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Lorraine S. Chua, Hua-Tao Xie, Szu-Yu Chen, Scheffer C. Tseng; A New Method of Isolating and Expanding Human Limbal Stroma Niche Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5113.
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As a niche, the limbal stroma modulates basal epithelial progenitors in the direction favoring stemness. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stem cell (SC) function is controlled. As a first step, we propose to develop a new method of isolating and expanding human limbal stroma niche cells.
Human corneaoscleral rim was cut into 12 segments with each segment yielding a limbal cluster after digestion with 1 mg/ml collagenase A for 18h at 37°C in expansion medium constituting of DMEM/F-12 (1:1) supplemented with 10% knockout serum, 4ng/ml bFGF, 5ug/ml insulin, 5ug/ml transferrin, 5ng/ml sodium selenite, and 10ng/ml hLIF. Such clusters containing the entire limbal basal epithelial progenitors together with their niche cells were rendered into single cells by trypsin and EDTA and seeded on a 5% Matrigel-coated dish at 1x105/cm2. These cells were replated every 5-7 days at a dilution of 1:3. At P4, the expanded cells were seeded at 4x104/cm2 onto 2mm thick gel made by adding 150ml of 50% Matrigel per 8-chamber slide. The resultant phenotype was analyzed by real-time qPCR for the expression of markers such as Nanog, Sox-2, Oct-4, CD34, Rex1, and p63 using total mRNA harvested before the end of each passage.
The collagenase digestion method enriched a population of PCK-/Vim+ non-epithelial cells subjacent to the basement membrane. The single cells gave rise to a monolayer of 1x105 cells at D6 on the Matrigel-coated dish. At D1, spindle cells emerged among small round "epithelial" cells. From P2 onward, almost all cells were uniformly spindle shaped. When re-seeded onto thick Matrigel, P4 cells turned from spindle to dendritic shape at D1 and formed aggregates at D3. Cells in the aggregate were quiescent and non-proliferating. Compared to that of D0 cluster immediately isolated by collagenase, qPCR revealed significantly lower expression of Nanog, Oct-4,Sox-2, and CD34(P<0.01,n=3) at P0, P1, P2 and P3 and a gradual decline of p63 expression which was undetectable by P2. When reseeded onto thick Matrigel, the mRNA expression of Nanog, Oct-4, Sox-2, CD34, and Rex1 in the aggregate significantly increased (P<0.01,n=3, compared to P3 cells). Compared to D0 clusters, expression of CD34 and Rex1 was even higher (P<0.01,n=3) while that of other markers had no statistical difference (P>0.05).
These results collectively indicate that limbal stromal niche cells can be isolated and expanded while maintaining their ESC phenotype markers and can be utilized to study how limbal epithelial SC quiescence, self renewal, and fate decision are controlled
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