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

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Blood group type A secretors are associated with a higher risk of COVID-19 cardiovascular disease complications.

The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or secretor status was associated with COVID-19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non-erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non-secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS-CoV-2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% [1.05, 1.47], p = 0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR = 2.56, CI 95% [1.43, 4.55], p = 0.0011) which is independent of gender. Molecular analysis revealed that group A non-secretors are significantly less likely to be hospitalized than secretors. Testing of convalescent plasma donors, among whom the majority displayed COVID-19 symptoms and only a small minority required hospitalization, group A non-secretors were slightly over-represented. Our findings showed that group A non-secretors are not resistant to infection by SARS-CoV-2, but are more likely to experience a less severe form of associated disease.

Large red cell-derived membrane particles are major contributors to hypercoagulability in sickle cell disease.

Sickle cell disease (SCD) is one of the most common inherited single gene disorders. Polymerisation of sickle hemoglobin results in erythrocytes that are inflexible and adherent, leading to coagulation, vascular and cellular activation and resultant blood vessel blockage. Previous studies have observed elevated numbers of red cell-derived particles (RCDP), also denoted extracellular vesicles, in SCD plasma. Here, imaging flow cytometry was used to quantify all RCDP in SCD plasma. A more heterogenous population of RCDP was observed than previously reported. Significantly, large right side-out red cell macrovesicles (MaV), 7 µm in diameter, were identified. Most RCDP were right side-out but a minor population of inside-out vesicles was also present. Electron micrographs confirmed the heterogenous nature of the RCDP detected. All MaV are decorated with prothrombotic phosphatidylserine (PS) and their removal from plasma lengthened clotting times by more than three-fold. Removal of all right side-out RCDP from SCD patient plasma samples resulted in a seven-fold increase in clotting time. These results indicate that MaV comprise a large area of prothrombotic membrane and are thus major contributors to hypercoagulation in SCD. Consequently, controlled removal of MaV and PS exposed RCDP from plasma could provide a novel therapy for managing this disease.

Disruption of the tumour-associated EMP3 enhances erythroid proliferation and causes the MAM-negative phenotype.

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.

Vimentin expression is retained in erythroid cells differentiated from human iPSC and ESC and indicates dysregulation in these cells early in differentiation.

BACKGROUND: Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. METHODS: In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. RESULTS: We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. CONCLUSION: As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies.

Non-muscle myosin II drives vesicle loss during human reticulocyte maturation.

The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.

Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing.

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle-cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S-s-U-). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells.

With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.

Superior survival of ex vivo cultured human reticulocytes following transfusion into mice.

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

The ins and outs of reticulocyte maturation revisited: The role of autophagy in sickle cell disease.

Autophagy plays an important role in the removal of membrane bound organelles during the last stage of erythropoiesis as the enucleate reticulocyte matures into the erythrocyte. Autophagic vesicles are expelled from the reticulocyte as intact, inside-out, phosphatidylserine (PS) decorated vesicles and are subsequently removed during splenic passage. Failure to remove these vesicles causes the elevation in PS exposed red cells in Sickle Cell Disease.

Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation.

Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin.

Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.

Induction of adult levels of ß-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL.

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on ß-globin expression. In contrast, transduction with both transcription factors stimulated ß-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of ß-globin equivalent to that of adult erythroid cells. Interestingly, this increase in ß-globin was coincident with a decrease in ε- and ζ-, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of ß-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin.

Qualitative and quantitative comparison of the proteome of erythroid cells differentiated from human iPSCs and adult erythroid cells by multiplex TMT labelling and nanoLC-MS/MS.

Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10-15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

Tetraspanins CD81 and CD82 facilitate α4ß1-mediated adhesion of human erythroblasts to vascular cell adhesion molecule-1.

The proliferation and terminal differentiation of erythroid progenitors occurs in human bone marrow within erythroblastic islands, specialised structures consisting of a central macrophage surrounded by developing erythroid cells. Many cell-cell and cell-matrix adhesive interactions maintain and regulate the co-ordinated daily production of reticulocytes. Erythroid cells express only one integrin, α4ß1, throughout differentiation, and its interactions with both macrophage Vascular Cell Adhesion Molecule-1 and with extracellular matrix fibronectin are critical for erythropoiesis. We observed that proerythroblasts expressed a broad tetraspanin phenotype, and investigated whether any tetraspanin could modulate integrin function. A specific association between α4ß1 and CD81, CD82 and CD151 was demonstrated by confocal microscopy and co-immune precipitation. We observed that antibodies to CD81 and CD82 augmented adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 but not to the fibronectin spliceoforms FnIII12-IIICS-15 and FnIII12-15. In contrast, different anti-CD151 antibodies augmented or inhibited adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 and the fibronectin spliceoform FnIII12-IIICS-15 but not to FnIII12-15. These results strongly suggest that tetraspanins have a functional role in terminal erythropoiesis by modulating interactions of erythroblast α4ß1 with both macrophages and extracellular matrix.

Autophagy facilitates organelle clearance during differentiation of human erythroblasts: evidence for a role for ATG4 paralogs during autophagosome maturation.

Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B (C74A) ; ATG4D (C144A) ) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B (C74A) -expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.