title: Spatial distribution of niche and stem cells in ex vivo human limbal cultures.
abstract: Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA indumathi@lvpei.org.
authors.forename: Indumathi
authors.initials: I
authors.lastname: Mariappan
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA.
authors.forename: Santhosh
authors.initials: S
authors.lastname: Kacham
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Purushotham
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA.
authors.forename: Savitri
authors.initials: S
authors.lastname: Maddileti
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA.
authors.forename: Jamila
authors.initials: J
authors.lastname: Siamwala
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, C-TRACER, and Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India; University of Rochester, Rochester, New York, USA.
authors.forename: Virender Singh
authors.initials: VS
authors.lastname: Sangwan
mesh_terms: D000943:Antigens, Differentiation / Q000096:biosynthesis; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism; D008297:Male; D055153:Stem Cell Niche; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation
keywords: Epithelial spirals; Limbal niche; Limbal stem cells; Self-organizing 3D niche