Red Blood Cell Production Research Articles

Accessory-cell-free differentiation of hematopoietic stem and progenitor cells into mature red blood cells

Background aimsThe culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis, i.e., the terminal maturation of reticulocytes into erythrocytes, so that it can occur without feeder or accessory cells and animal-derived components. MethodsHere, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and xeno-free conditions. ResultsThe approach improves post-enucleation cell integrity and cell survival, and it enables subsequent storage of cRBCs for up to 42 days in classical additive solution conditions without any specialized equipment. ConclusionsWe foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs.

Defects in Bone and Bone Marrow in Inherited Anemias: the Chicken or the Egg.

Recently, there has been an increasing number of studies on the crosstalk between the bone and the bone marrow and how it pertains to anemia. Here, we discuss four heritable clinical syndromes contrasting those in which anemia affects bone growth and development, with those in which abnormal bone development results in anemia, highlighting the multifaceted interactions between skeletal development and hematopoiesis. Anemia results from both inherited and acquired disorders caused by either impaired production or premature destruction of red blood cells or blood loss. The downstream effects on bone development and growth in patients with anemia often constitute an important part of their clinical condition. We will discuss the interdependence of abnormal bone development and growth and hematopoietic abnormalities, with a focus on the erythroid lineage. To illustrate those points, we selected four heritable anemias that arise from either defective hematopoiesis impacting the skeletal system (the hemoglobinopathies β-thalassemia and sickle cell disease) versus defective osteogenesis resulting in impaired hematopoiesis (osteopetrosis). Finally, we will discuss recent findings in Diamond Blackfan anemia, an intrinsic disorder of both the erythron and the bone. By focusing on four representative hereditary hematopoietic disorders, this complex relationship between bone and blood should lead to new areas of research in the field.

Oral 3-hydroxybuturate ingestion acutely lowers circulating testosterone concentrations in healthy young males.

Ketone bodies, such as 3-hydroxybutyrate (3-OHB), have been frequently used by endurance athletes, such as cyclists, to enhance performance and recovery and are recognized for their health benefits and therapeutic effects for decades. Testosterone is a potent regulator of red blood cell production. Evidence suggests that ketone bodies can increase the production of erythropoietin, which stimulates red blood cell production. Therefore, we investigated whether an acute increase in 3-OHB levels affects testosterone levels in healthy young men. We studied six healthy, young male participants who fasted overnight and were tested twice: (i) after drinking 37.5 g of Na-D/L-3-OHB dissolved in 500 mL of distilled water (KET), and (ii) after drinking 500 mL of placebo saline water (0.9% NaCl) (CTR). During the KET trial, 3-OHB levels increased to approximately 2.5 mM. Testosterone levels decreased significantly by 20% during KET compared to 3% during CTR. A simultaneous increase in luteinizing hormone was observed in KET. We observed no changes in other adrenal androgens, such as androstenedione and 11-keto androgens. In conclusion, an acute increase in 3-OHB levels decreases testosterone levels. Concomitantly, an increase in luteinizing hormone was observed. This suggests that 3-OHB may counteract some of the beneficial effects of endurance training. Further studies, involving larger sample sizes and performance outcomes, are required to fully understand this phenomenon.

Molecular regulatory mechanisms of erythropoiesis and related diseases.

The metabolism of cells and blood circulation allow for the constant production and destruction of red blood cells. Erythrocyte formation allows red blood cells to regenerate, which is crucial for maintaining the equilibrium of the organism. Erythrocyte formation is a multi-step, intricate process with distinct structural and functional characteristics at each stage. Erythropoiesis is regulated by a number of signaling pathways; malfunctional regulatory mechanisms may result in disease and aberrant erythropoiesis. Therefore, this article focuses on a review of the erythroid formation process, related signaling pathways, and red blood cell lineage diseases.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

PCR38 Contextualizing Health-State Utilities in Patients with Transfusion-Dependent eta-Thalassemia (TDT)

TDT is a rare genetic disease with impaired production of healthy red blood cells due to insufficient adult hemoglobin (HbA) production. The resulting ineffective erythropoiesis, and iron overload from chronic transfusions, makes this a complex disease. Cost-utility analyses in TDT to-date use health-state utility values that have not been compared to disease-specific quality of life (QoL) measures. This study aimed to contextualize TDT utility-value estimates with disease-relevant data.

Effect of durable hematocrit control and minimal thrombotic risk in real-world polycythemia vera patients treated with ruxolitinib.

e19080 Background: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by unregulated red blood cell production. Most patients (pts) harbor a JAK2 mutation and must have erythrocytosis or elevated red cell mass for diagnosis (DX). Pts are at increased risk of thrombosis. Current treatments include aspirin, therapeutic phlebotomy (TP), and cytoreductive therapy such as hydroxyurea (HU). In 2014, based on the RESPONSE study, ruxolitinib (rux), a selective JAK inhibitor, was approved for second line treatment after intolerance or inadequate response to HU. A recent real-world analysis of rux in PV showed durable hematocrit (HCT) control and decreased need for TP (Coltoff 2020). Despite emerging clinical reports, real-world data on HCT control, dose adjustment, and thrombotic risk with rux are extremely limited. Methods: A retrospective chart review from three centers was performed to evaluate PV pts treated with rux between Dec 2014 and Dec 2019. Pts age <18 at rux start, who received rux through investigational studies, and/or who had myelofibrosis were excluded. Data cutoff was May 30, 2022. Results: 69 patients were identified. Median time from PV DX to rux start was 4.4 yrs (range 0.1 to 40.2), and median follow-up from start of rux was 3.3 yrs (range 0.1 to 6.3). Pt characteristics: 37 (54%) Male, 59 pts (86%) White, 4 (6%) Black, and 64 (93%) Non-Hispanic. The most common reasons for starting rux were HU intolerance (46%), uncontrolled platelet count (28%), and symptom burden (19%). Median time on rux was 2.4 yrs (range 0.1 to 6.3); 43 pts (62%) remained on rux at data cutoff. A total of 6 pts (9%) received less than 3 months of rux before discontinuation, 8 pts (12%) received less than 6 months of rux. 41% pts (27/66), did not have HCT control (HCT <45%) within 1 month prior to rux start. Of evaluable pts, HCT control rates at 3 and 6 months were 88% (52/59) and 89% (47/53) respectively. Phlebotomy frequency in the 3-month periods before and after rux start were known for 48 pts; 21 (44%) experienced a decrease in phlebotomies within 3 months after initiation of rux, 20 (42%) remained stable, and 7 (16%) saw an increase (ranging from +1 to +3). Twelve pts (17%) required dose reductions/suspensions due to cytopenias. One pt experienced an acute pulmonary embolism while on rux (4.4 yrs after rux initiation). The pt was diagnosed with T cell lymphoma while on rux and the thrombotic event was attributed to lymphoma. This pt achieved HCT control on rux at 3 and 6 months. No arterial thromboses were observed. Conclusions: In this real-world analysis, rux was highly effective in controlling HCT in the vast majority of PV pts by 3 and 6 months. PV pts treated with rux rarely developed thrombosis. Rux was well tolerated, and few patients required dose reduction or discontinuation. [Table: see text]

Brief exposure to intermittent hypoxia increases erythropoietin levels in older adults.

Eight 4-min cycles of intermittent hypoxia represent the shortest hypoxic exposure to increase erythropoietin (EPO) levels in young adults. The impact of aging on the EPO response to a hypoxic stimulus remains equivocal. Thus, the objective of this study was to determine the effect of the same intermittent hypoxia protocol on EPO levels in older adults. Twenty-two participants (12 women, age: 53 ± 7 yr) were randomly assigned to an intermittent hypoxia group (IH, n = 11) or an intermittent normoxia group (IN, n = 11). Intermittent hypoxia consisted of eight 4-min cycles at a targeted oxygen saturation of 80% interspersed with normoxic cycles to resaturation. Air was made hypoxic by titrating nitrogen into a breathing circuit. Intermittent normoxia consisted of the same protocol, but nitrogen was not added to the breathing circuit. EPO levels were measured before and 4.5 h after the beginning of each protocol. Intermittent hypoxia lowered oxygen saturation to 82 ± 3%, which corresponded to a fraction of inspired oxygen of 10.9 ± 1.0%. There was a greater increase in EPO levels following intermittent hypoxia than intermittent normoxia (IH: 3.2 ± 2.2 vs. IN: 0.7 ± 0.8 mU/mL, P < 0.01). A single session of eight 4-min cycles of hypoxia increased EPO levels, the glycoprotein stimulating red blood cell production, in older adults. Exposure to intermittent hypoxia has therefore the potential to increase oxygen-carrying capacity in a population with reduced red blood cell volume.NEW & NOTEWORTHY We previously identified the shortest intermittent hypoxia protocol necessary to increase erythropoietin levels in young adults. The objective of this study was to determine whether the same intermittent hypoxia protocol increases erythropoietin levels in older adults. Eight 4-min bouts of hypoxia, representing a hypoxic duration of 32 min at a targeted oxygen saturation of 80%, increased erythropoietin levels in older adults, suggesting that exposure to intermittent hypoxia has the potential to increase oxygen-carrying capacity in an aging population.

Global profile of anemia during pregnancy versus country income overview: 19 years estimative (2000–2019)

Anemia is a worldwide public health problem that is worst in low- and middle-income countries (LMICs), reaching 60% of prevalence. The etiology of anemia is diverse and multifactorial, with iron deficiency being the most prevalent, and often found in pregnant women. Iron is indispensable for the production of red blood cells and approximately 80% of the available heme iron is used for hemoglobin synthesis in mature erythroblasts. Iron deficiency affects oxygen transport, compromising energy and muscle metabolism and can occur with depletion of iron storage, defective erythropoiesis, and low hemoglobin levels. We analyzed anemia prevalence in pregnant women from 2000 to 2019 worldwide correlating them with current (2022) country income, with especial attention to LMICs using WHO dataset. Our analysis indicates that pregnant women from LMICs had a higher probability (40%) of anemia during pregnancy especially those from Africa and South Asia. Africa and the Americas showed a higher decrease in the prevalence of anemia from 2000 to 2019. The Americas and Europe have a lower prevalence, concentrated in 57% of most upper-middle- and high-income countries. Black women are also more prone to develop anemia during pregnancy, especially if they are from LMICs. However, the prevalence of anemia appears to decrease with an increase in educational level. In conclusion, anemia prevalence fluctuated from 5.2 to 65.7% worldwide in 2019, validating it as a public health problem.

Current Trends in the Diagnosis and Evidence-Based Management of Patients with Myeloproliferative Disorders

Myeloproliferative disorders (MPD) are clonal hematopoietic disorders characterized by abnormal clone expansion of haematological progenitor cells, which can lead to thrombosis, bleeding, and/or transformation to leukaemia. Chronic myelogenous leukaemia (CML) is characterised by increasing granulocytic cell proliferation with no loss of differentiation, leading to an increased number of granulocytes and their immature progenitors, with occasional blast cells on peripheral blood film. Polycythaemia Vera (PV) is a disorder of stem cells that presents a total hyperplasia, malignant, and cancerous marrow. Its most prominent feature is an increased absolute red blood cell mass due to uncontrolled red blood cell production, followed by increased myeloid and megakaryocytic production due to an abnormal clone of the haematopoietic stem cells with increased sensitivity to growth factors. Essential thrombocythemia (ET) is defined by a persistent increase in platelet count with a proclivity for thrombosis and haemorrhage. Expansion of megakaryocytes in the cell lineage leads to high platelet count, and the condition is thought to be clonal. Primary myelofibrosis (PMF) results in aberrant megakaryocyte development. Extramedullary haematopoiesis and reactive connective tissues are found in fully mature bone marrow (BM). PMF is linked to uncontrolled megakaryopoiesis as well as atypical, fibrosis, and hypercellularity. Haematological and morphological abnormalities in combinations with mutations (BCR-ABL, CARL, JAK2 or MPL) represent major diagnostic criteria for MPD. The WHO criteria for tumours of the hemopoietic and lymphoid tissues was revised in 2016. Prognostic models rely on clinical and haematological data, but new models with genetic information are being created for clinical trial environments. Management of most patients with MPDs involves treatment or monitoring symptoms associated with presenting disease and/or preventing other events such as thrombosis. Treatment modalities include symptomatic treatments, surgery, therapeutic radiation, therapeutic phlebotomy, therapeutic apheresis, cytoreductive therapy and haematopoietic stem cells transplantations.

Unpacking the renal system component of the 'Structure and Function' Core Concept of Physiology by an Australian team.

Australia-wide consensus was reached on seven core concepts of physiology, one of which was 'Structure and Function' with the descriptor 'Structure and function are intrinsically related to all levels of the organism. In all physiological systems the structure from a microscopic level to an organ level dictates its function'. As a framework for the Structure and Function core concept, the renal system was unpacked by a team of five Australian Physiology educators from different universities and with extensive teaching experience into hierarchical levels, with five themes and twenty-five sub-themes up to 3 levels deep. Within Theme 1, the structures that comprise the renal system were unpacked. Within Theme 2, the physiological processes within the nephron such as filtration, reabsorption, and secretion were unpacked. Within Theme 3, the processes involved in micturition were unpacked. In Theme 4, the structures and processes involved in regulating renal blood flow and glomerular filtration were unpacked; and within Theme 5, the role of the kidney in red blood cell production was unpacked. Twenty-one academics rated the difficulty and importance of each theme/subtheme and results were analyzed using a one-way ANOVA. All identified themes were validated as 'essential' to 'important'/'moderately important' and rated between 'difficult' to 'not difficult'. A similar framework consisting of Structure, Physiological processes, Physical processes and Regulation can be used to unpack other body systems. Unpacking of the body systems will provide a list of what students should be taught in curricula across Australian universities and inform assessment and learning activities.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Sex differences in the erythropoietin response to hot tub treatment and carbon monoxide inhalation

The kidney increases erythropoietin (EPO) when oxygen delivery is reduced, and when reductions in oxygen delivery are sustained, increased EPO results in increased red blood cell (RBC) production. More recently, carbon monoxide (CO) and heat have been used chronically to increase RBC production and therefore hemoglobin (Hb) mass. However, studies examining the acute effects of these interventions are limited, and no studies have examined these interventions in women. The purpose of this study was to examine the effects of CO inhalation and heat treatment alone and in combination on circulating EPO and determine if there are sex differences in these responses. 11 healthy subjects completed the study (6 women). Baseline blood was drawn to measure iron, ferritin, and transferrin concentrations. Hb mass was measured via the 10-minute CO-rebreathe method. Subjects completed three interventions – CO inhalation (carboxyhemoglobin 13.01±2.5%) hot tub immersion (40 degrees C for 45 minutes), and combined CO and hot tub – in a randomized crossover design. Renal blood velocity (RBV) was measured via ultrasound during all interventions. Venous blood was drawn at baseline and every hour for 6 hours post-intervention for measurements of EPO. All interventions reduced renal oxygen delivery (Δ47.5±21mL O2/min), and there were no sex differences in the reduction in oxygen delivery. All three treatments similarly increased EPO from baseline to peak concentration (Δ4.94±5.6mIU/mL). Women had a significantly greater increase in EPO than men to all three interventions (Δwomen 8.22±5.6mIU/mL; Δmen 1±2.06mIU/mL). The degree of EPO increase on all three days negatively correlated with Hb mass (R2=0.64) and positively correlated with transferrin (R2=0.73). Women may respond better to these interventions aimed to increase EPO, but the degree of responsiveness may depend on baseline Hb mass as well as transferrin concentrations. Wu-Tsai Human Performance Alliance at Oregon This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impaired Erythropoietin Response to Intermittent Hypoxia in Patients with Type 2 Diabetes

Patients with type 2 diabetes (T2D) exhibit, on average, a 20% decline in maximal oxygen consumption when compared to healthy adults. Hemoglobin mass strongly correlates to maximal oxygen consumption. A reduced total blood volume has been observed in patients with T2D, suggesting that a reduced hemoglobin mass contributes to the decreased maximal oxygen consumption in this population. Hypoxia stimulates the release of erythropoietin (EPO), the hormone regulating red blood cell production. We previously showed that intermittent hypoxia, consisting of alternating short bouts of breathing hypoxic and normoxic air, increases EPO levels. Thus, the objective of this study was to determine the effect of a single session of intermittent hypoxia on serum EPO levels and hemoglobin mass in patients with T2D. We hypothesized that a single session of intermittent hypoxia would raise serum EPO levels and lead to an increase in hemoglobin mass in patients with T2D. Ten patients with T2D (4 women, age: 53 ± 10 years, body mass index: 36.2 ± 8.5 kg/m2, HbA1c: 7.2 ± 1.2%) were exposed to an intermittent hypoxia protocol consisting of eight 4-min cycles at a targeted oxygen saturation of 80% interspersed with normoxic cycles to resaturation. Air was made hypoxic by titrating nitrogen into a breathing circuit. Pulmonary gas exchange, oxygen saturation, and hemodynamics were continuously measured throughout the protocol. EPO levels were measured before and 4.5 hours after the beginning of the protocol. Hemoglobin mass was assessed via carbon monoxide rebreathing before and seven days following intermittent hypoxia. Intermittent hypoxia lowered oxygen saturation (&amp;shy;&amp;shy;97 ±&amp;shy; 2 to 81 ± 2%, p&lt;0.01), which resulted in a lower fraction of inspired oxygen (20.8 ±&amp;shy; 0.1 to 11.1 ± 1.0%, p&lt;0.01). There was no significant change in EPO levels following exposure to intermittent hypoxia (11.9 ± 5.3&amp;shy; to 12.1 ± 4.3 mU/ml, p=0.83). There was also no change in hemoglobin mass in response to intermittent hypoxia (864 ± 152&amp;shy; to 850 ± 150 g, p=0.64). Intermittent hypoxia did not affect mean arterial pressure (94 ± 5 to 97 ± 7 mmHg, p=0.18) but increased cardiac output (9.1 ± 2.7 to 9.8 ± 2.8 L/min, p=0.03) due to an increase in heart rate (78 ± 9 to 84 ± 10 bpm, p&lt;0.01). In conclusion, a single session of intermittent hypoxia did not increase serum EPO levels or hemoglobin mass in patients with T2D. These findings suggest an impaired EPO response to decreased oxygen levels in patients with T2D, which may contribute to the reduced hemoglobin mass and total blood volume observed in this population. College of Education Small Grants Program This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Iron-loaded deferiprone can support full hemoglobinization of cultured red blood cells

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.

In vitro erythrocyte production using human-induced pluripotent stem cells: determining the best hematopoietic stem cell sources

BackgroundBlood transfusion is an essential part of medicine. However, many countries have been facing a national blood crisis. To address this ongoing blood shortage issue, there have been efforts to generate red blood cells (RBCs) in vitro, especially from human-induced pluripotent stem cells (hiPSCs). However, the best source of hiPSCs for this purpose is yet to be determined.MethodsIn this study, hiPSCs were established from three different hematopoietic stem cell sources—peripheral blood (PB), cord blood (CB) and bone marrow (BM) aspirates (n = 3 for each source)—using episomal reprogramming vectors and differentiated into functional RBCs. Various time-course studies including immunofluorescence assay, quantitative real-time PCR, flow cytometry, karyotyping, morphological analysis, oxygen binding capacity analysis, and RNA sequencing were performed to examine and compare the characteristics of hiPSCs and hiPSC-differentiated erythroid cells.ResultshiPSC lines were established from each of the three sources and were found to be pluripotent and have comparable characteristics. All hiPSCs differentiated into erythroid cells, but there were discrepancies in differentiation and maturation efficiencies: CB-derived hiPSCs matured into erythroid cells the fastest while PB-derived hiPSCs required a longer time for maturation but showed the highest degree of reproducibility. BM-derived hiPSCs gave rise to diverse types of cells and exhibited poor differentiation efficiency. Nonetheless, erythroid cells differentiated from all hiPSC lines mainly expressed fetal and/or embryonic hemoglobin, indicating that primitive erythropoiesis occurred. Their oxygen equilibrium curves were all left-shifted.ConclusionsCollectively, both PB- and CB-derived hiPSCs were favorably reliable sources for the clinical production of RBCs in vitro, despite several challenges that need to be overcome. However, owing to the limited availability and the large amount of CB required to produce hiPSCs, and the results of this study, the advantages of using PB-derived hiPSCs for RBC production in vitro may outweigh those of using CB-derived hiPSCs. We believe that our findings will facilitate the selection of optimal hiPSC lines for RBC production in vitro in the near future.

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.

Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic (“don’t eat me”) CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6Chi monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more “pro-phagocytic,” suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages.

Structural and physiological changes of the kidney with age and its impact on chronic conditions and COVID-19

The kidney is an essential organ that removes waste products, balances the body's fluids, releases hormones that regulate blood pressure, produces an active form of vitamin D, promotes healthy bones, and controls the production of red blood cells. Structural and functional abnormalities occur in kidney with age. Alterations in kidney structure are based on physiological functions and environmental pressures. Variations in its structure across vertebrates are primarily due to the nature of alterations in number, complexity, arrangement, and location of the kidney tubules. Globally, individuals aged 65 and older are part of the fastest expanding population demographic, and as a result, a greater number of older patients are receiving a diagnosis of impaired renal function. The purpose of our mini-review is to summarize recent findings of the structural and functional differences between the normal and aging kidney, examine the evolutionary biology of the kidney across species, and demonstrate the role of aging in conditions such as diabetes, chronic kidney disease, and hypertension, along with their impact on SARS-CoV-2. Additional aims include discussing the potential therapeutic strategies to treat aged individuals with kidney health issues and how the impact of a healthy lifestyle, diet, and exercise can improve health conditions with aged kidneys.

Comprehensive pediatric reference intervals for 79 hematology markers in the CALIPER cohort of healthy children and adolescents using the Mindray BC-6800Plus system.

Hematological parameters vary significantly throughout growth and development due to physiological processes such as fetal-to-adult erythropoiesis and puberty. Pediatric age- and sex-specific reference intervals (RIs) are thus essential for appropriate clinical decision-making. The current study aimed to establish RIs for both common and novel hematology parameters on the Mindray BC-6800Plus system. Six hundred and eighty-seven healthy children and adolescents (30 days to 18 years) were enrolled. Participants were recruited as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals Program upon informed consent or identified from apparently healthy outpatient clinics. Whole blood was collected and assayed for 79 hematology parameters on the BC-6800Plus system (Mindray). Age- and sex-specific RIs were established as per Clinical and Laboratory Standards Institute EP28-A3c guidelines. Dynamic reference value distributions were observed for several hematology parameters, including erythrocytes, leukocytes, platelets, reticulocytes, and research-use-only markers. Age partitioning was required for 52 parameters, demonstrating changes in infancy and puberty. Sex partitioning was required for 11 erythrocyte parameters (i.e., red blood cell (RBC), hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, RBC distribution width coefficient of variation, hemoglobin distribution width, macrocyte count, macrocyte percentage, RBC (optical), and reticulocyte production index). Few parameters had undetectable levels in our healthy cohort (i.e., nucleated RBC count and immature granulocyte count). The current study completed hematological profiling for 79 parameters on the BC-6800Plus system in a healthy cohort of Canadian children and adolescents. These data emphasize the complex biological patterns of hematology parameters in childhood, particularly at the onset of puberty, and support the need for age- and sex-specific RIs for clinical interpretation.