Universal Red Blood Cells Research Articles

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Recent advances on the development of a universal blood type

Blood transfusion is the most common form of tissue transplant, and correct blood type matching is decisive for the success of this procedure. However, the availability of different blood types depends on each population, leading to a shortage of blood units from uncommon types. Then, it represents a problem for patients who need a blood transfusion because the supply for their blood type is scarce. Fortunately, researchers have been developing different techniques to engineer universal red blood cells (RBC) that could be transplanted to any human being independent of its blood type. This paper details the main features of blood transfusion and red blood cells maintenance and the two more recent procedures used to produce universal RBCs, the Enzymatically Converted Group O (ECO) and the antigen masking methods.

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

SummaryUniversal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs.We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs.The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Surface-anchored framework for generating RhD-epitope stealth red blood cells.

Rhesus D (RhD) is one of the most important immunogenic antigens on red blood cells (RBCs). However, the supply of RhD-negative blood frequently faces critical shortages in clinical practice, and the positive-to-negative transition of the RhD antigen remains a great challenge. Here, we developed an alternative approach for sheltering the epitopes on RhD-positive RBCs using a surface-anchored framework, which is flexible but can achieve an optimal shield effect with minimal physicochemical influence on the cell. The chemical framework completely obstructed the RhD antigens on the cell surface, and the assessments of both blood transfusion in a mouse model and immunostimulation with human RhD-positive RBCs in a rabbit model confirmed the RhD-epitope stealth characteristics of the engineered RBCs. This work provides an efficient methodology for improving the cell surface for universal blood transfusion and generally indicates the potential of rationally designed cell surface engineering for transfusion and transplantation medicine.

Development of a scalable agitation suspension culture differentiation platform for generating erythroid cells from O-negative human induced pluripotent stem cells

Background & Aim Universal red blood cells (RBCs) derived from differentiation of O-negative (neg) human induced pluripotent stem cells (hiPSCs) can potentially supplement the emergency transfusion needs of the healthcare industry. Given that each unit of blood requires 1 trillion RBCs, there is a need to develop efficient bioprocesses that could allow for large-scale differentiation and generation of very high numbers of RBCs. While many groups have described means to differentiate hiPSCs into erythroid cells, most of these have not yet been demonstrated to be amenable for scale-up. Methods, Results & Conclusion Using multifactorial process optimization, we have developed a continuous suspension agitation culture platform for differentiating hiPSCs-microcarrier aggregates towards erythroid cells. We have efficiently scaled-up the process starting from 5 ml ultra-low attachment 6 well plates to 125 ml spinner culture flasks. The best performing hiPSC line was efficiently differentiated, generating close to 1.5 billion erythroblasts in 100 ml culture volumes with peak cell densities of erythroblasts approaching 1.5 × 107 cells/ml. By flow cytometry, as much as 60% of hiPSC derived erythroblasts were shown to be enucleated following an optimized scalable co-culture system that we developed. Functional (oxygen binding, hemoglobin characterization and membrane deformability) and transcriptomics evaluation show minimal differences between hiPSC derived RBCs as compared to cord-blood and adult derived RBCs, making them potentially useful for future transfusion applications. The agitation suspension culture differentiation bioprocess developed by our group could serve as a key platform for future scale-up of hiPSCs differentiation towards RBCs and other hematopoietic lineages in controlled bioreactors.

Production of Universal Group O Red Blood Cells by Alpha-N-Acetylgalactosaminidase Enzyme Expressed in Pichia pastoris.

Enzymatic removal of blood groups antigens A and B is an efficient method for production of universal red blood cells. In this research, an α-N-acetylgalactosaminidase (NAGA) enzyme was expressed in Pichia pastoris for digestion of the A blood antigen. DNA sequence of the gene NAGA, originally expressed in Elizabethkingia meningosepticum (NAGA-EM), was ordered for optimization and synthesis. It was then expressed in P. pastoris (KM71H and GS115 strains). Expression of the recombinant NAGA was evaluated by dot blot, SDS-PAGE, and Western blotting. The activity of the enzyme was measured using a synthetic substrate in addition to the conversion of group A red blood cells to the O cells. Expression of NAGA-EM with an apparent molecular mass of 55kDa was verified by dot blot, SDS-PAGE and Western blot analysis. The maximum enzyme activity in the supernatant of KM71H was higher than that in the GS115 (250 vs. 200 U/ml). Treated group A RBCs did not react with the anti-A antiserum or with the sera from individuals with blood groups B and O. The results of this study indicated that NAGA-EM is an efficient enzyme for production of universal O blood cells.

Bacterial glycosidases acting on red blood cells

Glycosidases represent the major catalytic machinery for the breakage of glycosidic bonds. A classification system for glycosyl hydrolases, based on sequence similarity, is available on the dedicated carbohydrate‐active enzymes (CAZy) database and has led to the definition of more than 130 different families (1). Among them, two families of bacterial glycosyl hydrolases, identified by a combination of screening and bioinformatics approaches, perform efficiently and specifically in conversion of red blood cells for the production of universal red blood cells (2,3). Most of the enzymes are found in symbiotic or soil‐borne bacteria. Because members of one family have essentially identical kinetic properties with both branched and linear substrates, they can also act in the enzymatic removal of immunodominant alpha3Gal xenotransplantation epitope (3). We will describe the molecular determinants responsible for the fine substrate specificity of these enzymes and emphasize the potential biomedical applications in remodeling glycan structures.Support or Funding InformationSupported by the CNRS and DGA

Antigenically shielded universal red blood cells by polydopamine-based cell surface engineering

A strategy of surface engineering by using polydopamine was developed to shelter antigens on red blood cells and prevent coagulation reactions.

Large‐scale production of red blood cells from stem cells: What are the technical challenges ahead?

Blood-transfusion centers regularly face the challenge of donor blood shortages, especially for rare blood groups. The possibility of producing universal red blood cells from stem cells industrially has become a possible alternative since the successful injection of blood generated in vitro into a human being in 2011. Although there remains many biological and regulatory issues concerning the efficacy and safety of this new product, the major challenge today for future clinical applications is switching from the current limited 2-dimensional production techniques to large-scale 3-dimensional bioreactors. In addition to requiring technological breakthroughs, the whole process also has to become at least five-fold more cost-efficient to match the current prices of high-quality blood products. The current review sums up the main biological advances of the past decade, outlines the key biotechnological challenges for the large-scale cost-effective production of red blood cells, proposes solutions based on strategies used in the bioindustry and presents the state-of-the-art of large-scale blood production.

Poly(2-dimethylamino ethylmethacrylate)-Based Polymers To Camouflage Red Blood Cell Antigens

Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA) is a cationic polymer when dissolved in a 7.4 pH fluid. Owing to its ionic nature, this polycation interacts with the negatively charged cell membrane surface of red blood cells (RBCs). The electrostatic self-assembly of PDMAEMA on RBCs membrane can be employed for inducing the formation of a polymeric shield camouflaging blood group antigens on RBCs as a valuable strategy for developing "universal RBCs" for blood transfusion. The purpose of this research was to evaluate the camouflaging ability of PDMAEMA homopolymers and PDMAEMA-co-poly(ethylene glycol) copolymers differing in molecular weight and architecture. Surprisingly, the PDMAEMAs caused a partially masking, no masking, and sensitization of the same RBCs population. The MW and architecture of the polymers as well as temperature of PDMAEMA-RBCs treatment influenced the results observed. Herein, the very particular reactivity of PDMAEMAs and RBCs is analyzed and discussed.

Properties of a Novel <italic>α</italic>-galactosidase from <italic>B. Fragilis</italic> and Its Potential for Human Blood-type B to O Conversion

Enzymatic removal of blood group B antigen is an effective method to develop universal red blood cells (RBCs) by α-galactosidase. Here we investigated the physicochemical properties of a novel α-galactosidase from B. Fragilis and the optimization of enzymatic conversion for RBC of blood group B to O. The results showed that α-galactosidase exhibited maximum activity at a broad optimal pH of 5.6-6.0 and an optimum temperature of 41°C. Furthermore, the enzyme was efficient in complete removal of B antigen. The conditions for B to O blood group conversion were 26°C, pH 6.8 (250 mmol/L glycine and 3 mmol/L NaCl) for 1 h. The structure and function indexes of the converted red blood cells showed no significant difference from those of normal RBCs. Consequently, the novel α-galactosidase described here was more suitable for enzymatic conversion for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Fatal cold agglutinin-induced haemolytic anaemia: a case report

IntroductionCold agglutinin disease usually develops as a result of the production of a specific immunoglobulin M auto-antibody directed against the I/i and H antigens, precursors of the ABH and Lewis blood group substances, on red blood cells. Autoimmune and lymphoproliferative disorders, Mycoplasma pneumoniae and other infections can be associated with the production of cold agglutinins. In its classic presentation with haemolytic anaemia and Raynaud's syndrome, cold agglutinin disease is usually idiopathic. Several factors play a role in determining the ability of a cold agglutinin to induce a haemolytic anaemia such as antibody concentration and temperature range, in particular the highest temperature at which antibodies interact with red blood cells.Case presentationA 48-year-old Caucasian man presented to our hospital with symptoms of extreme asthenia caused by severe anaemia. The transfusion of red blood cells (O Rh-positive), started as prescribed by the emergency guidelines in force without pre-transfusion tests, induced fatal haemolysis because of the presence of high levels of anti-H antibodies in his blood, that reacted with the large amount of H antigen in universal (0) red blood cells.ConclusionEmergency transfusion of universal red blood cells (0 Rh-positive or negative) is usually accepted by the international guidelines in force in emergency departments. In this report we describe a rare complication caused by the very high concentration in the recipient of cold agglutinins and the activation of the complement system, responsible for red blood cell lysis and consequent fatal cardiovascular shock. We conclude that emergency transfusion of universal red blood cells (0 Rh-positive or negative) may be dangerous and its risk should be assessed against the risk of delaying transfusion until the pre-transfusion tests are completed.

Bacterial glycosidases for the production of universal red blood cells

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Thiolation Mediated Pegylation Platform to Generate Functional Universal Red Blood Cells

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Surface decoration of red blood cells with maleimidophenyl‐polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells

The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.

Universal red blood cells—enzymatic conversion of blood group A and B antigens

Accidental transfusion of ABO-incompatible red blood cells (RBCs) is a leading cause of fatal transfusion reactions. To prevent this and to create a universal blood supply, the idea of converting blood group A and B antigens to H using specific exo-glycosidases capable of removing the immunodominant sugar residues was pioneered by Goldstein and colleagues at the New York Blood Center in the early 1980s. Conversion of group B RBCs to O was initially carried out with α-galactosidase extracted from coffee beans. These enzyme-converted O (ECO) RBCs appeared to survive normally in all recipients independent of blood group. The clinical trials moved from small infusions to single RBC units and finally multiple and repeated transfusions. A successful phase II trial utilizing recombinant enzyme was reported by Kruskall and colleagues in 2000. Enzymatic conversion of group A RBCs has lagged behind due to lack of appropriate glycosidases and the more complex nature of A antigens. Identification of novel bacterial glycosidases with improved kinetic properties and specificities for the A and B antigens has greatly advanced the field. Conversion of group A RBCs can be achieved with improved glycosidases and the conversion conditions for both A and B antigens optimized to use more cost-efficient quantities of enzymes and gentler conditions including neutral pH and short incubation times at room temperature. Of the different strategies envisioned to create a universal blood supply, the ECO concept is the only one, for which human clinical trials have been performed. This paper discusses some biochemical and clinical aspects of this developing technology.