Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro

https://doi.org/10.1016/j.parint.2013.11.011Get rights and content

Highlights

  • Modified stem cell culture produced reticulocytes with higher efficiency.

  • Aotus-grown Plasmodium vivax infection on cultured reticulocytes was demonstrated.

  • Invasion inhibition was quantitated using P. vivax infection on cultured reticulocytes.

  • Selective infection on cultured reticulocytes was demonstrated using immunostaining.

Abstract

Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for practical use in research have been difficult to produce from this system. Here, we report an improved method of hematopoietic stem cell culture for P. vivax infection, which requires less time and produces higher or equivalent percentage of reticulocytes than previously reported systems. Reticulocytes were cultured from cryopreserved erythroblasts that were frozen after 8 day-cultivation of purified CD34 + cells from human umbilical cord blood. This method of production allowed the recovery of reticulocytes in a shorter time than with continuous stem cell culture. We obtained a relatively high percentage of peak reticulocyte production by using co-cultivation with a mouse stromal cell line. Using P. vivax mature stage parasites obtained from infected Aotus monkeys, we observed substantial numbers (up to 0.8% of the total number of the cells) of newly invaded reticulocytes 24 h after initial cultivation. The addition of fresh reticulocytes after 48 h culture, however, did not result in significant increase of second cycle reticulocyte invasion. Assays of invasion inhibition with specific antibodies were successful with this system, demonstrating potential for study of biological processes as well as the conditions necessary for long-term maintenance of P. vivax in vitro.

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.

Cited by (15)

  • A Way Forward for Culturing Plasmodium vivax

    2020, Trends in Parasitology
    Citation Excerpt :

    The reticulocytes from Day 15 were suitable for P. vivax merozoite invasion [38]. Although there was successful P. vivax merozoite invasion in the first cycle, the invasion efficiency dropped even after continuous supplementation with fresh reticulocytes [15,16,38]. Attempts have been made to develop an immortalized hematopoietic progenitor cell line for genetic manipulations and to provide a continuous supply of reticulocytes [39,40].

  • Advancing Research Models and Technologies to Overcome Biological Barriers to Plasmodium vivax Control

    2018, Trends in Parasitology
    Citation Excerpt :

    Data suggest that, while differential centrifugation is more laborious, reticulocytes enriched using this technique better support P. vivax invasion than density gradients [18,20] or immunomagnetic purification [15,16]. More recent advances in regard to hematopoietic stem cells (HSCs) provided a reproducible in vitro source of fresh reticulocytes to support P. vivax culture [21–23]. Relatively large quantities of HSCs can be enriched from human cord blood by magnetic microbead selection on the basis of their expression of either CD34 or CD133, and then used immediately or cryopreserved as aliquots for initiating new cultures as needed.

  • Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art

    2016, Pharmacology and Therapeutics
    Citation Excerpt :

    Accordingly, these questions should be addressed by the malaria community using the various malaria animal models and controlled human clinical trials. Recent advances in in vitro drug metabolism and malaria infection models will hopefully allow for more complete and expedited investigations of such questions (Furuya et al., 2014; Ng et al., 2015; Yang et al., 2015). The CYP 2D6/primaquine discovery also provides clinicians an additional avenue to address possible causes of relapse despite primaquine/8-aminoquinoline therapy.

  • Cell based assays for anti-Plasmodium activity evaluation

    2016, European Journal of Pharmaceutical Sciences
    Citation Excerpt :

    To date, no consistent hypnozoite efficacy assays are available, as P. vivax selectively infects reticulocytes (Mons et al., 1988a, 1988b), which are difficult to obtain and culture in vitro. However, improved methods of hematopoietic stem cell cultures for P. vivax infections, using reticulocytes that have been cultured from cryopreserved erythroblasts from human umbilical cord blood, have demonstrated large potential for maintaining P. vivax cultures in vitro (Furuya et al., 2014; Udomsangpetch et al., 2007). In addition, it was recently demonstrated that induced pluripotent stem cells that had differentiated into hepatocyte-like cells, were also able to support liver stage malaria infection in vitro by using P. vivax, and could these cells support EEF maturation (Ng et al., 2015).

  • Erythropoiesis and Malaria, a Multifaceted Interplay

    2022, International Journal of Molecular Sciences
View all citing articles on Scopus
View full text