Large Scale Culture and Differentiation of Erythroblasts from PBMC and iPSC
Authors: Claessen Maria , Varga Eszter , Heshusius Steven , Heideveld Esther , Hansen Martin , Visser Anna , Thiel-Valkhof Marijke , Sellink Erica , Burger Patrick , Zeerleder Sacha , Kersten Marie Jose , van den Akker Emile , von Lindern Marieke 
Published: October 2019

Abstract
Transfusion of donor-derived red blood cells to aleviate anemia is the most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs or enzymes. However, the source depends on donor availability and carries a potential risk of alloimmunization and blood borne diseases. More than 30 bloodgroup systems encode >300 bloodgroup antigens and bloodgroup matching becomes increasingly challenging in a multiethnic society. Particularly the chronically transfused patients are at risk for alloimmunisation. In vitro cultured, customizable red blood cells (cRBC) would negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications. We aim to produce human cRBC at large-scale and cost effective, for which we need to optimize culture conditions and reduce cost-drivers. We adapted our protocols to GMP culture requirements, which reproducibly provided pure human erythroid cultures within 25 days with a 3.4x107 times expansion from peripheral blood mononuclear cells without prior CD34+ isolation. This expansion depended on the serum free medium we produce, which is supplemented with erythropoietin (Epo, 1 U/ml), stem cell factor (SCF) and glucocorticoids. Expanded erythroblasts CD71 highCD235low/- were differentiated for 10 days in medium supplemented with 5% human plasma, heparin and a higher concentration of Epo (10U/ml) yielding CD71dimCD235a+CD44+CD117-DRAQ5- cRBC. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability was measured by an Automated Rheoscope and Cell Analyser (ARCA), and oxygen equilibrium curves were measured with a Hemox analyzer. Both parameters were similar in cRBC and freshly isolated reticulocytes. RNA sequencing was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, and erythroid specific metabolic enzymes, concommittant with loss of expression of genes involved in the formation of organelles, and cell proliferation. The culture process is compatible with upscaling using 5L G-Rex bioreactors., Currently we are preparing a clinical study using biotinylated cRBC. Ultimately, however, large scale production requires an immortal source, for which we aim to use human induced pluripotent stem cells (iPSC) established from rare donors that lack most blood group antigens. Using single cell passaging of iPSC and differentiation in colonies, we generate at average 2x105 cRBC per single iPSC. However, the cRBC cultured from iPSC were less stable following enucleation, and expressed embryonic type globins. Comparison of transcriptome data from iPSC-derived erythroid cells at distinct differentiation stages with erythroid cells at similar stages that were cultured from adult- or cord blood mononuclear cells, or from fetal liver confirmed that most iPSC-derived erythroid cells largely express an embryonic RNA profile. In conclusion, our current protocols enable us to test cRBC cultured from adult peripheral blood for their stability after transfusion. Concurrently, we develop novel bioreactors to upscale the production, and we optimise the protocol to generate cRBC from immortal iPSC lines with near 'universal donor' genotypes.

Disclosures
No relevant conflicts of interest to declare.