Abstract
Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.
Highlights
RKM, OK, JT, PM, CR, LV, and EMP performed the research described in the article and all data analysis
Generation of Fy receptor-expressing K562 cell lines It is well known that P. vivax [19] and its closely related non-human primate malarias P. knowlesi [20] and P. cynomolgi [21] require the Duffy blood group antigen receptor to invade their host red blood cells
Two transcription variants are known for Fya or Fyb differing in the 50 UTR and coding sequence, where isoform “short” has a shorter and distinct N-terminus compared with isoform “long.” As parasite binding and invasion may vary depending on the specific Duffy variant, different Duffy alleles, alone and in combination, have been transduced into K562 cells, resulting in the generation of five different Fy blood group-expressing K562 cell lines (Fya-short; Fya-long; Fyb-short; Fyb-long and Fya/b long/long) (Figure 1A)
Summary
RKM, OK, JT, PM, CR, LV, and EMP performed the research described in the article and all data analysis. To tackle the need for a P. vivax-permissive, reproducible, and stable reticulocyte population and gain a better understanding of the regulatory factors behind the proliferation, differentiation, and enucleation of erythroid precursors, we set out to study the potential for differentiation of the erythroleukemia K562 cell line. We reasoned that this stable, continuously proliferating, immortal erythroleukemia line could provide the continuous source of reproducible and stable reticulocytes needed for long-term P. vivax in vitro culture. A 150-fold increase in a-globin after treatment with mithramycin A in the double knockdown was noteworthy given that the a-globin increased only 50fold in the control
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