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Szu-Yu Chen, Hua-Tao Xie, Scheffer C. G. Tseng; Optimization of Ex Vivo Outgrowth Containing Limbal Epithelial Stem Cells and Their Niche Cells on Epithelially-denuded AM. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5130.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we have developed a novel method of isolating limbal clusters containing entire limbal basal epithelial progenitors and their closely associated niche cells expressing embryonic stem cell (ESC) markers. Such a limbal cluster generates a circular monolayer outgrowth with a diameter >15 mm in 7 days when cultured in SHEM on epithelially-denuded amniotic membrane (dAM). We would like to optimize such ex vivo expansion by changing the medium components.
1/12 piece of the human corneoscleral ring was cut from 1 mm within and beyond the anatomic limbus. A limbal cluster was obtained from each piece after digestion in 1 mg/ml collagenase A for 18 h at 37ºC, transferred to dAM, and cultured in SHEM (control), ESC medium supplement with or without 40ng/ml bFGF, 10ng/ml EGF, 30ng/ml BMP4 or 4ng/ml bFGF plus 10ng/ml LIF. On Day 8, epithelial sheets were removed by dispase (10ml/ml, 2h) and subjected to real-time qPCR and immunostaining for expression of pan-cytokeratin (PCK), p63α, vimentin (Vim), CD34, Oct4, nanog, SSEA4, Sox2, Rex1, Nestin, and N-cadherin. 500 single cells were seeded on 6-well dish containing MMC-treated 3T3 for 12-14 days before rhodamine B staining.
Compared to dispase-isolated sheets, qPCR revealed that collagenase-isolated clusters contained significantly higher mRNA expression of Vim, CD34, Oct4, nanog, SSEA, Sox2, nestin, and N-cad. Immunostaining further confirmed such positive expression in Vim+/PCK-/p63α- cells. When compare to the control cultured in SHEM, a significant smaller outgrowth in diameter were observed in ESCM (8.6±1.7mm) alone or with bFGF (6.5±2.0mm), EGF (10.8±3.1mm), BMP4 (6.3±1.1mm), or bFGF/LIF (8.1±1.9 mm) (n=3, p<0.01). Cells were smaller and more cuboidal in ESCM than in SHEM. Maintenance of ESC marker expression was achieved by ESCM in either bFGF or BMP4 (n=3, P<0.01). On 3T3, addition of EGF in ESCM yielded monolayers with significantly higher CFU and more holoclones than all other culture media.(n=3, p<0.005).
These data collectively suggest that expression of ESC markers is best preserved by monolayers cultured on dAM in ESCM containing bFGF or BMP4, suggesting SC quiescence is best achieved under BMP4 or bFGF. However, monolayers cultured in ESCM containing EGF yielded more clonal growth on 3T3, suggesting SC renewal is best facilitated by EGF. Further optimization will help engineer a surgical graft containing limbal SCs and their niche cells for treating corneal blindness caused by limbal stem cell deficiency.
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