Goh Hwee Ngee, Rathjen Peter D, Familari Mary, Rathjen Joy
Department of Zoology, University of Melbourne , Victoria, Australia .
The Menzies Research Institute Tasmania, University of Tasmania , Tasmania, Australia .
Biores Open Access. 2014 Jun 1;3(3):98-109. doi: 10.1089/biores.2014.0010.
Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell-derived endoderm, and in a band of endoderm immediately distal to the embryonic-extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.
哺乳动物胚胎中的内胚层形成首先发生在囊胚期，此时原始内胚层和多能细胞分化为不同的谱系，并且在原肠胚形成期间再次发生，此时确定的内胚层祖细胞群体从原条中出现。确定的内胚层的形成可以通过培养中的多能细胞分化来模拟。早期原始外胚层样（EPL）细胞是一种由胚胎干细胞形成的多能细胞群体，其分化被用于鉴定和表征确定的内胚层形成。在EPL细胞衍生的内胚层中，以及在原肠胚前期和原肠胚形成期胚胎中胚胎-胚外边界远端紧邻的一条内胚层带中，检测到丝氨酸蛋白酶抑制剂Kazal型3（Spink3）的表达。后期表达标记了将卵黄囊与发育中的肠道分隔开的内胚层区域。在胚胎中，通过内吞作用测定确定，Spink3表达标记了一个由远端内脏内胚层和确定的内胚层近端区域组成的内胚层区域。该区域与由促甲状腺激素释放激素（Trh）标记的更远端的确定内胚层群体不同。在EPL细胞分化过程中可以形成表达Spink3或Trh的内胚层，并且这些群体的比例可能受培养基和生长因子添加的影响。此外，进一步的分化表明这些群体的潜能不同。这些方法揭示了确定的内胚层谱系中意想不到的复杂性，这种复杂性在未来的分化方案中需要加以考虑，以确保形成合适的确定内胚层祖细胞。
