Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.
Journal
American journal of hematology
ISSN: 1096-8652
Titre abrégé: Am J Hematol
Pays: United States
ID NLM: 7610369
Informations de publication
Date de publication:
09 2019
09 2019
Historique:
received:
04
03
2019
revised:
20
05
2019
accepted:
28
05
2019
pubmed:
31
5
2019
medline:
11
3
2020
entrez:
1
6
2019
Statut:
ppublish
Résumé
Malaria pathogenesis is caused by the replication of Plasmodium parasites 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 both P. vivax and P. 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 of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. 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.
Identifiants
pubmed: 31148215
doi: 10.1002/ajh.25543
pmc: PMC6984401
mid: NIHMS1065142
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
963-974Subventions
Organisme : Broad Institute
Pays : International
Organisme : NIAID NIH HHS
ID : R01 AI140751
Pays : United States
Organisme : Swiss National Science Foundation
Pays : Switzerland
Organisme : Conselho Nacional de Desenvolvimento Científico e Tecnológico
Pays : International
Organisme : NHLBI NIH HHS
ID : R01 HL139337
Pays : United States
Organisme : National Science Foundation
Pays : International
Informations de copyright
© 2019 Wiley Periodicals, Inc.
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