# Single-cell RNA sequencing reveals novel regulatory pathways in maintaining limbal epithelial stem cell homeostasis

> **NIH NIH R01** · NORTHWESTERN UNIVERSITY · 2022 · $388,000

## Abstract

PROJECT SUMMARY/ABSTRACT
The epithelial stem cells for the corneal epithelium and their immediate progeny, the early TA cells, are
preferentially located in the basal layer of the limbal epithelium and are key for maintaining corneal epithelial
homeostasis. The early TA cells migrate toward central cornea and populate the peripheral corneal epithelial
basal layer. The more mature or late TA cells are believed to reside in the central corneal epithelial basal layer.
This paradigm of corneal epithelial stem/TA cell/post-mitotic cell is over 30 years old and some of the features
of these three populations have been defined. However, due to the heterogeneity of the limbal and peripheral
corneal epithelial basal layer, it has been extremely difficult to isolate the stem/early TA cell population. Thus, it
was technically difficult for the characterization of stem vs early TA versus late TA cells. We have conducted
single cell RNA sequencing and established a single cell transcriptome for discrete limbal/corneal epithelial cell
populations. Unbiased clustering identified stem/early TA, late TA and differentiated epithelial cells. Using this
single cell transcriptome data, we discovered that ID3 was one of the highest expressed genes in stem cell
population. ID3 is a key regulator for maintaining stem cell homeostasis in several non-ocular tissues and not
previously recognized in corneal/limbal epithelium. ID3 proteins were specifically detected in stem cell-enriched
limbal epithelial basal layer and nearly undetectable in corneal epithelium. Depletion of ID3 in limbal epithelium
reduced the numbers of cells expressing putative limbal epithelial stem cell (LESC) markers, suggesting that
ID3 plays a positive role in maintaining LESCs, which will be investigated in Aim1. Our preliminary data also
lead to the idea that LRRK1 is a positive regulator of ID3 in limbal epithelium via targeting CaMKIIb and/or
CTCF (Aim2) and LRRK1/CaMKIIb/CTCF/ID3 axis may help maintain LESCs (Aims1 and 2). To accomplish
these goals, we will capitalize on our ability to modulate LRRK1, ID3, CaMKIIb and CTCF levels in
complimentary model systems that include submerge cultured primary cells, 3-D organotypic raft cultures, and
mice. Dysregulation of ID3 is associated with the pathogenesis of various diseases (e.g., Sjögren's Syndrome).
Knowledge on how ID3 is regulated will translate into a better understanding of the pathogenesis of the
diseases with dysregulation of ID3. Since abnormal LESC homeostasis is involved in various corneal epithelial
disorders (e.g., diabetic cornea), the knowledge gained from this proposal may provide a rationale for
developing novel therapeutics based on modulating the expression of ID3 for treating these cornea diseases
associated with compromised LESCs.

## Key facts

- **NIH application ID:** 10470883
- **Project number:** 5R01EY032922-02
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Han Peng
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $388,000
- **Award type:** 5
- **Project period:** 2021-09-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10470883

## Citation

> US National Institutes of Health, RePORTER application 10470883, Single-cell RNA sequencing reveals novel regulatory pathways in maintaining limbal epithelial stem cell homeostasis (5R01EY032922-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10470883. Licensed CC0.

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