A pilot study on indunction and differentiation of human fibroblasts to iPS cells and iPS-RPE cells mediated by retrovirus infection

Authors: Tian Yuanyuan,  Jiang Chao,  Chen Xue,  Ding Sijia,  Xu Min,  Zhao Chen

DOI: 10.3760/cma.j.issn.2095-0160.2016.09.006
Published 2016-09-10
Cite as Chin J Exp Ophthalmol, 2016,34(9): 793-798.

Abstract

Background

Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition, iPS-RPE cells may provide a personalized treatment platform for the patient’s own cells treatment.

Objective

This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4, Sox2, c-Myc and KLF4 genes.

Methods

Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p. S1087L and individual without SNRNP200 p. S1087L mutation, respectively, with the size 2 cm×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cell-conditioned medium to generate and induce iPSCs, and then the iPSCs were identified by morphology, alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method, and iPS-RPE cells were identified by detecting the expression of RPE65, zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).

Results

Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement, and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses, and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation, and the positive expressions of hESC-specific surface antigens including SSEA3, SSEA4, TRA-1-60, TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation, and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65, ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.

Conclusions

Mutation SNRNP200 p. S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.

Key words:

Dermal fibroblasts, human/cytology; induced pluripotent stem cells/cytology; Retinal pigment epithelium cells/cytology; Retinitis pigmentosa/therapy; Cells, cultured; Cell differentiation; Biomarkers

Contributor Information

Tian Yuanyuan
Department of Ophthalmology, Affiliated First Hospital of Nanjing Medical University, Nanjing 210029, China
Jiang Chao
Chen Xue
Ding Sijia
Xu Min
Zhao Chen
(Read 24 times, 1 visits today)
Updated: September 30, 2019 — 9:29 am