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

饲养细胞通常需要用于建立和维持胚胎干（ES）细胞/诱导性多能干（iPS）细胞。从胎儿分离的小鼠胚胎成纤维细胞（MEFs）和STO小鼠基质细胞系是用于该目的的最广泛使用的细胞。本研究的目的是确定哪种细胞适合从人乳牙牙髓细胞（HDDPC）和多生牙（SNT）中建立iPS细胞。采用NeonR Transfection系统，将人OCT 3/4、SOX 2/KLF 4、LMYC/LIN 28和pmaxGFP（作为报告基因）共3种质粒转染原代培养的HDDPC和SNT（5 × 104）。然后将它们在用于维持ES细胞的特定培养基中培养15天。通过胰蛋白酶消化收集集落，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HDDPC重编程的状态。维持在MEFs上的HDDPC转化为具有几种未分化特性的iPS细胞，但STO细胞上的HDDPC未能转化。在MEF上生长的建立的HDDPC衍生的iPS细胞成功地维持在STO细胞上，而不丧失其多能性。我们的研究结果表明，HDDPC可以使用重编程因子进行重编程，并且MEFs作为饲养细胞比STO细胞更好地用于建立和维持HDDPC衍生的iPS细胞，这表明饲养细胞的选择是有效产生iPS细胞的关键因素。

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.