July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Establishment of non-integrated iPSCs from urine-derived cells of a Chinese patient with X-linked retinoschisis
Author Affiliations & Notes
  • Xin Yan
    Aier School of Ophthalmology, Central South University, Changsha, China
    Aier Eye Institute, Changsha, China
  • Yonglong Guo
    Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, China
  • Shengru Mao
    Aier School of Ophthalmology, Central South University, Changsha, China
    Aier Eye Institute, Changsha, China
  • Yalan Zhou
    Aier School of Ophthalmology, Central South University, Changsha, China
    Aier Eye Institute, Changsha, China
  • Jiansu Chen
    Aier Eye Institute, Changsha, China
    Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, China
  • Shibo Tang
    Aier School of Ophthalmology, Central South University, Changsha, China
    Aier Eye Institute, Changsha, China
  • Footnotes
    Commercial Relationships   Xin Yan, None; Yonglong Guo, None; Shengru Mao, None; Yalan Zhou, None; Jiansu Chen, None; Shibo Tang, None
  • Footnotes
    Support  National Natural Scientific Fund of China (81570876)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2867. doi:https://doi.org/
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      Xin Yan, Yonglong Guo, Shengru Mao, Yalan Zhou, Jiansu Chen, Shibo Tang; Establishment of non-integrated iPSCs from urine-derived cells of a Chinese patient with X-linked retinoschisis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2867. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : X-linked retinoschisis (XLRS) is one of the most severely affected genetic causes of irreversible retinal degeneration in young males. However, the mechanism of XLRS still remains unknown. Here, we generated induced pluripotent stem cells (iPSCs) from a Chinese 10-year-old male with XLRS for future pathological and mechanical study on XLRS.

Methods : The specific mutation of the XLRS patient was confirmed by blood sample for DNA-sequencing. Urine cells were collected by centrifugation (200g, 3min) in isolation medium. After cell culture, urine-derived cells underwent the reprogramming procedure using the Sendai Reprogramming Kit containing four Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc. The single iPSC-like clone was selected for picking and expansion to accomplish the generation of this XLRS patient specific iPSC line. The pluripotency was verified by immunofluorescence staining and embryoid body (EB) assay. The analysis of short tandem repeat (STR) and karyotype were performed.

Results : We confirmed a heterozygous C>T mutation in exon 6 of the RS1 gene (Fig. 1A). Non-integrated iPSC line was generated by Sendai Virus Vectors reprogramming in the urine-derived cells collected from the XLRS patient (Fig. 1B, C). RT-PCR was processed for the up-regulation of endogenous pluripotent genes (Nanog and Sox2, Fig. 2A) and elimination of the exogenous genes (SeV, Klf4 and c-Myc) after passage 20. The pluripotency was further verified by immunofluorescence staining of pluripotency marker (OCT4 and SSEA4, Fig. 2B). Embryoid body (EB) assay showed the ability of differentiation to three germ layers (Fig. 2C). The analyses of STR and karyotype indicated the accordant DNA fingerprinting and normal ploidy.

Conclusions : We generate XLRS-iPSC line from a 10-year-old Chinese male with XLRS. Our study enables to offer a useful resource to investigate pathogenic mechanisms in XLRS, as well as a plentiful source of cells for future individual cell-based therapies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Fig. 1 Mutation of the XLRS patient(A) and morphology of urine cells(B) and XLRS-UiPSCs(C) .

Fig. 1 Mutation of the XLRS patient(A) and morphology of urine cells(B) and XLRS-UiPSCs(C) .

 

Fig. 2 Characterization of XLRS-UiPSC line.

Fig. 2 Characterization of XLRS-UiPSC line.

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