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.

Key Points FCA is a novel flow cytometry–based platelet aggregation assay that allows single receptor analysis in small volume/thrombocytopenic samples FCA facilitates platelet studies in experimental animal models even during gestation and allows kinetic measurements in individual animals

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 (K 0), 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.

Key Points• Megakaryocyte-specific Sp1/ Sp3 double-knockout mice display thrombocytopenia, platelet dysfunction, and defects in megakaryocyte maturation.• Selective Mylk inhibition by ML7 affects proplatelet formation and stabilization and ITAM receptor-mediated platelet aggregation.Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation. (Blood. 2015; 125(12):1957-1967 IntroductionPlatelets are the blood cells responsible for maintaining the body hemostasis and, in humans, ;10 11 platelets are produced by megakaryocytes daily.1 Megakaryopoiesis is the process whereby hematopoietic progenitor cells differentiate into mature megakaryocytes, which produce platelets in a process known as thrombopoiesis. 2,3 This takes place at sites of primary hematopoiesis, ie, the bone marrow and, in mice, also in the spleen. 4 The hormone thrombopoietin (TPO), produced by the liver, regulates platelet production. TPO levels in plasma are inversely proportional to the megakaryocyte/ platelet mass. 5 Megakaryocyte differentiation is characterized by highly specialized cellular changes including endomitosis (leading to polyploid cells), accumulation of a and dense granules, and development of a demarcation membrane system (DMS), which is required as a membrane reservoir to produce large numbers of platelets.2,3 Mature megakaryocytes migrate toward the proximity of blood vessels through which they protrude cytoplasmic microtubulerich extensions into the blood stream, known as proplatelets, from which platelets are shed.6 Coordinated interactions between the membrane, cytoskeletal machinery and intracellular signaling are essential for prop...