Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Korea.
Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Korea.
J Cell Mol Med. 2019 Mar;23(3):2052-2063. doi: 10.1111/jcmm.14111. Epub 2019 Jan 4.
Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-β inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.
在移植前，需要对神经祖细胞（NPCs）的安全性和有效性进行临床前研究。因此，在猪等大动物模型中生成用于神经细胞分化的高效体外平台非常重要。在这项研究中，将猪诱导多能干细胞（iPSCs）以高细胞密度接种到含有双 SMA 和 Mad 相关蛋白（SMAD）抑制剂、TGF-β抑制剂和 BMP4 抑制剂的神经诱导培养基中。与对照组相比，dSMADi 衍生的 NPC 显示出 NPC 标志物，如 PLAG1、NESTIN 和 VIMENTIN，以及 Sox1 的 mRNA 表达更高。发现视黄酸处理组中 HOXB4 的 mRNA 表达显著增加。在体外增殖的 NPC 生成神经球，能够进一步分化为神经元和神经胶质细胞。含有损伤相关细胞因子的神经胶质瘤培养培养基可使体外培养的猪 iPSC-NPC 存活良好，与标准对照培养基相比，其神经元标志物表达更高。总之，本研究开发了一种有效的方法，可将猪 iPSCs 向 NPC 进行神经定向分化。
