PAGES: DOI: 10.1590/0074-0276108062013019 Short communication
Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

Carmen Fernandez-Becerra1, Joel Lelievre2, Mireia Ferrer1, Nuria Anton1, Richard Thomson1, Cristina Peligero1, Maria Jesus Almela2, Marcus VG Lacerda3,4, Esperanza Herreros2, Hernando A del Portillo1,5,+

1Barcelona Centre for International Health Research, Hospital Clinic-Universitat de Barcelona, Barcelona, Spain
2Tres Cantos Medicines Development Campus, GlaxoSmithKline, Tres Cantos, Spain
3Fundação de Medicina Tropical Dr Heitor Vieira Dourado, Manaus, AM, Brasil
4Universidade do Estado do Amazonas, Manaus, AM, Brasil
5Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain


The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+ hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

The use of human haematopoietic stem cells (hHSCs) and specific factors to drive their differentiation into reticulocytes and mature red blood cells (RBCs) (Giarratana et al. 2005, 2011) represented a major improvement to develop a continuous in vitro culture system for blood stages of Plasmodium vivax as it provided a constant source of reticulocytes derived from progenitors cells isolated from cord blood (Panichakul et al. 2007). Unfortunately, this system was not highly reproducible and it yielded extremely low percentages of infected reticulocytes (< 0.0015%) presumably and partly due to the foetal haemoglobin content of reticulocytes from cord blood (Panichakul et al. 2007). More recently, however, Noulin et al. (2012) described a new method for the production and cryopreservation of cord blood HSC-derived reticulocytes to be used for both Plasmodium falciparum and P. vivax invasion tests, with substantially higher percentage of infected hHSC-derived reticulocytes (< 2.5%).

To determine whether peripheral blood (PB) (from buffy coats of healthy donors) and bone marrow (BM) (Lonza Group Ltd) CD34+ hHSCs could be used as another source of reticulocytes production for furthering development of an in vitro culture system for P. vivax, we set to demonstrate that RBCs differentiated from these progenitors cells express adult haemoglobin and that reticulocytes and erythrocytes derived from them are permissive to invasion, respectively, by P. vivax and P. falciparum parasites. To do so, we purified through Ficoll-Hypaque gradient centrifugation PB mononuclear cells from buffy coats of blood from healthy donors. Next, CD34+ hHSCs were isolated through magnetic beads couple to anti-CD34 antibodies (Miltenyi Biotec) according to the manufacturers' instructions. Using this procedure, we usually obtained 104-105 CD34+ hHSCs starting from approximately 1-3 x 108 cells from 50 mL of buffy coat. Cells were subsequently expanded and differentiated to reticulocytes in a three-step protocol using NIH 3T3 cells (ATCC) to sustain expansion and differentiation of CD34+ hHSCs to reticulocytes and mature RBCs. These cells had been previously shown to have the capacity of stimulating the generation of haematopoietic colony-forming cells (Bigas et al. 1995), but never used before as feeder stromal cells in this differentiation process. Results demonstrated that NIH 3T3 cells help to support differentiation, enucleation and cell culture viability of PB CD34+ hHSCs to reticulocytes and mature RBCs (A in Figure). In addition, corresponding cell expansion levels as those reported for MS5 stromal cells of around 16,000-fold were obtained with HIH 3T3 cells. As expected (Giarratana et al. 2005), at day 15 after differentiation started, we obtained the maximum percentage of reticulocytes (83.5 ± 12.1%). Of note, similar results were obtained from CD34+ hHSCs from BM (not shown).

Whereas adult haemoglobin is composed of two alpha and two beta subunits, foetal haemoglobin is composed of two alpha and two gamma subunits, commonly denoted as α2γ2. To determine the haemoglobin composition of reticulocytes derived from PB and BM CD34+ hHSCs, we used Maldi TOF Mass Spectrometry. As controls, we used reticulocytes derived from cord blood. Samples were analysed in a linear positive mid mass mode to determine the molecular mass of the intact chains. Three signals corresponding to the globin chains α, β and γ (around 15,130, 15,870 and 16,000 Da respectively) were detected (B in Figure). As expected, PB and BM CD34+ hHSCs synthesised adult haemoglobin (with α and β chains) whereas reticulocytes derived from cord blood CD34+ hHSCs synthesised foetal haemoglobin (with α and γ chains).

Invasion experiments of ex-vivo generated RBCs by P. falciparum (3D7 strain) and P. vivax (wild isolates from the Brazilian Amazon Region) were then assayed as previously described (Trager & Jensen 1976, Costa et al. 2011, Russell et al. 2011) to demonstrate that these cells can be used for in vitro culture. To do so, we first demonstrated by flow cytometry that day 14 growing RBCs from CD34+ hHSCs from PB (C in Figure) and BM (not shown) expressed surface markers required for invasion of malaria parasites, namely the Duffy blood group and the glycophorin A antigen. Next, we performed invasion assays using parasites from the 3D7 P. falciparum strain and P. vivax isolates directly obtained from patients at the Tropical Medicine Foundation Dr Heitor Vieira Dourado (FMT-HVD) [all patients were assisted by the Ethical Review Board from FMT-HVD (approval 257.815)]. Results demonstrated that erythrocytes and reticulocytes differentiated from PB CD34+ hHSCs were permissive for invasion of P. falciparum and P. vivax (D1, 2 in Figure, respectively). Similar results were obtained with CD34+ hHSCs from BM (not shown). Moreover, as previously reported in RBCs obtained from CD34+ hHSCs from cord blood (Panichakul et al. 2007) or PB (Tamez et al. 2011), invasion was also observed in nucleated cells (erythroblasts).

Here, we show for what we believe is the first time, the permissiveness of RBCs produced from human PB and BM CD34+ hHSCs to be invaded by P. falciparum and P. vivax. Moreover, these cells express adult haemoglobin and the Duffy receptor, which is the main receptor for entrance of P. vivax (Chitnis & Miller 1994). To facilitate implementation of this method, we have used buffy coats from blood of healthy donors. Furthermore, in addition to MS5 feeder supporting cells (Giarratana et al. 2005), we also tested fibroblast NIH 3T3 cells (ATCC) for expansion and differentiation of CD34+ hHSCs. These cells seem to stimulate haematopoietic proliferation and differentiation through soluble molecule(s) (Bigas et al. 1995) suggesting the possibility of production of reticulocytes with no stromal support as has been recently shown with other factors (Timmins et al. 2011). Of notice, a method for cryopreservation of cord blood hHSC-derived reticulocytes that can be successfully invaded by both cryopreserved P. falciparum and P. vivax isolates has been recently described (Noulin et al. 2012). A combination of these methods and reticulocytes from PB and BM CD34+ hHSCs is therefore amenable for testing whether they allow exponential growth of P. vivax parasites.



To all patients participating of this study, and to Lorena Martin-Jaular, for discussions and advice in FACS analysis.



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Received 21 June 2013
Accepted 12 July 2013
Financial support: Open Lab Initiative (GSK-Tres Cantos)
CF-B and JL contributed equally to this study.
+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.


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