MSPTM 2019 Annual Scientific Conference
13 - 14 March 2019
InterContinental Kuala Lumpur

Abstract

Title
GENERATION OF RETICULOCYTES DERIVED FROM HUMAN PERIPHERAL BLOOD CD34+ HAEMATOPOIETIC STEM CELLS (FOR PLASMODIUM KNOWLESI LONG-TERM IN VITRO CULTURE)
Type
Oral Presentation
Theme
Scaling Up Efforts in Tropical Disease and Vector Control through Evidence-Based Research
Topic
Malaria

Authors

Main Author
Fatin Sofia Mohamad1
Presenting Author
Fatin Sofia Mohamad1
Co-Author
Muhammad Syahmi Khairuzzaman Che Mohd Nasir1
Maryam Azlan1
Suat Cheng Tan1
Nurhidanatasha Abu Bakar1

Authors' Institution

Department / Institution / Country
School of Health Sciences / Universiti Sains Malaysia / Malaysia1
Content
Abstract Content
Plasmodium knowlesi, found in wild primate populations, is now recognised as the fifth human malaria parasite. The predilection of P. knowlesi for reticulocytes has currently hampered the establishment of its long-term in vitro culture system. Here, we demonstrated that the peripheral blood-derived CD34+ (PB-CD34+) cells cultured in the presence of appropriate cytokines and growth factors could be expanded and differentiated to generate higher production of reticulocytes for P. knowlesi in vitro culture. After five days expansion in serum-free expansion medium II supplemented with stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 and interleukin-6, the PB-CD34+ cells were subjected to differentiation with erythropoietin to differentiate the cells into the erythroid lineage. The haematopoietic stem cell (HSC) maturation stages were characterised in terms of the expression of stage specific markers (CD34, CD45, CD36, CD71 and CD235a) and cell morphology. The morphology of reticulocytes was assessed with Brilliant Cresyl Blue staining. The results show that the PB-CD34+ cells subjected to expansion and differentiation had an increase in the reticulocytes number (6.8 fold) (from three independent experiments), indicating the cells were survived and proliferated. Analysis of the cell surface markers using flow cytometry shows the differentiation of PB-CD34+ cells into reticulocytes (CD45-, CD36-, CD235a+ and CD71+), which was confirmed by the presence of enucleated cells with three dots of Cresyl Blue-stained RNA on day 14. The cells were then cryopreserved that allow the storage of reticulocytes for later use in P. knowlesi in vitro culture. In conclusion, peripheral blood-derived HSCs can be an alternative to cell sources besides from cord blood or bone marrow to generate a large number of reticulocytes required for the long-term P. knowlesi in vitro culture.
Keywords: Plasmodium knowlesi; peripheral blood-derived CD34+ cells; reticulocytes; erythroid lineage; flow cytometry
Requires Audio or Video system for Presentation?: No