Cloned human embryonic stem cells for tissue repair and transplantation
Cloned human embryonic stem cells for tissue repair and transplantation
复制标题
DOI：
10.1385/scr:1:2:099
复制
发表时间：
2005-01-01
期刊：
影响因子：
--
通讯作者：
Kang, Sung Keun
中科院分区：
文献类型：
--
作者：
Hwang, Woo Suk;Lee, Byeong Chun;Kang, Sung Keun
克服人类胚胎干（ES）细胞的移植排斥的一种方法是从患者自身细胞的核转移中获得ES细胞。由于一个有效的协议，人类体细胞核移植（SCNT）尚未报告，几个关键因素需要确定和优化。我们对家畜的经验表明，重编程时间（细胞融合和卵母细胞激活之间的时间段），激活方法和体外培养条件在染色质重塑和SCNT胚胎的发育能力中均起着关键作用。本文综述了人SCNT的优化和人ES细胞的克隆化。在我们的研究中，当我们允许2小时的重编程以支持适当的胚胎发育时，大约25%的人类重建胚胎发育成胚细胞。由于在SCNT中不存在精子介导的激活，因此需要人工刺激来启动胚胎发育。用10 μ M钙离子载体孵育5分钟，然后用2.0 μ M 6-二甲基氨基嘌呤孵育，被认为是最有效的化学激活方案SCNT使用人卵母细胞。为了克服胚胎培养中的低效率，我们通过分别用血清白蛋白和果糖代替牛血清白蛋白和葡萄糖补充mSOFaa来制备具有氨基酸的人改良合成输卵管液（hmSOFaa）。在G1.2培养基中培养人SCNT衍生的胚胎前48小时，然后在G2.2培养基中培养或在hmSOFaa培养基中连续培养。本文所述的方案以19- 29%的比率产生克隆囊胚，这与使用已建立的SCNT方法在牛（约25%）和猪（约26%）中的比率相当。共培养30个SCNT衍生的胚细胞，通过免疫手术去除滋养层分离出20个内细胞团（ICM），并衍生出一个具有典型ES细胞形态和多能性的人克隆ES细胞系（SCNT-hES 1）。我们的方法为在移植医学中使用来自核转移ES（ntES）衍生细胞的自体干细胞打开了大门。
One approach to overcome transplant rejection of human embryonic stem (ES) cells is to derive ES cells from nuclear transfer of the patient's own cells. Because an efficient protocol for human somatic cell nuclear transfer (SCNT) has not been reported, several critical factors need to be determined and optimised. Our experience with domestic animals indicate that reprogramming time (the period of time between cell fusion and oocyte activation), activation method and in vitro culture conditions each play a critical role in chromatin remodeling and the developmental competence of SCNT embryos. In this review, we describe the optimization of human SCNT and derividation of human cloned ES cells. In our study, about approx 25% of human reconstructed embryos developed into blastocytes when we allowed 2h for reprogramming to support proper embryonic development. since sperm mediated activation is absent in SCNT, an artificial stimulus is needed to initiate embryo development. Incubation with 10 mu M calcium ionophore for 5 min followed by incubation with 2.0 mu M 6-dimethyl amino purine was found to be the most efficient chemical activation protocol for SCNT using human oocytes. In order to overcome inefficiencies in embryo culture, we prepared human modified synthetic oviductal fluid with amino acids (hmSOFaa) by supplementing mSOFaa with serum albumin and fructose instead of bovine serum albumin and glucose, respectively. Culturing human SCNT-derived embryos in G1.2 medium for the first 48 h followed by a culture in G2.2 medium or culturing continuously in hmSOFaa medium. The protocol described here produced cloned blastocysts at rates of 19-29%, which is comparable with the rates in cattle (approx 25%) and pigs (approx 26%) using established SCNT methods. A total of 30 SCNT-derived blastocytes were cultured, 20 inner cell masses (ICMs) were isolsted by immunosurgical removal of the trophoblast, and one human cloned ES cell line (SCNT-hES1) with typical ES cell morphology and pluripotency was derived. Our approach opens the door for the use of autulogous cells derived from nuclear transfer ES (ntES)-derived cells in transplantation medicine.