Ocular Tissue Engineering Research Center, Shahid Beheshti University of medical sciences, Tehran, Iran.
Sabzevar University of Medical Sciences, Sabzevar, Iran.
J Cell Biochem. 2018 Mar;119(3):2666-2678. doi: 10.1002/jcb.26434. Epub 2017 Nov 30.
Ex vivo culture of limbal stem cells (LSCs) is a current promising approach for reconstruction of the ocular surface. In this context, 3T3 feeder layer cells (mouse embryo fibroblast) are generally utilized to maintain and expand LSCs. The aim of this study is to develop a novel culture method (animal-derived products free) to expand LSCs, using umbilical cord derived human unrestricted somatic stem cells (hUSSCs) instead of 3T3 cell with an emphasis on maintaining of the Stemness in LSCs. Using flow-cytometer, isolated hUSSCs were characterized for CD105, CD90, CD166, CD34, CD45, CD31 cell surface markers and their differentiation capability into adipogenic as well as osteogenic lineages were evaluated. In addition to colony-forming efficiency (CFE), epithelial lineage differentiation and karyotyping, LSC properties were evaluated for ABCG2, ΔNP63-α, CK19, CK3, and CK12 mRNA and protein expressions using quantitative RT-PCR (qRT-PCR) and immunocytochemistry, when these cells were co-cultured with hUSSCs (in comparison with 3T3 feeder layer). LSCs, co-cultured with hUSSCs, showed normal karyotype (46, XX), while they could efficiently form colony (86 ± 3) and display up-regulation of the genes associated with stemness and down-regulation of corneal epithelial differentiation genes. Consistent with 3T3 feeder cells, hUSSCs with spindle-shaped morphology and quick splitting up properties had ability to preserve the stem like-cell phenotype of LSCs. These findings were confirmed by qRT-PCR and flow-cytometer. Findings of present study suggest hUSSCs as a promising alternative method for 3T3 feeder layer cells, to preserve growth and stemness of LSCs ex vivo culture.
体外培养角膜缘干细胞（LSCs）是目前重建眼表的一种很有前途的方法。在这种情况下，通常使用 3T3 饲养层细胞（小鼠胚胎成纤维细胞）来维持和扩增 LSCs。本研究的目的是开发一种新的培养方法（无动物源性产品），使用脐带来源的人类无限制体干细胞（hUSSCs）代替 3T3 细胞来扩增 LSCs，重点是保持 LSCs 的干性。使用流式细胞仪，对分离的 hUSSCs 进行 CD105、CD90、CD166、CD34、CD45、CD31 细胞表面标志物的鉴定，并评估其向脂肪和成骨谱系分化的能力。除了集落形成效率（CFE）、上皮谱系分化和核型分析外，还通过定量 RT-PCR（qRT-PCR）和免疫细胞化学评估 LSC 特性，当这些细胞与 hUSSCs 共培养时（与 3T3 饲养层相比），评估 ABCG2、ΔNP63-α、CK19、CK3 和 CK12 mRNA 和蛋白的表达。与 3T3 饲养层共培养的 LSCs 具有正常的核型（46，XX），而它们能够有效地形成集落（86±3），并显示与干性相关的基因上调和角膜上皮分化基因下调。与 3T3 饲养细胞一致，具有纺锤形形态和快速分裂特性的 hUSSCs 具有保持 LSCs 类似干细胞表型的能力。这些发现通过 qRT-PCR 和流式细胞术得到了证实。本研究的结果表明，hUSSCs 是替代 3T3 饲养层细胞的一种很有前途的方法，可用于体外培养 LSCs 的生长和干性。
