Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing DOI Creative Commons
Meri Vattulainen, Jos G.A. Smits, Dulce Lima Cunha

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 24, 2023

Summary A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for development hPSC-based therapeutics. In this study, single-cell RNA-sequencing (scRNA-seq) was performed to decipher heterogeneity during three hPSC lines towards corneal limbal cells (LSCs). The scRNA-seq data revealed nine clusters encompassing entire process, among which five followed anticipated path LSCs. remaining four were previously undescribed states that annotated either mesodermal-like or undifferentiated subpopulations, and their prevalence line-dependent. Distinct cluster-specific marker genes identified in study confirmed by immunofluorescence analysis employed purify hPSC-derived LSCs, effectively minimized variation line-dependent efficiency. summary, offered molecular insights into hPSC-LSC differentiation, allowing data-driven strategy consistent robust generation essential future advancement toward clinical translation. Highlights hPSCs LSCs spans epithelial, mesodermal, states. reveals heterogeneity. ITGA6 AREG can be used select pure LSC-like subpopulation.

Language: Английский

Identification of the regulatory circuit governing corneal epithelial fate determination and disease DOI Creative Commons
Jos G.A. Smits, Dulce Lima Cunha,

Maryam Amini

et al.

PLoS Biology, Journal Year: 2023, Volume and Issue: 21(10), P. e3002336 - e3002336

Published: Oct. 19, 2023

The transparent corneal epithelium in the eye is maintained through homeostasis regulated by limbal stem cells (LSCs), while nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, precise cell fates of these two types epithelial cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis human LSCs from cornea and characterized molecular signatures, highlighting similarities differences. Through gene regulatory network analyses, we identified shared type-specific transcription factors (TFs) that define specific established hierarchy. Single-cell RNA-seq (scRNA-seq) analyses confirmed TFs. Notably, LSC-specific TFs can cooperatively target genes associated with opacity. Importantly, discovered FOSL2, direct PAX6 gene, novel candidate opacity, it regulates implicated diseases. By characterizing our study unveils circuitry governing LSC fate its association

Language: Английский

Citations

12
Effects of miR-204-5p modulation on PAX6 regulation and corneal inflammation DOI Creative Commons
Mojdeh Abbasi,

Maryam Amini,

Petros Moustardas

et al.

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: Nov. 2, 2024

Abstract Congenital aniridia is a rare eye disease characterized by loss of PAX6 protein leading to aniridia-associated keratopathy that significantly reduces vision. The miR-204-5p possible regulator function and here we evaluate its effect in multiple vitro vivo models. In vitro, overexpression suppressed vascular factor ANGPT1 human limbal stem cells (T-LSC) Pax6-knockdown LSC (mut-LSC), primary epithelial (LEC) at the gene levels following LPS stimulation. However, inhibited VEGFA expression only mut-LSCs LPS-stimulated LEC. Also, increased mut-LSC differentiated corneal cells, but not Topical after LPS-induced keratitis mice failed suppress Vegfa , Angpt1 Il-1β Tnf-α or rescue Pax6 contrast results, although it reduced inflammatory infiltrate cornea. Sey/+ mice, did inhibit ERK1/2 pathway. While short-term treatment effectively suppresses enhances epithelia, effects are variable across immortalized cells. Effects longer-term treatment, however, require further study.

Language: Английский

Citations

4
Network analysis reveals potential mechanisms that determine the cellular identity of keratinocytes and corneal epithelial cells through the Hox/Gtl2-Dio3 miRNA axis DOI Creative Commons
Yanjie Guo,

Weini Wu,

Haoyu Chen

et al.

Frontiers in Cell and Developmental Biology, Journal Year: 2025, Volume and Issue: 13

Published: Jan. 17, 2025

During embryonic development, both corneal epithelial cells (CECs) and keratinocytes (KCs) originate from the surface ectoderm. As a result of this shared origin, may exhibit same characteristics as skin epidermis in pathological situations, while are ideal seed for tissue-engineered corneas. However, how identities determined is currently unclear. In study, to investigate molecular mechanisms determining identity cells, small RNA mRNA sequencing analyses these two cell types were performed. Analysis data revealed that almost all miRNAs Gtl2-Dio3 imprinting region highly expressed accounted 30% differentially (DEMs). Since genes form long polycistronic under control Gtl2 promoter, we next examined expression transcription factors their binding near locus. The findings indicated homeobox family dominated factors, Hox silenced cells. Transcription site prediction ChIP-seq proteins Gtl-Dio3 miRNA target mainly regulate Wnt signaling pathway PI3K-Akt pathway. crucial Pax6, Otx2, Foxc1, also targets miRNAs. Our study potential determine cellular through Hox/Gtl2-Dio3 axis, which provides new perspective understanding developmental regulation opacity, well establishing groundwork promoting transdifferentiation into

Language: Английский

Citations

0
Effect of MiRNA 204-5P Mimics and Lipopolysaccharide-Induced Inflammation on Transcription Factor Levels, Cell Maintenance, and Retinoic Acid Signaling in Primary Limbal Epithelial Cells DOI Open Access

Maryam Amini,

Tanja Stachon, Shao‐Lun Hsu

et al.

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(8), P. 3809 - 3809

Published: April 17, 2025

MicroRNA-204-5p (miR-204-5p) is a critical regulator of differentiation, structural maintenance, and inflammation in limbal epithelial cells (LECs). This study examined the role miR-204-5p modulating gene expression related to transcription factors, cell structure, extracellular matrix remodeling, retinoic acid signaling under normal lipopolysaccharide (LPS)-induced inflammatory conditions. Using qPCR, we analyzed mRNA levels FOSL2, FOXC1, Meis2, PPARγ, ABCG2, PTGES2, IL-1β, IL-6, KRT3, KRT12, MMP2, MMP9, RARA, RARB, RXRA, RXRB, CRABP2, RBP1, RDH10, ADH7, ADH1A1, FABP5, CYP1B1, CYP26A1, while changes protein were assessed via Western blot or ELISA. Our data revealed that overexpression reduced RDH10 conditions (p ≤ 0.039). Additionally, it decreased FOSL2 RXRA = 0.006, p 0.011) KRT3 FABP5 0.010, 0.001). The IL-6 was significantly increased following LPS treatment overexpressing 0.029). A analysis significant reductions FOXC1 miR-204-5p-transfected during LPS-induced 0.020, 0.030). These findings suggest modulates genes migration, response LECs. modulation by highlights these proteins as novel targets

Language: Английский

Citations

0
S100A9-TLR4 axis aggravates dry eye through the blockage of autophagy DOI
Lihong Liang,

Xue Yang,

Hao Zeng

et al.

Experimental Eye Research, Journal Year: 2024, Volume and Issue: 247, P. 110052 - 110052

Published: Aug. 14, 2024

Language: Английский

Citations

3
Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing DOI Creative Commons

Di Sun,

Xiaowen Zhang, Rong Chen

et al.

Stem Cell Research & Therapy, Journal Year: 2024, Volume and Issue: 15(1)

Published: July 6, 2024

Abstract Background Dysfunction or deficiency of corneal epithelium results in vision impairment blindness severe cases. The rapid and effective regeneration epithelial cells relies on the limbal stem (LSCs). However, molecular functional responses LSCs their niche to injury remain elusive. Methods Single-cell RNA sequencing was performed tissues from normal mice defect models. Bioinformatics analysis confirm distinct characteristics cell fates LSCs. Knockdown Creb5 OSM treatment experiment were determine roles wound healing. Results Our data defined signatures reconstructed pseudotime trajectory cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, identified a transcription factor Creb5, expressed significantly upregulated after injury. Loss-of-function experiments revealed silencing delayed healing mobilization. Through cell–cell communication analysis, we 609 candidate regeneration-associated ligand-receptor interaction pairs between cells, discovered unique subset Arg1 + macrophages infiltrated injury, which present as source Oncostatin M (OSM), an IL-6 family cytokine, demonstrated effectively accelerate Conclusions This research provides valuable single-cell resource reference for discovery mechanisms potential clinical interventions aimed at ocular surface reconstruction.

Language: Английский

Citations

1
Effect of Ritanserin and Duloxetine on the Gene Expression of Primary Aniridia and Healthy Human Limbal Stromal Cells, In Vitro DOI Creative Commons
Zhen Li, Nóra Szentmáry, Fabian N. Fries

et al.

Ophthalmology and Therapy, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 21, 2024

Language: Английский

Citations

1
The Effect of Lens Properties on Visual Acuity, Aniridia Associated Keratopathy and Secondary Glaucoma in Congenital Aniridia Subjects DOI
Annamária Náray, Fabian N. Fries, Cristian Munteanu

et al.

Current Eye Research, Journal Year: 2024, Volume and Issue: unknown, P. 1 - 13

Published: Dec. 17, 2024

The potential risks and benefits of cataract surgery, in context congenital aniridia (CA), are not widely understood, yet. Our purpose was to investigate the effect lens properties on visual acuity (VA), aniridia-associated keratopathy (AAK) stage presence glaucoma at Homburg Aniridia Center.

Language: Английский

Citations

1
Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA sequencing DOI Creative Commons
Meri Vattulainen,

Jos G.A. Smits,

J.A. Arts

et al.

Stem Cell Reports, Journal Year: 2024, Volume and Issue: 19(7), P. 1010 - 1023

Published: June 27, 2024

A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for development hPSC-based therapeutics. In this study, single-cell RNA sequencing (scRNA-seq) was performed to decipher heterogeneity during three hPSC lines toward corneal limbal cells (LSCs). The scRNA-seq data revealed nine clusters encompassing entire process, among which five followed anticipated path LSCs. remaining four were previously undescribed states that annotated either mesodermal-like or undifferentiated subpopulations, and their prevalence line dependent. Distinct cluster-specific marker genes identified in study confirmed by immunofluorescence analysis employed purify hPSC-derived LSCs, effectively minimized variation line-dependent efficiency. summary, offered molecular insights into hPSC-LSC differentiation, allowing data-driven strategy consistent robust generation essential future advancement clinical translation.

Language: Английский

Citations

0
Single-cell RNA sequencing of murine limbal epithelia reveals Gas1 as a novel stem/progenitor cell marker for the corneal epithelium DOI
Alexander Richardson, Susan Corley, Naomi C. Delic

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 16, 2024

Abstract The corneal epithelium is endowed with a rare population of stem cells that reside within the limbus, circumferential transition zone partitions cornea from conjunctiva, thus referred to as limbal epithelial (LESC). Despite surge in investigations using single-cell RNA sequencing (scRNA-seq) ocular surface, unifying marker(s) distinguish these their progeny yet be identified. We used keratin (K)-14-driven lineage-tracing system and SmartSeq-2 transcriptomics 5-60-week-old mice interrogate identity epithelia. Four cell clusters were identified, derived both Confetti+ Confetti− (clusters 0–3), cluster 3 designated harbor progenitor cells. found one gene interest 3, growth arrest-specific 1 (Gas1) coding for cell-surface protein. PCR, flow cytometry immunofluorescence disclosed this rarely expressed Gas1 was also co-expressed K14 young old upregulated following mild mechanical debridement injury central cornea. expression antigen can identify, extract enrich downstream molecular generating better-quality cell-based grafts treat severe disease.

Language: Английский

Citations

0