Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.
Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.
复制标题
从外周血中生成永生化红系祖细胞系：疟原虫属功能分析的模型系统。
DOI：
10.1002/ajh.25543
复制
发表时间：
2019
影响因子：
12.8
通讯作者：
Duraisingh,ManojT
中科院分区：
文献类型：
--
作者：
Scully,ErikJ;Shabani,Estela;Rangel,GabrielW;Grüring,Christof;Kanjee,Usheer;Clark,MarthaA;Chaand,Mudit;Kurita,Ryo;Nakamura,Yukio;Ferreira,MarceloU;Duraisingh,ManojT
Malaria pathogenesis is caused by the replication ofPlasmodiumparasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long‐term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by bothP. vivaxandP. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor ofP. vivaxin humans. Invasion ofP. vivaxinto this DARC‐knockout cell line was strongly inhibited providing direct genetic evidence thatP. vivaxrequires DARC for RBC invasion. Further, genetic complementation of DARC restoredP. vivaxinvasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood‐stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.
