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

Yunfan Xiong,Ying Liu,Jian Ge

doi:10.5935/0004-2749.20180075

Yunfan Xiong, Ying Liu + Show 1 more

Open Access

https://doi.org/10.5935/0004-2749.20180075

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Abstract

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. 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. 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. 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.

Highlights

In 2007, Takahashi et al successfully reprogrammed human cells into induced pluripotent stem cells[1]. iPSCs can differentiate into various types of terminal cells harboring the same genetic make-up as donor patients. iPSCs can be used for in vitro studies pertaining to the pathogenesis of human diseases and are capable of unlimited in vitro proliferation-the latter characteristic suggesting their potential use in targeted cell transplantation therapies for human patients[2,3]
Primary cultures of human ocular fibroblasts Fibroblasts were successfully obtained from six human pterygium tissues-HPF1-HPF6-and five human Tenon’s capsule tissues-HTF1-HTF5
IPSCs should not, in theory, carry tissue-specific markers unique to terminally differentiated cells, the reprogramming process that encompasses the transition of adult cells to iPSCs resulting in the removal of tissue-specific markers

Summary

Introduction

In 2007, Takahashi et al successfully reprogrammed human cells into induced pluripotent stem cells (iPSCs)(1). iPSCs can differentiate into various types of terminal cells harboring the same genetic make-up as donor patients. iPSCs can be used for in vitro studies pertaining to the pathogenesis of human diseases and are capable of unlimited in vitro proliferation-the latter characteristic suggesting their potential use in targeted cell transplantation therapies for human patients[2,3].before iPSC technology can be adapted for large-scale clinical applications, at least two basic conditions need to be satisfied: first, the induction efficiency of iPSCs needs to be sufficiently high and second, the absolute safety of patients during transplantation procedures must be ensured[4]. The traditional iPSC culture system contains various animal-derived components, including animal-derived bFGF and bovine serum. In the traditional method of culturing iPSCs, an animal-sourced feeder cell layer, such as mouse embryonic fibroblast cells (MEFCs)(1), is required to maintain pluripotency and self-renewal capacity. Non-human sialic acids secreted by feeder cells, when absorbed by stem cells, can cause immunogenicity[6,7]. This phenomenon can cause immune rejection during cell transplantation. To avoid these safety issues, animal-derived components in traditional iPSC culture medium can be replaced by corresponding human or recombinant source components. As part of the present analysis, the relative effec tiveness of animal- and non-animal-derived culture systems in the generation of iPSCs was compared

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