Palis James, Koniski Anne
Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY, 14642, USA.
Methods Mol Biol. 2018;1698:117-132. doi: 10.1007/978-1-4939-7428-3_7.
The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.
红系祖细胞在半固体培养基中形成成熟红细胞集落的能力，为这些祖细胞提供了一种功能检测方法，并极大地促进了我们对红细胞生成的理解。自20世纪70年代以来的研究促成了红细胞生成单位模型的发展，据此，最早的红系定向祖细胞，即不成熟的爆式红系集落形成单位（BFU-E），依次产生晚期BFU-E和红系集落形成单位（CFU-E）。CFU-E继而产生血红蛋白化的成熟成红细胞前体。正是这些终末细胞构成了源自半固体培养基中培养的祖细胞的红系细胞成熟集落。红系集落的体外生成需要细胞因子支持，最显著的是促红细胞生成素（EPO），它对CFU-E的存活以及促进成红细胞成熟至关重要。在小鼠胚胎发育过程中，原始红系集落形成祖细胞（EryP-CFC）的短暂群体出现在卵黄囊中，并在胎儿血液中产生一波成熟的原始成红细胞。这一波EryP-CFC之后紧接着卵黄囊中出现的一波BFU-E，它们进入血液并定植于胎儿肝脏，以在胎儿体内产生第一批定型红细胞。与成年骨髓中的BFU-E不同，胎儿肝脏中的BFU-E在单独与EPO培养时可在体外形成集落，并且对EPO水平更敏感。在此，我们描述了在半固体培养基中体外培养小鼠胚胎（原始）和胎儿/成年（定型）红系祖细胞的方法。
