A New Isolation Method of Human Limbal Progenitor Cells by Maintaining Close Association with Their Niche Cells

Abstract

In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63a + and small in the size (8 mm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin -/p63a -/vimentin + cells with the size as small as 5 mm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serumfree, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.

In Vivo Confocal Microscopic Analysis of Normal Human Anterior Limbal Stroma

“…9,10 MSCs in the limbal stroma are more primitive than those in corneal stroma and support LESCs, demonstrating that these stromal stem cells constitute a component of the limbal-niche (unpublished data). Therefore, it is possible that the hyperreflective structures on IVCM represent the limbal stromal niche.…”

“…Other researchers similarly described high cellularity in the anterior limbal stroma in phalloidin green and propidium iodide stained confocal sections. 19 The cellular components in this region include (i) ABCG2 and PAX6 positive multipotent stromal stem cells, 20 (ii) vimentin positive mesenchymal cells, expressing ESC markers 9 , and (iii) CD34, CD31, Flk-1,VWF positive stromal cells. 12 In another study, we demonstrated the presence of clusters of CD90 and CD105 positive MSCs in the native anterior limbal stroma, along with vimentin positive mature keratocytes distributed singly (unpublished data).…”

“…Recent studies using human cadaver tissue examined the cellular components of the anterior limbal stroma and their role in the maintenance of stemness in the basal epithelium. [9][10][11][12] However, the structure and function of the anterior limbal stroma has not been studied in vivo in normal individuals or patients with LSCD. Hence the former is the objective of the present study using IVCM.…”

Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

“…Finally, Chen SY et al provided evidence, that dissociation of cells from sheets or clusters by incubation in 0.25% trypsin and 1 mM EDTA at 37°C for 15 min may render the isolated cells dependent on 3T3 feeder layers for maintained clonal formation capacity [21].…”

“…ISSN: proliferative potential, and percentage of cells presenting markers associated with stemness [20][21][22][23][24][25][26][27]. While optimal results for clinical purposes reportedly depend on adherence to the above outlined protocol [4], the results from experimental studies may indicate an adverse effect of trypsin-EDTA on viability [25], and on the proliferative potential of the dissociated cells [23,27], and such dissociation may also render the cells dependent on animal feeder cells for proper colony forming efficiency.…”