Modeling macular corneal dystrophy using corneal organoids from patient derived induced pluripotent stem cells

Abstract

Purpose : Mutations in carbohydrate sulfotransferase 6 (CHST6) gene are the causal factor for macular corneal dystrophy (MCD). Lack of understanding about the molecular mechanisms involved in the disease process has hindered target identification and development of novel therapeutics to address MCD. Hence, we aimed to develop a relevant invitro MCD model using patient derived induced pluripotent stem cells.

Methods : Blood samples were obtained from MCD patients and healthy controls with written informed consent and prior approval of the Institutional Ethics Committee. Sequencing of the CHST6 gene was performed in clinically identified MCD patients. iPSC lines were generated from MCD and non-diseased control lymphocytes from peripheral blood using Sendai virus mediated transduction and reprogramming by Klf4–Oct3/4–Sox2 (KOS), c-Myc, and Klf4. The iPSC lines were characterized by immunofluorescence and qPCR to detect pluripotent markers. Embryoid bodies were generated to evaluate the tri-lineage potential of the iPSCs. Corneal organoids were generated by sequential rounds of directed differentiation from the MCD patient derived and control iPSCs from healthy donors and validated by qPCR and immunofluorescence assays.

Results : Mutations in the exon 3 coding region of the CHST6 gene were identified in the MCD patients. iPSC lines were generated from 3 MCD patients and 2 non-diseased controls. All iPSCs lines expressed significantly higher levels pluripotency markers – SSEA4, Tra-1-60, Nanog and possessed tri-lineage differentiation potential compared to the parental cells. The patient and control iPSCs derived corneal organoids expressed typical corneal cell markers such as vimentin, transforming growth factor induced protein (TGFBI), keratan sulphate and aquaporin 0. We observed dysregulation in the mRNA expression of TGFBI in the patient derived iPSCs compared with the controls. Expression data revealed a fold increase in GRP78/BiP, the key regulator for endoplasmic reticulum (ER) stress in patient derived iPSCs compared with the controls.

Conclusions : Our data from the patient derived iPSC model indicates ER stress to be one of the key pathological drivers of MCD. Furthermore, the corneal organoids derived from the patient iPSCs can be used as a primary 3D model to screen for novel drugs in the management of MCD.

This is a 2020 ARVO Annual Meeting abstract.