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Yutong Jing, Yalan Zhou, Shengru Mao, Yini Wang, Shibo Tang, Jiansu Chen; Establishment of non-integrated iPSCs from urine-derived cells of a Chinese family with macular corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4661.
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© ARVO (1962-2015); The Authors (2016-present)
Macular corneal dystrophy (MCD) is an autosomal recessive, which is related to carbohydrate sulphotransferase (CHST6) gene mutations. CHST6 gene mutations lead to abnormal keratin sulfate (KS) synthesis and corneal opacity. However, the mechanism of MCD still is still unclear. Here, we generated induced pluripotent stem cells (iPS) via a non-integrative method using urine-derived cells (UCs) from a Chinese family with MCD (MCD-UiPS) for future pathological and mechanical study.
We chose a Chinese family of three siblings with MCD identified by blood sample for DNA-sequence. Extraction of urine cells from 200ml fresh midstream urine. Reprogramming was performed by plasmids of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. The single iPSC-like clone was selected for picking and expansion. The pluripotency was confirmed by immunofluorescence staining and real time polymerase chain reaction (qPCR).
Slit lamp photograph showed two of macular keratopathy in three siblings with MCD patients (two female) (Fig.1A). We confirmed their CHST6 gene mutations in exon 3 of frameshift deletion/insertion at c.62_63delTinsGA and c.C892T (Fig.1B). Non-integrated MCD-UiPS line was generated by plasmid Vectors reprogramming in the urine-derived cells collected from above MCD patient. The pluripotency was confirmed by immunofluorescence staining of pluripotency marker (OCT4 and SSEA4) (Fig.2A). Real time polymerase chain reaction (qPCR) was further verified that endogeneous pluripotency genes OCT4, SOX2, and Nanog were fully activated, with expression levels comparable to those of the normal human control-iPS cell line (Fig.2B).
The CHST6 gene mutation of c.62_63delTinsGA in exon 3 is first reported for MCD. We generate MCD-iPS line from a Chinese family of three siblings with MCD, which can offers a useful resource to investigate pathogenic mechanisms in MCD, as well as a plentiful source of cells for future individual gene-based therapies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Fig. 1 Slit lamp photographs and DNA sequence
Fig. 2 Characterization of MCD-UiPS cellline
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