Hypoxic culture in the isolation of corneal stem cells

缺氧培养在角膜干细胞分离中的应用

基本信息

批准号：
17390471
负责人：
SHIMMURA Shigeto
金额：
$ 6.53万
依托单位：
Tokyo Dental Collage
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390471/
关键词：
Corneal epithelium Stem Cells Hypoxia Cell Culture Cytokeratin Regenerative Medicine Immunohistochemistry Progenitor Cells

项目摘要

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.

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源性祖细胞（COP），并发表在《干细胞》上。 COP 是神经嵴衍生的多能干细胞，能够分化为角膜角膜细胞、成纤维细胞、神经细胞以及神经胶质细胞和脂肪细胞。 COP 在无血清培养基中传代超过 10 代。我们还开始了人类COP的分离，并计划找到一种允许长期培养人类COP的方案。我们还设计了一种在缺氧条件下有效培养角膜上皮祖细胞的培养方法。在 2% O2 下，细胞角蛋白 12 (K12) 阴性祖细胞比在常氧下增殖更有效。缺氧条件下集落形成效率（CFE）也较高，表明未成熟细胞被选择性培养。角膜上皮祖细胞和干细胞的高效培养将对再生医学产生影响。我们已经拥有使用祖细胞设计分层上皮片的技术。我们还从当前的研究中发现，使用 2 层饲养细胞产生类似于角膜缘上皮表型的上皮片。我们还报道K15可用作角膜缘祖细胞的标记物，可用于表征分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，我们计划在接下来的项目中进一步研究这一点。