Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34⁺ isolation, and a 3 × 10⁷-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71^dimCD235a⁺CD44⁺CD117⁻DRAQ5⁻ RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.

The classical central macrophage found in erythroblastic islands plays an important role in erythroblast differentiation, proliferation and enucleation in the bone marrow. Convenient human in vitro models to facilitate the study of erythroid-macrophage interactions are desired. Recently, we demonstrated that cultured monocytes/macrophages enhance in vitro erythropoiesis by supporting hematopoietic stem cell survival. Here, we describe that these specific macrophages also support erythropoiesis. Human monocytes cultured in serum-free media supplemented with stem cell factor, erythropoietin, lipids and dexamethasone differentiate towards macrophages expressing CD16, CD163, CD169, CD206, CXCR4 and the phagocytic TAM-receptor family. Phenotypically they resemble both human bone marrow and fetal liver resident macrophages. This differentiation was dependent on glucocorticoid receptor activation. Proteomic studies confirm that glucocorticoid receptor activation differentiates monocytes to anti-inflammatory tissue macrophages with a M2 phenotype, termed GC-macrophages. Proteins involved in migration, tissue residence and signal transduction/receptor activity were upregulated whilst lysosome and hydrolase activity GO-categories were down regulated. Functionally, we demonstrate that GC-macrophages are highly mobile and can interact to form clusters with erythroid cells of all differentiation stages and phagocytose the expelled nuclei recapitulating aspects of erythroblastic islands. In conclusion, glucocorticoid-directed monocyte differentiation to macrophages represent a convenient model system to study erythroid-macrophage interactions.

The final steps of erythropoiesis involve unique cellular processes including enucleation and reorganization of membrane proteins and the cytoskeleton to produce biconcave erythrocytes. Surprisingly this process is still poorly understood. In vitro erythropoiesis protocols currently produce reticulocytes rather than biconcave erythrocytes. In addition, immortalized lines and iPSC-derived erythroid cell suffer from low enucleation and suboptimal final maturation potential. In light of the increasing prospect to use in vitro produced erythrocytes as (personalized) transfusion products or as therapeutic delivery agents, the mechanisms driving this last step of erythropoiesis are in dire need of resolving. Here we review the elusive last steps of reticulocyte maturation with an emphasis on protein sorting during the defining steps of reticulocyte formation during enucleation and maturation.

Expansion of erythroblasts from human peripheral blood mononuclear cells is 4- to 15-fold more efficient than that of CD34⁺ cells purified from peripheral blood mononuclear cells. In addition, purified CD34⁺ and CD34⁻ populations from blood do not reconstitute this erythroid yield, suggesting a role for feeder cells present in blood mononuclear cells that increase hematopoietic output. Immunodepleting peripheral blood mononuclear cells for CD14⁺ cells reduced hematopoietic stem and progenitor cell expansion. Conversely, the yield was increased upon co-culture of CD34⁺ cells with CD14⁺ cells (full contact or transwell assays) or CD34⁺ cells re-constituted in conditioned medium from CD14⁺ cells. In particular, CD14⁺⁺CD16⁺ intermediate monocytes/macrophages enhanced erythroblast outgrowth from CD34⁺ cells. No effect of CD14⁺ cells on erythroblasts themselves was observed. However, 2 days of co-culturing CD34⁺ and CD14⁺ cells increased CD34⁺ cell numbers and colony-forming units 5-fold. Proliferation assays suggested that CD14⁺ cells sustain CD34⁺ cell survival but not proliferation. These data identify previously unrecognized erythroid and non-erythroid CD34⁻ and CD34⁺ populations in blood that contribute to the erythroid yield. A flow cytometry panel containing CD34/CD36 can be used to follow specific stages during CD34⁺ differentiation to erythroblasts. We have shown modulation of hematopoietic stem and progenitor cell survival by CD14⁺ cells present in peripheral blood mononuclear cells which can also be found near specific hematopoietic niches in the bone marrow.

Key Points: P vivax Duffy binding protein associates exclusively with a small population of immature reticulocytes. Increased DARC-DBP binding site accessibility in immature reticulocytes compared with erythrocytes is a key to infection by P vivax.

The non-selective mechanosensitive ion channel PIEZO1 controls erythrocyte volume homeostasis. Different missense gain-of-function mutations in PIEZO1 gene have been identified that cause Hereditary Xerocytosis (HX), a rare autosomal dominant haemolytic anemia. PIEZO1 expression is not limited to erythrocytes and expression levels are significantly higher in erythroid precursors, hinting to a role in erythropoiesis. During erythropoiesis, interactions between erythroblasts, central macrophages, and extracellular matrix within erythroblastic islands are important. Integrin α4β1 and α5β1 present on erythroblasts facilitate such interactions in erythroblastic islands. Here we found that chemical activation of PIEZO1 using Yoda1 leads to increased adhesion to VCAM1 and fibronectin in flowing conditions. Integrin α4, α5, and β1 blocking antibodies prevented this PIEZO1-induced adhesion suggesting inside-out activation of integrin on erythroblasts. Blocking the Ca²⁺ dependent Calpain and PKC pathways by using specific inhibitors also blocked increased erythroid adhesion to VCAM1 and fibronectins. Cleavage of Talin was observed as a result of Calpain and PKC activity. In conclusion, PIEZO1 activation results in inside-out integrin activation, facilitated by calcium-dependent activation of PKC and Calpain. The data introduces novel concepts in Ca²⁺ signaling during erythropoiesis with ramification on erythroblastic island homeostasis in health and disease like Hereditary Xerocytosis.