Cells for Sight Transplantation & Research Programme, Department of Ocular Biology & Therapeutics, UCL Institute of Ophthalmology, London, UK.
Regen Med. 2010 Nov;5(6):877-89. doi: 10.2217/rme.10.73.
To evaluate a serum-free system where mitotically active subconjunctival fibroblasts were co-cultured with conjunctival epithelial cells to mimic a niche environment for conjunctival progenitor cells.
Human conjunctival epithelial cells were expanded in vitro and evaluated for their colony-forming efficiency and clonal ability. The cells were then transferred to a serum-free co-culture system and cultured in the presence of mitotically active subconjunctival fibroblasts (human conjunctival epithelial cells and human bulbar subconjunctival fibroblasts [HCEC-HCF]). Cells were evaluated by Ki67 staining, total colony-forming efficiency and the number of colonies with a surface area of more than 10 mm(2). The expression of putative progenitor cell markers p63α, ABCG2 and CK15, and the presence of MUC5AC- and periodic acid-Schiff-positive cells was compared with standard culture conditions (HCEC-3T3).
Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions demonstrated strong immunoreactivity to p63α and ABCG2. Co-localization of CK15 and p63α revealed a subpopulation of CK15-positive cells under HCEC-3T3 conditions compared with only a few CK15-positive cells found under HCEC-HCF conditions. MUC5AC- and periodic acid-Schiff-positive cells were much more common under HCEC-3T3 conditions than under HCEC-HCF conditions. These results were confirmed by reverse transcription-PCR. Cells in HCEC-HCF conditions demonstrated a significantly higher total colony-forming efficiency and a significantly higher percentage of colonies with holoclone-like morphology.
The simulation of a niche environment in vitro by co-culturing mitotically active subconjunctival fibroblasts with conjunctival epithelial cells supports the maintenance of conjunctival cells with progenitor cell characteristics and therefore might be a useful tool to expand conjunctival epithelial progenitor cells in vitro for clinical use.
评估一种无血清系统，其中有丝分裂活跃的结膜下成纤维细胞与结膜上皮细胞共培养，以模拟结膜祖细胞的生态位环境。
体外扩增人结膜上皮细胞，评估其集落形成效率和克隆能力。然后将细胞转移到无血清共培养系统中，并在有丝分裂活跃的结膜下成纤维细胞（人结膜上皮细胞和人球结膜下成纤维细胞[HCEC-HCF]）存在的情况下培养。通过 Ki67 染色、总集落形成效率和面积大于 10mm²的集落数量来评估细胞。比较了祖细胞标记物 p63α、ABCG2 和 CK15 的表达以及 MUC5AC-和过碘酸希夫阳性细胞的存在情况与标准培养条件（HCEC-3T3）。
在 HCEC-HCF 和 HCEC-3T3 条件下培养的结膜上皮细胞对 p63α 和 ABCG2 表现出强烈的免疫反应性。CK15 和 p63α 的共定位显示，与 HCEC-3T3 条件下仅发现少数 CK15 阳性细胞相比，HCEC-3T3 条件下 CK15 阳性细胞存在亚群。与 HCEC-HCF 条件相比，MUC5AC-和过碘酸希夫阳性细胞在 HCEC-3T3 条件下更为常见。这些结果通过逆转录-PCR 得到了证实。HCEC-HCF 条件下的细胞表现出明显更高的总集落形成效率和具有全克隆样形态的集落的百分比明显更高。
通过有丝分裂活跃的结膜下成纤维细胞与结膜上皮细胞共培养，在体外模拟生态位环境，支持维持具有祖细胞特征的结膜细胞，因此可能是体外扩增结膜上皮祖细胞用于临床应用的有用工具。
