Chen Szu-Yu, Mahabole Megha, Tseng Scheffer C G
Research and Development Department, Tissue Technology, Incorporated, Miami, FL.
Transl Vis Sci Technol. 2013 Nov;2(7):1. doi: 10.1167/tvst.2.7.1. Epub 2013 Nov 7.
Transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on epithelially denuded amniotic membrane (dAM) in supplemented hormonal epithelial medium (SHEM) is an alternative solution for treating corneal blindness due to limbal stem cell (SC) deficiency. Because the phenotype of limbal niche cells (NCs) is preserved better in serum-free modified embryonic stem cell (ESC) medium (MESCM) than SHEM, we question whether the aforementioned expansion protocol can be further optimized by maintaining limbal NCs using MESCM.
Collagenase-isolated limbal clusters were cultured on dAM in SHEM or MESCM for 8 to 10 days. Epithelial outgrowth sheets removed by dispase were subjected to real-time quantitative polymerase chain reaction (qPCR) and immunostaining for expression of corneal epithelial markers (p63, pax6, and K12) and NC markers (FLK-1, CD34, CD31, PDGFR-B, and -SMA). A total of 1000 single cells were seeded on 6-well dish containing 3T3 feeder layers for 12 to 14 days before rhodamine B staining.
Epithelial outgrowth in SHEM showed a significant loss of corneal SC and ESC markers when compared with freshly collagenase-isolated limbal clusters. Although the epithelial outgrowth was slower in MESCM, epithelial cell size was consistently smaller than that found in SHEM. Furthermore, MESCM maintained a significantly higher percentage of PCK-/ Vim+ cells and exhibited a significant upregulation of NC markers and corneal epithelial SC markers (K15, Bmi-1, and Msi-1) than SHEM. Furthermore, the number of purported holoclones was significantly promoted in MESCM than SHEM.
These data collectively suggest that MESCM can be used to replace SHEM to further promote expansion of LEPC by maintaining limbal native NCs.
Effective ex vivo expansion of limbal epithelial SC is a first and important step toward the success of treating corneal blindness caused by limbal stem cell deficiency and paves the way for future applications in regenerative medicine.
在补充激素的上皮培养基（SHEM）中，将体外扩增的角膜缘上皮祖细胞（LEPCs）移植到上皮剥脱的羊膜（dAM）上，是治疗因角膜缘干细胞（SC）缺乏导致的角膜盲的一种替代解决方案。由于角膜缘生态位细胞（NCs）在无血清改良胚胎干细胞（ESC）培养基（MESCM）中比在SHEM中能更好地保持其表型，我们质疑上述扩增方案是否可以通过使用MESCM维持角膜缘NCs来进一步优化。
用胶原酶分离的角膜缘细胞团在SHEM或MESCM中的dAM上培养8至10天。用dispase去除的上皮生长片进行实时定量聚合酶链反应（qPCR）和免疫染色，以检测角膜上皮标志物（p63、pax6和K12）和NC标志物（FLK-1、CD34、CD31、PDGFR-B和α-SMA）的表达。在含有3T3饲养层的6孔板上接种总共1000个单细胞，培养12至14天，然后进行罗丹明B染色。
与新鲜胶原酶分离的角膜缘细胞团相比，SHEM中的上皮生长显示角膜SC和ESC标志物显著丢失。虽然MESCM中的上皮生长较慢，但上皮细胞大小始终小于SHEM中的。此外，MESCM维持的PCK-/Vim+细胞百分比显著更高，并且与SHEM相比，NC标志物和角膜上皮SC标志物（K15、Bmi-1和Msi-1）显著上调。此外，MESCM中所谓的全克隆数量比SHEM中显著增加。
这些数据共同表明MESCM可用于替代SHEM，通过维持角膜缘天然NCs来进一步促进LEPC的扩增。
角膜缘上皮SC的有效体外扩增是成功治疗因角膜缘干细胞缺乏导致的角膜盲的首要且重要的一步，并为再生医学的未来应用铺平了道路。
