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S.-Y. Chen, S. C. G. Tseng; A New Ex-Vivo Expansion Method of Limbal Epithelial Progenitors by Maintaining Close Contact With Their Niche Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3736.
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© ARVO (1962-2015); The Authors (2016-present)
Since 1997, total of 6 protocols have been practiced to transplant ex vivo expanded human limbal epithelial progenitors derived from a small limbal biopsy to patients with unilateral limbal stem cell deficiency (LSCD). We hypothesize that a more effective protocol can be developed by preserving the close contact between progenitors and their NCs.
Human corneoscleral rim was cut into 12 segments 1mm within and beyond the anatomic limbus. Each segment yielded a limbal cluster after digestion with 10 mg/ml Dispase 16h/4ºC (D) or 1 mg/ml collagenase A 18h/37 ºC (Coll) ± D 2h/37ºC (Coll+D) or trypsin EDTA (Coll-T/E) in SHEM. Each cluster was transferred to a fibronectin-coated plastic (PL) or epithelially denuded AM (dAM). 500 cells from each cluster were seeded on 3T3 fibroblast feeder layers for 14 days. CFE, clonal growth and outgrowth size were assessed. Phenotypes were characterized by immunostaining to pancytokeratin(PCK), vimentin(Vim), N-cadherin(N-cad), p63α, pax6, and K12 and 15.
Vim+/PCK- cells from cytospin was significantly higher in Coll than D (4% vs. 0.3%, n=3, P<0.01); these cells subjacent to the epithelial mass were also Vim+/N-cad+. Coll yielded a significantly higher CFE of holoclones and paraclones than D (4.5 ± 0.4%/3.9 ± 0.3% vs. 0.8 ± 0.2%/1.7 ± 0.2%, respectively, n=3, P<0.01). By D7, Coll reached a monolayer of 19.9 ± 2.7mm in diameter on dAM, much bigger than 5.6 ± 1.1mm on PL (n=6, P<0.001), while peripheral cornea showed negligible outgrowth. The percentage of successful outgrowth with a transplantable size >8 mm in 7 days was significantly higher in Coll (84.2%, n=38) than D (46.1%, n= 26, P <0.01). Importantly, such a high success was significantly affected by subsequent T/E to disrupt the interaction with niche cells in Coll-T/E (11%, n=9, P<0.001), but not by subsequent removal of the remaining basement matrix in Coll+D (72%, n=12, P>0.1). The resultant monolayer had uniformly small cells in both central and peripheral regions, expressed p63α, K15, and pax6 with K12 (by some large superficial cells), and had surprising islands of negative BrdU labeling in the center and the periphery. In contrast, D generated diffuse BrdU labeling.
These results indicate that the collagenase digestion method retains close contact of progenitor cells with subjacent niche cells, and results in a higher success in rapid expansion to a transplantable size on dAM. This model may also be used to study how human limbal epithelial progenitors are modulated by their niche cells.
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