Liang Haydn C-Y, Holmes Roxanne, Zúñiga-Pflücker Juan Carlos
Department of Immunology, Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2013;1029:119-28. doi: 10.1007/978-1-62703-478-4_9.
Hematopoiesis is the highly regulated and complex process by which blood cells are formed. Hematopoiesis can be achieved in vitro by the differentiation of embryonic stem cells (ESCs) into hematopoietic lineage cells. Differentiation of ESCs initially gives rise to mesoderm colonies that go on to form hemangioblast cells, which possess endothelial and hematopoietic lineage potential. While the differentiation of several hematopoietic lineages from ESCs, such as erythrocytes and macrophages, can be easily recapitulated in vitro, T-cell differentiation requires additional Notch-dependent signals for their generation. Keeping with this, ESCs induced to differentiate with OP-9 cells, a bone marrow-derived stromal cell line, give rise to erythro-myeloid cells and B lymphocytes, while the expression of an appropriate Notch ligand, such as Delta-like 1, on OP-9 cells (OP9-DL1) is required to support the generation of T-cells in vitro. Here, we describe an updated and streamlined protocol for the generation of T-lineage cells from mouse ESCs cultured on OP9-DL1 cells. This approach can facilitate studies aimed to assess the effects of environmental and genetic manipulations at various stages of T-cell development.
造血作用是一个受到高度调控的复杂过程，通过这个过程血细胞得以形成。造血作用可以在体外通过胚胎干细胞（ESC）分化为造血谱系细胞来实现。ESC的分化最初产生中胚层集落，这些集落进而形成造血干细胞，造血干细胞具有内皮细胞和造血谱系潜能。虽然从ESC分化出几种造血谱系，如红细胞和巨噬细胞，在体外很容易重现，但T细胞分化需要额外的Notch依赖性信号才能产生。与此一致的是，用OP-9细胞（一种骨髓来源的基质细胞系）诱导ESC分化，会产生红系-髓系细胞和B淋巴细胞，而在OP-9细胞（OP9-DL1）上表达适当的Notch配体，如Delta样1，是在体外支持T细胞产生所必需的。在这里，我们描述了一种更新的、简化的方案，用于从小鼠ESC在OP9-DL1细胞上培养产生T谱系细胞。这种方法可以促进旨在评估环境和基因操作在T细胞发育各个阶段的影响的研究。
