Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA counterpart in terminal hematopoiesis.

Each day, about 1012 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.

LSD1/KDM1A and GFI1B repress endothelial fate and induce hematopoietic fate in induced pluripotent stem cell-derived hemogenic endotheliums.

Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors.

Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55-2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2 /L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.

Rapid, Sensitive, and Specific Severe Acute Respiratory Syndrome Coronavirus 2 Detection: A Multicenter Comparison Between Standard Quantitative Reverse-Transcriptase Polymerase Chain Reaction and CRISPR-Based DETECTR.

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.

Hemoglobin switching in mice carrying the Klf1Nan variant.

Haploinsufficiency for transcription factor KLF1 causes a variety of human erythroid phenotypes, such as the In(Lu) blood type, increased HbA2 levels, and hereditary persistence of fetal hemoglobin. Severe dominant congenital dyserythropoietic anemia IV (OMIM 613673) is associated with the KLF1 p.E325K variant. CDA-IV patients display ineffective erythropoiesis and hemolysis resulting in anemia, accompanied by persistent high levels of embryonic and fetal hemoglobin. The mouse Nan strain carries a variant in the orthologous residue, KLF1 p.E339D. Klf1Nan causes dominant hemolytic anemia with many similarities to CDA-IV. Here we investigated the impact of Klf1Nan on the developmental expression patterns of the endogenous beta-like and alpha-like globins, and the human beta-like globins carried on a HBB locus transgene. We observe that the switch from primitive, yolk sac-derived, erythropoiesis to definitive, fetal liver-derived, erythropoiesis is delayed in Klf1wt/Nan embryos. This is reflected in globin expression patterns measured between E12.5 and E14.5. Cultured Klf1wt/Nan E12.5 fetal liver cells display growth- and differentiation defects. These defects likely contribute to the delayed appearance of definitive erythrocytes in the circulation of Klf1wt/Nan embryos. After E14.5, expression of the embryonic/fetal globin genes is silenced rapidly. In adult Klf1wt/Nan animals, silencing of the embryonic/fetal globin genes is impeded, but only minute amounts are expressed. Thus, in contrast to human KLF1 p.E325K, mouse KLF1 p.E339D does not lead to persistent high levels of embryonic/fetal globins. Our results support the notion that KLF1 affects gene expression in a variant-specific manner, highlighting the necessity to characterize KLF1 variant-specific phenotypes of patients in detail.

SMIM1 missense mutations exert their effect on wild type Vel expression late in erythroid differentiation.

BACKGROUND: Vel expression on erythrocytes is variable due to polymorphisms, complicating Vel typing. Weak Vel expression can be caused by mutations within SMIM1 in a heterozygous setting, suggesting a dominant negative effect of SMIM1 mutants on wild type (wt)SMIM1 expression. Here we report how SMIM1 expression is regulated during erythropoiesis, to understand its variable expression on erythrocytes. STUDY DESIGN AND METHODS: Peripheral blood reticulocytes at different stages, cultured erythroid precursors and HEK293T cells were used to investigate expression and putative competition between wtSMIM1 and mutated SMIM1 VEL*01W.01, (c.152T>A (p.Met51Lys)), VEL*01W.02 (c.152T>G (p.Met51Arg)), and VEL*01W.03 (c.161T>C (p.Leu54Pro)). RESULTS: Depending on the mutations in SMIM1 an effect on total and membrane expression of SMIM1 was observed in transfected HEK293T cells, but co-expression of wtSMIM1 and mutatedSMIM1 did not have an effect on wtSMIM1 membrane expression. During differentiation of donors expressing VEL*01W.01, VEL*01W.03, Vel-positive, Vel-negative (homozygote SMIM1*64_80del), and Vel-heterozygote SMIM1*64_80del primary human erythroblasts no overt defect was found in Vel expression dynamics or total SMIM1 expression levels when compared with wtSMIM1 erythroblasts. However, during enucleation, total Vel expression was significantly lower on reticulocytes of Vel-weak donors expressing heterozygote mutated SMIM1 compared to Vel-positive or Vel-heterozygote SMIM1*64_80del donors, while Vel expression on extruded nuclei was maintained. In addition, reticulocyte maturation in vivo showed further loss of Vel expression in these individuals and nearly absent on erythrocytes. CONCLUSION: These results suggest that SMIM1 mutations exert a dominant negative effect on wtSMIM1 probably by affecting SMIM1 multimerization and thereby Vel epitope presentation at the latest stages of erythroid differentiation.

Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis.

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

Identification of a novel DI*02(2558T) allele associated with weakened expression of DI2 antigen.

BACKGROUND: The distribution of DI1/DI2 antigens of the Diego blood group system is polymorphic in Mongoloid populations and the corresponding alloantibodies are clinically significant. Here a novel DI variant was found by donor screening, and the effect of the novel and previously reported mutations on expression of DI1/DI2 antigens and Band 3 protein was explored. STUDY DESIGN AND METHODS: DNA samples of 1150 Chinese donors were collected. DI*01/DI*02 genotyping was determined by Sanger sequencing. For the carrier of novel allele, the expression of Band 3 and DI1/DI2 antigens on red blood cells (RBCs) was detected by Western blot and flow cytometry, respectively. in vitro expression studies were conducted by transfecting the mutant (including the novel and three reported DI*02(2534T), DI*02(2358_2359insCAC), and DI*02(2572T) alleles) or wild-type DI*02 constructs into HEK 293T cells, the expression of Band 3 and DI1/DI2 antigens was analyzed. RESULTS: A novel heterozygous mutation (c.2558C>T, p.Thr853Met), which is located near the DI1/DI2 polymorphism site (c.2561T>C), was identified in a donor with DI:-1,2 phenotype. Reduced expression of DI2 antigen was observed on the RBCs, while weakened expression of Band 3 and absence of DI2 antigen were detected in cells transfected with the mutant DI*02(2558T) construct. In addition, absent or decreased expression of Band 3 and DI2 antigen was also detected in cells transfected with three reported mutant constructs. CONCLUSION: The novel DI*02(2558T) allele and three previously described DI mutations can affect the expression of Band 3 protein and/or DI2 antigen and/or interfere with DI*01/DI*02 genotyping result.

Circular RNA expression in human hematopoietic cells is widespread and cell-type specific.

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. CircRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

The RNA-Binding Protein ATXN2 is Expressed during Megakaryopoiesis and May Control Timing of Gene Expression.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

Trichothiodystrophy causative TFIIEß mutation affects transcription in highly differentiated tissue.

The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.

DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism.

Plasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

Molecular mechanisms of bleeding disorderassociated GFI1BQ287* mutation and its affected pathways in megakaryocytes and platelets.

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

A variant RhAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes.

BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c- E- e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.

Development of a recombinant anti-Vel immunoglobulin M to identify Vel-negative donors.

BACKGROUND: Alloimmunization against the high-frequency Vel blood group antigen may result in transfusion reactions or hemolytic disease of fetus and newborn. Patients with anti-Vel alloantibodies require Vel-negative blood but Vel-negative individuals are rare (1:4000). Identification of Vel-negative donors ensures availability of Vel-negative blood; however, accurate Vel blood group typing is difficult due to variable Vel antigen expression and limited availability of anti-Vel typing sera. We report the production of a recombinant anti-Vel that also identifies weak Vel expression. STUDY DESIGN AND METHODS: A recombinant anti-Vel monoclonal antibody was produced by cloning the variable regions from an anti-Vel-specific B cell isolated from an alloimmunized patient into a vector harboring the constant regions of immunoglobulin (Ig)G1-kappa or IgM-kappa. Antibody Vel specificity was tested by reactivity to SMIM1-transfected HEK293T cells and by testing various red blood cells (RBCs) of donors with normal, weak, or no Vel expression. High-throughput donor screening applicability was tested using an automated blood group analyzer. RESULTS: A Vel-specific IgM class antibody was produced. The antibody was able to distinguish between Vel-negative and very weak Vel antigen-expressing RBCs by direct agglutination and in high-throughput settings using a fully automated blood group analyzer and performed better than currently used human anti-Vel sera. High-throughput screening of 13,288 blood donations identified three new Vel-negative donors. CONCLUSION: We generated a directly agglutinating recombinant anti-Vel IgM, M3F5S-IgM, functional in manual, automated agglutination assays and flow cytometry settings. This IgM anti-Vel will improve diagnostics by facilitating the identification of Vel-negative blood donors.

Glucocorticoids induce differentiation of monocytes towards macrophages that share functional and phenotypical aspects with erythroblastic island macrophages.

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 and progenitor cell survival. Herein, 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 is 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 are upregulated whilst lysosome and hydrolase activity GO-categories are downregulated. 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 represents a convenient model system to study erythroid-macrophage interactions.