Hypoxic culture in the isolation of corneal stem cells

我们成功地从小鼠角膜基质中分离出干细胞。我们将这些细胞命名为角膜衍生的祖细胞（COP），并发表在《干细胞》上。COP是神经嵴来源的多能干细胞，其具有分化为角膜角膜基质细胞、成纤维细胞、神经细胞以及神经胶质细胞和脂肪细胞的能力。COP在无血清培养基中繁殖超过10代。我们还开始了人COPs的分离，并计划找到一种允许长期培养人COPs的方案。我们还设计了一种培养方法，可以在缺氧条件下有效培养角膜上皮祖细胞。在2%O2下，细胞角蛋白12（K12）阴性祖细胞比在常氧下更有效地增殖。集落形成效率（CFE）在缺氧下也更高，表明选择性地培养未成熟细胞。角膜上皮祖细胞和干细胞的高效培养将对再生医学产生重要影响。我们已经有了利用祖细胞设计分层上皮层的技术。我们还发现，从目前的调查，使用2层饲养细胞产生的上皮片，类似于角膜缘上皮表型。我们还报道了K15可作为角膜缘祖细胞的标记物，其可用于表征复层细胞片中的上皮细胞。在阐明角膜缘上皮干细胞生态位方面仍有许多问题需要澄清，我们计划在即将到来的项目中进一步探讨这一点。

We succeeded in the isolation of stem cells from the mouse corneal stroma. We named these cells as cornea-derived progenitors (COPs) that we published in Stem Cells. COPs are neural crest derived, multipotent stem cells that have the ability to differentiate into corneal keratocytes, fibroblasts, neural cells as well as glial cells and adipocytes. COPs were propagated for over 10 passages in serum-free medium. We have also started the isolation of human COPs, and plan to find a protocol that allows the long-term culture of human COPs.We also devised a culture method to efficiently culture corneal epithelial progenitor cells under hypoxia. Under 2% 02, cytokeratin 12 (K12) negative progenitor cells proliferated more efficiently than under normoxia. Colony forming efficiency (CFE) was also higher under hypoxia indicating that immature cells are selectively cultivated. The efficient culture of corneal epithelial progenitors and stem cells will have impact on regenerative medicine. We already have the technique to engineer stratified epithelial sheets using progenitor cells. We also found from the current investigation that using 2 layers of feeder cells produces epithelial sheets that resemble the limbal epithelial phenotype. We also reported that K15 could be used as a marker of limbal progenitors cells, which can be used to characterize epithelial cells in stratified cells sheets. There are still many points that require clarification in elucidating the limbal epithelial stem cell niche, and we plan to pursue this further in upcoming projects.