Established efficiency of iPS cells derived from impacted supernumerary tooth considering bio-recycle

考虑生物回收，确定源自受影响多生牙的 iPS 细胞的效率

基本信息

批准号：
23792364
负责人：
SAITO Yoko
金额：
$ 2.75万
依托单位：
Niigata University (2012)Kagoshima University (2011)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23792364/
关键词：
小児歯科学再生医学バイオリサイクル歯 iPS細胞 iＰＳ細胞

项目摘要

Feeder cells are generally required for establishing and maintaining embryonic stem (ES) cells/inducible pluripotent stem (iPS) cells. Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used cells for this purpose. The aim of this study was to determine which cells are suitable for establishing iPS cells from human deciduous tooth dental pulp cells (HDDPCs) and supernumerary teeth (SNT). Primarily cultured HDDPCs and SNT (5 × 104) wereco-transfected with 3 plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 together with pmaxGFP (used as a reporter) by using the NeonR Transfection system, a novel electroporation method involving a capillary and wire-type electrode. They were then cultured in a medium specified for maintaining ES cells for 15 days. Colonies were collected by trypsinization and re-seeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether they exhibit ES-like morphology and alkaline phosphatase activity to evaluate the state of HDDPC reprogramming. HDDPCs maintained on MEFs were transformed to iPS cells with several undifferentiated properties, but those on STO cells failed. Established HDDPC-derived iPS cells grown on MEFs were successfully maintained on STO cells without loss of their pluripotent nature. Our results indicate that HDDPCs can be reprogrammed using reprogramming factor, and that MEFs are better than STO cells as feeder cells for establishing and maintaining HDDPC-derived iPS cells, suggesting that the choice of feeders is a key factors enabling efficient generation of iPS cells.

建立和维持胚胎干 (ES) 细胞/诱导多能干 (iPS) 细胞通常需要饲养细胞，从胎儿中分离的小鼠胚胎成纤维细胞 (MEF) 和 STO 小鼠基质细胞系是用于此目的最广泛的细胞。本研究的目的是确定哪些细胞适合从人乳牙牙髓细胞 (HDDPC) 和多生牙 (SNT) 中建立 iPS 细胞。使用 NeonR 转染系统（一种新颖的电穿孔方法）将培养的 HDDPC 和 SNT (5 × 104) 与含有人 OCT3/4、SOX2/KLF4 或 LMYC/LIN28 的 3 个质粒以及 pmaxGFP（用作报告基因）共转染然后将它们在指定用于维持 ES 细胞的培养基中培养 15 天。胰蛋白酶消化并重新接种到丝裂霉素 C 处理的 MEF 或 STO 细胞上，将集落连续传代最多 26 代，在此期间，评估集落形态以确定它们是否表现出 ES 样形态和碱性磷酸酶活性，以评估MEF 上维持的 HDDPC 重编程状态被转化为具有多种未分化特性的 iPS 细胞，但 STO 细胞上的 HDDPC 重编程失败。在 MEF 上生长的 HDDPC 衍生 iPS 细胞成功地维持在 STO 细胞上，且不丧失其多能性，我们的结果表明 HDDPC 可以使用重编程因子进行重编程，并且 MEF 比 STO 细胞更适合作为建立和维持 HDDPC 的饲养细胞。衍生的 iPS 细胞，表明饲养层的选择是实现 iPS 细胞高效生成的关键因素。