Investigative Ophthalmology & Visual Science Cover Image for Volume 65, Issue 7
June 2024
Volume 65, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2024
Single-cell RNA sequencing reveals gene expression alterations in limbal epithelial subpopulations of human diabetic corneas
Ruchi Shah; Gianni Carraro; Adam J Poe; Yizhou Wang; Jake Inzalaco; Andrew Fealy; Cynthia Amador; Nagendra Verma; Onkar Sawant; Barry R Stripp; Alexander V Ljubimov; Mehrnoosh Saghizadeh
Author Affiliations & Notes
  • Ruchi Shah
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Gianni Carraro
    Lung Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Adam J Poe
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Yizhou Wang
    Applied Genomics, Computation and Translational Core, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Jake Inzalaco
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Andrew Fealy
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Cynthia Amador
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Nagendra Verma
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Onkar Sawant
    Center for Vision and Eye Banking Research, Eversight, Cleveland, Ohio, United States
  • Barry R Stripp
    Lung Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Alexander V Ljubimov
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Mehrnoosh Saghizadeh
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Ruchi Shah None; Gianni Carraro None; Adam Poe None; Yizhou Wang None; Jake Inzalaco None; Andrew Fealy None; Cynthia Amador None; Nagendra Verma None; Onkar Sawant None; Barry Stripp None; Alexander Ljubimov None; Mehrnoosh Saghizadeh None
  • Footnotes
    Support  NIH R01EY031377; NIH R01EY013431; NIH R01EY025377
Investigative Ophthalmology & Visual Science June 2024, Vol.65, 6172. doi:
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      Ruchi Shah, Gianni Carraro, Adam J Poe, Yizhou Wang, Jake Inzalaco, Andrew Fealy, Cynthia Amador, Nagendra Verma, Onkar Sawant, Barry R Stripp, Alexander V Ljubimov, Mehrnoosh Saghizadeh; Single-cell RNA sequencing reveals gene expression alterations in limbal epithelial subpopulations of human diabetic corneas. Invest. Ophthalmol. Vis. Sci. 2024;65(7):6172.

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Abstract

Purpose : Diabetes causes lasting corneal epithelial alterations such as impaired barrier function, stem cell dysfunction, delayed wound healing. Corneal epithelial homeostasis involves a complex coordination of limbal cell subpopulations. However, the transcriptomic changes in these cell populations in systemic diseases such as diabetes have never been addressed. In this study, we compared gene expression alterations of epithelial cell populations in non-diabetic vs. diabetic corneal epithelium using single-cell RNA sequencing (scRNAseq).

Methods : Central and limbal epithelial cells were isolated from 6 non-diabetic and 6 diabetic human donor corneas and fixed for scRNAseq. The scRNAseq analysis was validated by immunostaining in corneal tissue sections.

Results : scRNAseq revealed 24 cell clusters in diabetic and non-diabetic samples. The clusters were categorized as conjunctival, central corneal and limbal epithelium, limbal stromal cells, stem/progenitor cells and limbal niche/supportive cells. The t-distributed stochastic neighbor embedding (t-SNE) analysis revealed distinct clusters based on top expressed gene markers: KRT7, KRT13, S100A8, LYPD2, and KRT6A for conjunctival cells (CjC); KRT12 and KRT3 for corneal epithelial cells (CEC), ITGB1, GJB2, and CPVL for limbal basal cells (LBC); KRT14, KRT15, and KRT17 for limbal progenitor cells (LPC); MKI67, TOP2A, and BIRC5 for transit amplifying/proliferating cells (TAC); ABCG2, ITGA9, SOX17, SOX18 for limbal epithelial stem cells (LESC); NES and SOX2 for neural crest progenitor cells (NCPC); PDGFRB and LUM for limbal stromal/mesenchymal stem cells (LSC/MSC); MLANA, ABCB5 and TYRP1 for melanocytes (MEL); C1QC for immune/Langerhans cells (IC/LC); and CCL5 for immune cells (IC). scRNAseq revealed altered expression of several genes such as ITGA3, MMP10, WNT5A, NID1 in diabetic corneas, validated at the protein level as previously reported. In the diabetic limbus, we report for the first time significantly altered expression of marker genes GPHA2, IFITM3, and POSTN for LPC; PBK for TAC; SOX10 for MEL; and SOX18 for LESC, which were validated by immunostaining.

Conclusions : scRNAseq has revealed new markers of diabetes-altered genes in the limbal epithelium that may play an important role in regulating corneal homeostasis and wound healing.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Attribution-NonCommercial-NoDerivatives
Copyright © 2025 Association for Research in Vision and Ophthalmology.
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