R&D Department, TissueTech, Inc., Ocular Surface Center, and Ocular Surface Research and Education Foundation, Miami, Florida 33173, USA.
Invest Ophthalmol Vis Sci. 2012 Jan 25;53(1):279-86. doi: 10.1167/iovs.11-8441.
Limbal stromal niche cells heterogeneously express embryonic stem cell (SC) markers. This study was conducted to isolate and expand them and to prove that their phenotype is critical for supporting SCs.
Human limbus was isolated by dispase or collagenase. Single cells were seeded on coated, 2D, or 3D Matrigel and were serially passaged in modified embryonic SC medium (MESCM), supplemented hormonal epithelial medium (SHEM), or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (DF) before they were seeded in 3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in 3D Matrigel. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and Western blot; SC clonal growth was measured on 3T3 feeder layers.
Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-cadherin, and CD34 was promoted in MESCM more than SHEM or DF. Reunion of PCK+ and Vim+ cells generated spheres in 3D Matrigel, but spindle cells emerged on 2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which the expression of ESC markers had declined but could be regained after reseeding in 3D Matrigel in MESCM but not in SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63α, less CK12, and more holoclones than those mixed with spindle cells expanded in DF.
Limbal stromal niche cells expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors.
角膜缘基质巢细胞呈不均一性表达胚胎干细胞（SC）标志物。本研究旨在分离和扩增这些细胞，并证明其表型对于支持 SC 至关重要。
通过使用Dispase 或胶原酶分离人角膜缘。单细胞接种于涂有、2D 或 3D Matrigel 的培养板上，并在改良的胚胎干细胞培养基（MESCM）、补充有激素的上皮培养基（SHEM）或含有 10%胎牛血清的 Dulbecco 改良 Eagle 培养基（DF）中连续传代，然后接种于 3D Matrigel。通过在 3D Matrigel 中混合扩增的单细胞和Dispase 分离的上皮细胞来实现球体生长。通过 qRT-PCR、免疫荧光染色和 Western blot 分析 SC 标志物的表达；在 3T3 饲养层上测量 SC 克隆生长。
胶原酶而非 Dispase 分离出了位于下方的间充质细胞，这些细胞在 MESCM 中的 Oct4、Sox2、Nanog、Rex1、SSEA4、N-钙黏蛋白和 CD34 的表达较 SHEM 或 DF 更明显。PCK+和 Vim+细胞的融合在 3D Matrigel 中生成球体，但在 2D 或涂有 Matrigel 的培养板上出现了纺锤形细胞。在涂有 Matrigel 的培养板上连续传代可快速扩增纺锤形细胞，这些细胞中 ESC 标志物的表达下降，但在 MESCM 中重新接种于 3D Matrigel 后可恢复，但在 SHEM 或 DF 中则不能。在 MESCM 中扩增的与纺锤形细胞混合的上皮球体表达更多的 p63α、更少的 CK12 和更多的全克隆，而与在 DF 中扩增的纺锤形细胞混合的上皮球体则表达较少的 p63α、较多的 CK12 和更多的半克隆。
表达 SC 标志物的角膜缘基质巢细胞可被分离和扩增，以防止分化并维持角膜缘上皮祖细胞的克隆生长。
