Generation of an immortalised erythroid cell line from haematopoietic stem cells of a haemoglobin E/β-thalassemia patient

Abstract

The β-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent β-globin chain synthesis. HbE/β-thalassemia is a subtype of β-thalassemia with extremely high frequency in Asia. Studying molecular defects behind β-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/β-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient’s red blood cells. This is the first human cellular model for β-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics

“…In addition, the methodology can be applied to create lines with disease phenotypes for investigating underlying molecular defects and as drug screening platforms. 52 In particular, BEL-C and BEL-P provide valuable research tools with which to evaluate novel approaches for conversion of fetal to adult and adult to fetal globins in treatment of globin gene disorders, such as induction of fetal globin in patients with sickle cell disease and b-thalassemia.…”

Home Sweet Home: Plasmodium vivax-Infected Reticulocytes—The Younger the Better?

“…As for P. vivax, we are still missing a reliable method for its in vitro culture; the major impediment has been our inability to efficiently handle it under in vitro culture conditions its sole target cell for asexual blood-stage replication: the reticulocyte ( Thomson-Luque et al, 2019). Improved methods for reticulocyte enrichment from different sources have been provided (Vettore et al, 1980;Brun et al, 1990;Kumar et al, 2015;Shaw-Saliba et al, 2016), as well as the production of reticulocytes through better optimized hematopoietic stem cell (HSC) cultures (Giarratana et al, 2005, Noulin et al, 2013Scully et al, 2019) and immortalized lines (Satchwell et al, 2019;Heshusius et al, 2019;Trakarnsanga et al, 2020). The lack of a more efficient enucleation (Menon and Ghaffari, 2021) can be overcome by genetic complementation (Scully et al, 2019).…”

Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

“…HUDEP-2 [19] and BEL-A [20] generated by lentiviral transduction of HEE genes in cord blood and adult CD34 + cells have been extensively characterized for their erythroid progenitor properties, including erythroid differential potential. The method used for the generation of these iEPCs is suitable for creating an iEPC cell bank from patients with erythroid diseases for disease modeling and potential drug screening [34]. However, the major challenge for creating an iEPC bank using this method using HEE lentiviral vectors for immortalization is the requirement of bone marrow aspirates from the patients or treatment with drugs such as granulocyte-macrophage-colony-stimulating factor (GM-CSF) to obtain a sufficient number of CD34 + HSPCs.…”

“…This line was also engineered by gene-editing to produce enucleated RBCs with enhanced transfusion compatibility [33]. Recently, an iEPC was generated using a similar approach from the HSPCs of a patient with HbE/β-thalassemia for disease modeling [34].…”