Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro
Graphical abstract
Introduction
Plasmodium vivax research is limited by the lack of practical in vitro culture system. One of the obstacles to this system is that P. vivax mainly infects reticulocytes, which constitute only a small percentage of the red blood cells in adult donor blood [1]. Attempts to culture the parasites in vitro have therefore included provision of reticulocytes from Aotus monkeys [2], from human hemochromatosis patients [3] and human cord blood [4], [5], [6]. However, it is difficult to maintain these cultures for more than a few days and the sources for reticulocytes are not widely available.
Hematopoietic stem cells (HSCs) can be a useful source for reticulocytes when the cells are cultured in a condition that directs erythrocyte development [7]. Panichakul et al. [8] reported an in vitro culture system of P. vivax using reticulocytes obtained from HSCs. The system produced up to 0.5% of reticulocytes in the HSC culture and the percentage of P. vivax-infected cells that could be obtained was low (0.0013% maximum). Better yield of reticulocytes from HSC culture may be needed for P. vivax research. Also, obtaining reticulocytes from a HSC culture system takes 14 days, which can be very demanding, especially considering that new reticulocytes need to be continuously added to the cultures for successful parasite infection. Noulin et al. [9] recently reported the use of cryopreserved hematopoietic stem cells to produce reticulocytes for infection of P. vivax parasites from infected patients. They achieved relatively high percentage of reticulocytes which could be infected by P. vivax parasites. However, detailed information about subsequent status of the infected parasites, particularly the percentages of each stage during the culture was not demonstrated and infection with P. vivax parasites experimentally amplified in Aotus monkeys was not examined.
Here, we report an improved method for HSC cultures to produce reticulocytes for in vitro infection of P. vivax parasites. By using cryopreserved erythroblasts developed from CD34 + human cord blood, and adding to the culture a mouse stromal cell line, reticulocytes are consistently obtained within 7 to 8 days in high percentage (15–20%). Using these reticulocytes we demonstrate that P. vivax can efficiently infect these cells, allowing laboratory investigation of parasite host cell invasion.
Section snippets
Hematopoietic stem cell (HSC) culture
Purified CD34 + cells from human cord blood (AllCells, Emeryville, CA) were grown in Iscove's modified Dulbecco's medium (IMDM, Cedarlane) containing l-glutamine (4 mM, Invitrogen), penicillin and streptomycin (50 U/ml and 50 μg/ml, respectively, Invitrogen), monothioglycerol (160 μM), transferrin (120 μg/ml), insulin (10 μg/ml), ferrous nitrate (90 ng/ml), ferrous sulfate (900 ng/ml) and bovine serum albumin (10 mg/ml, StemCell Technologies, Vancouver, BC, Canada). All reagents are from Sigma-Aldrich
Results
To obtain substantial numbers of reticulocytes for P. vivax infection, we modified a previously reported method of HSC culture for the production of reticulocytes [7] (Fig. 1). In order to shorten the culture period of the original method, we cryopreserved erythroblasts after culturing the stem cells for 8 days and followed the rest of the culture schedule after thawing the cryopreserved cells. We did not find any detectable differences in morphology and erythrocyte surface marker expression
Discussion
To improve HSC cultures for P. vivax in vitro studies, we cryopreserved erythroblasts and employed a co-culture with a mouse stromal cell line that increased the yield of reticulocyte production to approximately 20% within 7 days of the culture. In addition, numbers of hematopoietic stem cells increase more than 300 times the initial number of CD34 + purified cells before cryopreservation (data not shown), making the culture easier to prepare since we can store a higher number of erythroblasts
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study was partly supported by the National Institutes of Health grant (1R03AI079475-01) and also partly by the Intramural Research Program of the NIH, NIAID. We thank Dr. Yves Colin Aronovicz for providing anti-FY6 monoclonal antibody and Dr. Louis H. Miller for critical advice on the study. We also thank Sarah Kaslow, Theresa Aguirre, Faith Sentz, and Ahlin Bruce for support on Aotus research.
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