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

饲养层细胞通常是建立和维持胚胎干细胞/可诱导多能干细胞所必需的。从胚胎中分离的小鼠胚胎成纤维细胞(MEF)和STO小鼠基质细胞系是应用最广泛的细胞。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)建立iPS细胞。将含有人Oct3/4、SOX2/KLF4和LMYC/LIN28的3种真核表达载体与pMaxGFP(以pMaxGFP为报告载体)通过NeonR电穿孔系统(一种新型的毛细管和线状电极)共同导入原代培养的HDDPC和SNT(5×104)。然后，他们在一种特定的维持ES细胞的培养液中培养15天。通过胰酶消化收集集落，重新接种到丝裂霉素C处理的MEF或STO细胞上。蜂群经过长达26次的连续传代。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。保持在MEF上的HDDPC被转化为具有多种未分化特性的iPS细胞，而在STO细胞上的HDDPC则失败了。在MEF上生长的已建立的HDDPC来源的iPS细胞成功地维持在STO细胞上，而不会失去其多能性。我们的结果表明，可以使用重编程因子对HDDPC进行重编程，并且MEF作为饲养层细胞比STO细胞更适合于建立和维持HDDPC来源的iPS细胞，提示饲养层的选择是高效生成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.