Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, Centre de Recherche en Reproduction Animale, University of Montreal, 3200 Sicotte, St-Hyacinthe, Quebec J2S 7C6, Canada.
Reproduction. 2011 Mar;141(3):321-32. doi: 10.1530/REP-09-0536. Epub 2011 Jan 5.
Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.
虽然最近已经从体内来源的胚胎中获得了假定的马胚胎干细胞（ES）样细胞系，但目前尚不清楚是否可以从体细胞核移植（SCNT）和孤雌胚胎中获得 ES 细胞（ESC）系。我们的目的是建立用于从马模型中进行细胞治疗研究的自体 ESC 系的培养条件。我们的结果表明，使用具有明显可见内细胞团（ICM）的早期囊胚和使用蛋白酶酶解 ICM 都可以从 PA 和 SCNT 胚胎中衍生出更高比例的原发性 ICM 外植体。原发性 ICM 外植体表达多能性的分子标记物POU 类 5 同源盒 1（POU5F1）和（性别决定区-Y）-盒 2（SOX2），在某些情况下，还表达 NANOG。PA 原发性 ICM 外植体传代后的细胞表现出碱性磷酸酶（AP）活性和 POU5F1、SOX2、尾相关同源盒-2（CDX2）和同源框蛋白 Eomesodermin（EOMES）的表达，但可能会失去 NANOG。这些细胞可以产生表达 POU5F1、CDX2 和 EOMES 的囊胚样结构。对马胚胎的免疫组织化学分析显示 POU5F1 存在于滋养外胚层、原始内胚层和 ICM 中。这些结果表明，经过原发性 ICM 外植体传代后的细胞对滋养层干细胞标记物呈阳性，同时表达 POU5F1 并表现出 AP 活性。因此，这些细胞很可能代表滋养细胞而不是真正的 ESC。本研究代表了在大型动物模型上进行临床前细胞治疗研究生产自体马 ESC 的重要第一步。
