系下，将原代培养的人翼状胬肉成纤维细胞与人眼球筋膜囊成纤维细胞诱导为诱导多能干细胞；本研究同时评估了传统非无异种成分重编程体系。通过活细胞染色筛选并计数诱导多能干细胞克隆，评估不同成纤维细胞间以及不同培养体系间的重编程效率。通过聚合酶链式反应（Polymerase Chain Reaction, PCR）、免疫荧光染色（immunofluorescence staining）等一系列实验对诱导多能干细胞进行鉴定表征。
研究结果：利用不同重编程体系成功构建了源自人翼状胬肉成纤维细胞与人眼球筋膜囊成纤维细胞的诱导多能干细胞，上述细胞均表现出诱导多能干细胞的特性。采用细胞疗法体系、传统体系及E6/E8体系的诱导多能干细胞重编程效率分别为0.014%、0.028%及0.001%；而源自人翼状胬肉成纤维细胞与眼球筋膜囊成纤维细胞的诱导多能干细胞采用上述体系时的重编程效率分别为0.018%与0.017%。
研究结论：仙台病毒（Sendai virus）可促进眼部成纤维细胞——包括人翼状胬肉成纤维细胞与人眼球筋膜囊成纤维细胞——的诱导多能干细胞重编程，二者用于诱导多能干细胞重编程均具备安全性与高效性。尽管无异种成分条件下眼部来源诱导多能干细胞的重编程效率未优于传统重编程体系，但当需在无异种成分条件下进行诱导多能干细胞诱导时，细胞疗法体系为重编程方案的优质选择。