Human Hematopoietic Progenitor Cells and Erythrocytes Generated from Human iPSCs Lacking CD55

Abstract

CD55 (OMIM*125240; also known as Decay-Accelerating Factor for complement or DAF) is a plasma membrane protein that is widely distributed on all blood cells and many other cell types. A critical physiologic role of CD55 is to inhibit the complement cascade at the level of the central C3 convertase step. Variation in CD55 (GPI-anchored) forms the basis of the Cromer blood group system (CROM). CD55 also serves as a receptor for certain strains of E. coli and certain types of enteroviruses. More recently, CD55 was identified as a receptor for Plasmodium falciparum, a deadly malaria parasite that infects human erythrocytes. This finding was reached by experiments of gene knock-down using RNA interference in human cord blood hematopoietic progenitor cells that were subsequently differentiated into erythrocytes (1). In cells with lower expression of CD55, there were lower rates of infection. CD55 null erythrocytes from patient samples were refractory to infection, implying that CD55 is essential for invasion. We decided to use the erythrocytes generated from human induced pluripotent stem cells (iPSCs), which can be expanded without limit and efficiently genetically modified in order to obtain cell lines completely lacking CD55 expression. This novel approach is feasible because we previously established a methodology (2014 ASH meeting presentation) for generating human erythrocytes after terminal differentiation of iPSC-derived hematopoietic cells, in which 5-10% of the cells became enucleated (2). In this study, we found that a lab strain of P. falciparum parasites is able to invade and propagate in the iPSC-derived erythrocytes.To knock out the CD55 gene and generate large numbers of erythrocytes that are resistant to the infection of P. falciparum parasites, we used two different CRISPR guide RNAs as well as SpCas9 in human iPSCs. We successfully isolated and expanded human iPSC clones that have 85-bp deletion in CD55 exon 1 of both alleles, resulting in the frameshift and the disappearance of CD55 expression on cell surface. To assure that the CRISPR mediated the knock-out (KO) is specific to the CD55 gene, we used a doxycycline-inducible expression vector to deliver a CD55 transgene. Upon doxycycline induction, CD55 cell surface expression was restored in the selected CD55 KO iPSC lines. The complete CD55 KO had little effects in the growth of human iPSCs or their differentiation into hematopoietic progenitor cells that co-express CD45 and CD34. However, we observed that erythroid differentiation was reduced in the absence of CD55 expression. This observation contradicts the previous publication where CD55 RNA interference technology was likely incomplete (1). The inducible CD55 expression system we constructed will be able to overcome the requirement for CD55 before the formation of mature erythrocytes and invasion by P. falciparum parasites.The present study provides a novel genetic model for studying the infection of P. falciparum malaria parasites into human erythrocytes, based on the robust technologies of genome-edited human iPSCs and derived erythrocytes. The genetically modified erythrocytes such ones lacking CD55 expression may provide a novel source of P. falciparum resistant erythrocytes for blood transfusion in areas that malaria is epidemic and many people are anemic. This study will also provide a proof-of-principle that we can generate a large number of human erythrocytes with gain of function from a small number of genetically modified stem cells.