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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.

Abstract Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe3+, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe3+ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe3+ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.

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.

Abstract Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

Abstract Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated β-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a−b−) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.

Abstract In β-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell–cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop β-hemoglobinopathies treatments.

Erythrocytes represent at least 60% of all cells in the human body. During circulation, they experience a huge variety of physical and chemical stimulations, such as pressure, shear stress, hormones or osmolarity changes. These signals are translated into cellular responses through ion channels that modulate erythrocyte function. Ion channels in erythrocytes are only recently recognized as utmost important players in physiology and pathophysiology. Despite this awareness, their signaling, interactions and concerted regulation, such as the generation and effects of “pseudo action potentials”, remain elusive. We propose a systematic, conjoined approach using molecular biology, in vitro erythropoiesis, state-of-the-art electrophysiological techniques, and channelopathy patient samples to decipher the role of ion channel functions in health and disease. We need to overcome challenges such as the heterogeneity of the cell population (120 days lifespan without protein renewal) or the access to large cohorts of patients. Thereto we will use genetic manipulation of progenitors, cell differentiation into erythrocytes, and statistically efficient electrophysiological recordings of ion channel activity.

Abstract: 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 CD71dimCD235a+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.

E-cadherin is a key regulator of epithelial cell–cell adhesion, the loss of which accelerates tumor growth and invasion. E-cadherin is also expressed in hematopoietic cells as well as epithelia. The function of hematopoietic E-cadherin is, however, mostly elusive. In this study, we explored the validity of mouse models to functionally investigate the role of hematopoietic E-cadherin in human hematopoiesis. We generated a hematopoietic-specific E-cadherin knockout mouse model. In mice, hematopoietic E-cadherin is predominantly expressed within the basophil lineage, the expression of which is dispensable for the generation of basophils. However, neither E-cadherin mRNA nor protein were detected in human basophils. In contrast, human hematopoietic E-cadherin marks the erythroid lineage. E-cadherin expression in hematopoiesis thereby revealed striking evolutionary differences between the basophil and erythroid cell lineage in humans and mice. This is remarkable as E-cadherin expression in epithelia is highly conserved among vertebrates including humans and mice. Our study therefore revealed that the mouse does not represent a suitable model to study the function of E-cadherin in human hematopoiesis and an alternative means to study the role of E-cadherin in human erythropoiesis needs to be developed.

Induced pluripotent stem cell (iPSC) line was generated from erythroblasts (EBLs) derived from a patient diagnosed with severe congenital neutropenia, caused by mutations in ELANE (c.614delG). Transgene-free iPSC line was generated using Sendai virus reprogramming. The iPSC line showed normal karyotype, expressed pluripotency associated genes and was capable of in vitro spontaneous differentiation towards the three germ layers. The generated iPSC line can be used to study severe congenital neutropenia and the role of neutrophil elastase protein.

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Gray Platelet Syndrome (GPS), caused by compound heterozygous NBEAL2 mutations (c.6568delT and c.7937T>C). GPS is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study GPS pathophysiology and the basic functions of NBEAL2 protein in different cell types.

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Chédiak-Higashi Syndrome (CHS), caused by mutations in LYST (c.4322_4325delAGAG and c.10127A>G). EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study CHS pathophysiology and the role of LYST in different cell types.

Induced pluripotent stem cells (iPSCs) were generated from blood outgrowth endothelial cells (BOECs) obtained from a healthy donor and from a patient diagnosed with Hermansky Pudlak Syndrome type 2 (HPS2), caused by compound heterozygous AP3B1 mutations (c.177delA and c.1839-1842delTAGA). BOECs were reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study HPS2 pathophysiology and the basic functions of AP3B1 protein in different cell types.

In vitro differentiation of red blood cells (RBCs) is a desirable therapy for various disorders. Here the authors develop a culture system using stem cell-derived macrophages to show that inducible expression of a transcription factor, KLF1, enhances RBC production, potentially through the induction of three soluble factors, ANGPTL7, IL33 and SERPINB2.

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.

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8–10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Keywords: iPSC, Differentiation, Erythroid, Hematopoietic, Megakaryocytic, Myeloid

Peripheral blood mononuclear cells were isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system.

Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C → T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causing mutation was shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B.

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.

Glutaraldehyde is a well-known substance used in biomedical research to fix cells. Since hemolytic anemias are often associated with red blood cell shape changes deviating from the biconcave disk shape, conservation of these shapes for imaging in general and 3D-imaging in particular, like confocal microscopy, scanning electron microscopy or scanning probe microscopy is a common desire. Along with the fixation comes an increase in the stiffness of the cells. In the context of red blood cells this increased rigidity is often used to mimic malaria infected red blood cells because they are also stiffer than healthy red blood cells. However, the use of glutaraldehyde is associated with numerous pitfalls: (i) while the increase in rigidity by an application of increasing concentrations of glutaraldehyde is an analog process, the fixation is a rather digital event (all or none); (ii) addition of glutaraldehyde massively changes osmolality in a concentration dependent manner and hence cell shapes can be distorted; (iii) glutaraldehyde batches differ in their properties especially in the ratio of monomers and polymers; (iv) handling pitfalls, like inducing shear artifacts of red blood cell shapes or cell density changes that needs to be considered, e.g., when working with cells in flow; (v) staining glutaraldehyde treated red blood cells need different approaches compared to living cells, for instance, because glutaraldehyde itself induces a strong fluorescence. Within this paper we provide documentation about the subtle use of glutaraldehyde on healthy and pathologic red blood cells and how to deal with or circumvent pitfalls.

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.

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.

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.

Control of gene expression in erythropoiesis has to respond to signals that may emerge from intracellular processes or environmental factors. Control of mRNA translation allows for relatively rapid modulation of protein synthesis from the existing transcriptome. For instance, the protein synthesis rate needs to be reduced when reactive oxygen species or unfolded proteins accumulate in the cells, but also when iron supply is low or when growth factors are lacking in the environment. In addition, regulation of mRNA translation can be important as an additional layer of control on top of gene transcription, in which RNA binding proteins (RBPs) can modify translation of a set of transcripts to the cell’s actual protein requirement. The 5′ and 3′ untranslated regions of mRNA (5′UTR, 3′UTR) contain binding sites for general and sequence specific translation factors. They also contain secondary structures that may hamper scanning of the 5′UTR by translation complexes or may help to recruit translation factors. In addition, the term 5′UTR is not fully correct because many transcripts contain small open reading frames in their 5′UTR that are translated and contribute to regulation of mRNA translation. It is becoming increasingly clear that the transcriptome only partly predicts the proteome. The aim of this review is (i) to summarize how the availability of general translation initiation factors can selectively regulate transcripts because the 5′UTR contains secondary structures or short translated sequences, (ii) to discuss mechanisms that control the length of the mRNA poly(A) tail in relation to mRNA translation, and (iii) to give examples of sequence specific RBPs and their targets. We focused on transcripts and RBPs required for erythropoiesis. Whereas differentiation of erythroblasts to erythrocytes is orchestrated by erythroid transcription factors, the production of erythrocytes needs to respond to the availability of growth factors and nutrients, particularly the availability of iron.

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.

Erythropoiesis is regulated at many levels, including control of mRNA translation. Changing environmental conditions, such as hypoxia or the availability of nutrients and growth factors, require a rapid response enacted by the enhanced or repressed translation of existing transcripts. Cold shock domain protein e1 (Csde1/Unr) is an RNA-binding protein required for erythropoiesis and strongly upregulated in erythroblasts relative to other hematopoietic progenitors. The aim of this study is to identify the Csde1-containing protein complexes and investigate their role in post-transcriptional expression control of Csde1-bound transcripts. We show that Serine/Threonine kinase receptor-associated protein (Strap/Unrip), was the protein most strongly associated with Csde1 in erythroblasts. Strap is a WD40 protein involved in signaling and RNA splicing, but its role when associated with Csde1 is unknown. Reduced expression of Strap did not alter the pool of transcripts bound by Csde1. Instead, it altered the mRNA and/or protein expression of several Csde1-bound transcripts that encode for proteins essential for translational regulation during hypoxia, such as Hmbs, eIF4g3 and Pabpc4. Also affected by Strap knockdown were Vim, a Gata-1 target crucial for erythrocyte enucleation, and Elavl1, which stabilizes Gata-1 mRNA. The major cellular processes affected by both Csde1 and Strap were ribosome function and cell cycle control.

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6−/− erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6−/− erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia – and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive “hook” bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of β-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.

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.

MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells and in mature megakaryocytes. This biphasic expression of MEIS1 suggests that the function of MEIS1 in stem cells is distinct from its function in lineage committed cells. Mouse models show that Meis1 is required for renewal of stem cells, but the function of MEIS1 in human hematopoietic progenitor cells has not been investigated. We show that two MEIS1 splice variants are expressed in hematopoietic progenitor cells. Constitutive expression of both variants directed human hematopoietic progenitors towards a megakaryocyte-erythrocyte progenitor fate. Ectopic expression of either MEIS1 splice variant in common myeloid progenitor cells, and even in granulocyte-monocyte progenitors, resulted in increased erythroid differentiation at the expense of granulocyte and macrophage differentiation. Conversely, silencing MEIS1 expression in progenitor cells induced a block in erythroid expansion and decreased megakaryocytic colony formation capacity. Gene expression profiling revealed that both MEIS1 splice variants induce a transcriptional program enriched for erythroid and megakaryocytic genes. Our results indicate that MEIS1 expression induces lineage commitment towards a megakaryocyte-erythroid progenitor cell fate in common myeloid progenitor cells through activation of genes that define a megakaryocyte-erythroid-specific gene expression program.

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5'UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5'UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5'UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.

β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) ‘responders’ who increase hemoglobin to therapeutic levels; (ii) ‘moderate-responders’ who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) ‘non-responders’ who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it had little impact on cells from responders. Part of the gene program up-regulated by hydroxyurea in non-responders was already highly expressed in responders before hydroxyurea treatment. Baseline HbF expression was low in non-responders, and hydroxyurea treatment induced significant cell death. We conclude that cells from responders have adapted well to constitutive stress conditions and display a propensity to proceed to the erythroid differentiation program.

The endonuclease complex Ercc1/Xpf is involved in interstrand crosslink repair and functions downstream of the Fanconi pathway. Loss of Ercc1 causes hematopoietic defects similar to those seen in Fanconi Anemia. Ercc1−/− mice die 3-4 weeks after birth, which prevents long-term follow up of the hematopoietic compartment. We used alternative Ercc1 mouse models to examine the effect of low or absent Ercc1 activity on hematopoiesis. Tie2-Cre-driven deletion of a floxed Ercc1 allele was efficient (>80%) in fetal liver hematopoietic cells. Hematopoietic stem and progenitor cells (HSPCs) with a deleted allele were maintained in mice up to 1 year of age when harboring a wt allele, but were progressively outcompeted when the deleted allele was combined with a knockout allele. Mice with a minimal Ercc1 activity expressed by 1 or 2 hypomorphic Ercc1 alleles have an extended life expectancy, which allows analysis of HSPCs at 10 and 20 weeks of age. The HSPC compartment was affected in all Ercc1-deficient models. Actively proliferating multipotent progenitors were most affected as were myeloid and erythroid clonogenic progenitors. In conclusion, lack of Ercc1 results in a severe competitive disadvantage of HSPCs and is most deleterious in proliferating progenitor cells.

Background In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established.Design and Methods To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene.Results We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts.Conclusions We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins.

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD(G602D/R722W)/XPA(-/-)) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80(-/-) mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.