ISOLATION AND CHARACTERIZATION OF RETICULOCYTES DIFFERENTIATED FROM HUMAN PERIPHERAL BLOOD-DERIVED CD34+ HEMATOPOIETIC STEM CELLS AND THE POTENTIAL OF RETICULOCYTE INVASION BY PLASMODIUM FALCIPARUM

Abstract

In vitro reticulocyte model may serve as a promising tool for understanding human erythropoiesis in health and disease. Yet, the availability of reticulocyte model is restricted by the limited number of these circulating cells in human peripheral blood (PB). Here, we described a method to isolate and characterize reticulocytes differentiated from human PB- derived CD34+ hematopoietic stem cells (HSCs) and evaluate the potential of reticulocyte invasion by the malaria parasite, Plasmodium falciparum. PB-derived CD34+ HSCs were isolated from a total of 200 mL PB of healthy volunteers (n=20), and were grown and expanded for five days in serum-free media supplemented with cytokines and growth factors followed by differentiation with erythroid-supporting cytokines. The expansion rate of PB- derived CD34+ HSCs, the phenotype and morphology of generated reticulocytes were studied. The susceptibility of functional reticulocytes to invasion by P. falciparum was determined. During expansion, the total cell population increased approximately 2.11 ± 0.10-fold in an optimized culture initiated with PB-derived CD34+ HSCs. The characteristics and morphology of reticulocytes were apparent on day 14 of differentiation. Moreover, the invasion assay also proved that P. falciparum invaded the HSC-derived reticulocytes generated in this study. The simplified and reproducible protocol using CD34+ HSCs from PB makes generated reticulocytes beneficial for fundamental research in erythroid development and provides the in vitro reticulocyte model for studying malaria parasites that target erythroid cells.