Optimized Beta-Globin Expression and Enucleation from Induced Red Blood Cells for In Vitro Modeling of Sickle Cell Disease

Abstract Human induced pluripotent stem cells (hiPSCs) hold remarkable capacity for disease modeling and the development of novel therapeutic treatments for sickle cell disease (SCD). hiPSCs can theoretically produce all cell types including induced red blood cells (iRBCs). Sickle cell patients, in particular, could benefit from autologous, engineered red blood cells (RBCs). Many patients possess rare Rh phenotypes, are allo-sensitized to blood products and are at risk of iron overload from recurrent transfusions. Therefore, the generation of personalized iRBCs is attractive. Yet, in vitro iRBC production has been hampered by an inability of these cells to differentiate into terminally-mature, enucleated, beta globin-expressing RBCs. Here, we describe updated strategies to improve in vitro production of iRBCs. hiPSCs from sickle cell patients as well as those with normal hemoglobin were differentiated into hematopoietic stem progenitor cells (HSPCs) and immortalized via the overexpression of a previously characterized set of transcription factors promoting self-renewal and multipotency under the control of a doxycycline-regulated promoter. Utilizing an in vitro protocol incorporating increasing concentrations of human plasma, HSPCs differentiated from these lines proceed through terminal erythroid differentiation, including the formation of CD71-/GlyA+/α4 integrin-/Band 3+ cells. Plasma-stimulated iRBCs achieved robust enucleation (11-60.7%) and underwent fetal to adult globin-switching. Further, nearly 21% of the enucleated iRBCs were RNA negative erythrocytes 5-8 microns in diameter. RNA sequencing analysis reveals similar transcriptional profiles between iRBCs and peripheral blood CD34+- derived cultured RBCs (cRBCs) yet distinct differences between SCD and WT iRBCs. SCA iRBCs have increased extracellular matrix organization, cell-cell adhesive properties and up-regulation of hypoxia-response genes. Heme metabolism, DNA repair, fatty acid metabolism and oxidative phosphorylation are all impaired in SCD iRBCs. Assessment of cell physiology exposes membrane damage in SCD iRBCs with increased phalloidin permeability in comparison to wild type controls. Intriguingly, SCD iRBCs co-expressing gamma and beta-globin also demonstrate sickling under hypoxic conditions. With the development of expandable source of erythroid progenitors capable of producing mature red cells, we now aim to utilize this platform for robust disease modeling and autologous cell therapy. Figure. Figure. Disclosures Heeney: Pfizer: Research Funding; Sancilio Pharmaceuticals: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee.

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In vitro investigation of the antisickling properties of aqueous fruits extracts of Citrus paradisi, Musa acuminata and Malus domestica

GSC Biological and Pharmaceutical Sciences ◽

10.30574/gscbps.2020.13.3.0412 ◽

2020 ◽

Vol 13 (3) ◽

pp. 203-209

Author(s):

Augustine O. Odibo ◽

Ifunanya R. Akaniro ◽

Emmanuel M. Ubah

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Malus Domestica ◽

Blood Cells ◽

Musa Acuminata ◽

Citrus Paradisi ◽

Cell Disease ◽

In Vitro Investigation

Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transportation of red blood cells (RBCs) in blood vessels, leading to various clinical complications. Many drugs that are available for treating the disease are insufficiently effective, toxic, or too expensive. Therefore, there is a pressing need for safe, effective, and inexpensive therapeutic agents from indigenous plants used in ethnomedicine. In the current study, the potentials of aqueous extracts of Citrus paradisi, Musa acuminata, Malusdomestica fruit in sickle cell disease management were investigated in vitro using P-hydroxybenzoic acid and normal saline as positive and negative control respectively. The method employed the inhibition of sodium metabisulphite induced sickling of HbSS red blood cells, collected from confirmed sickle cell patients. Results obtained showed that; 50 mg/ml aqueous extract of Musa acuminata showed the lowest sickling inhibition (80.3%) at 60 minutes while 1 mg/ml gave the highest inhibition of 97.3% at 90 minutes. For Citrus paradisi, lowest (83.3%) and highest (98%) sickling inhibitions were obtained with 25 mg/ml and 50 mg/ml fruit extracts at 30 minutes and 60 minutes respectively. Malus domestica had the highest antisickling activities of 99% and 99.33% respectively at 30 and 60 minutes. In all, the sickling inhibition was least with Musa acuminata but highest with Malus domestica. Also, highest inhibitions were observed at 60 minutes (optimum time) and 10 mg/ml (optimum concentration). This study has demonstrated that; Malus domestica, Citrus paradisi and Musa acuminata possess antisickling potentials useful in the management or therapy of sickle cell diseases.

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High-Throughput Mirna Analysis Suggests Pro-Inflammatory Profile in Sickle Cell Disease

Blood ◽

10.1182/blood-2018-99-110812 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1077-1077

Author(s):

Matthew Cannon ◽

Sarah Glass ◽

Sidney Smith ◽

Melanie Heinlein ◽

Rosa Lapalombella ◽

...

Keyword(s):

Chronic Pain ◽

Steady State ◽

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Research Funding ◽

Red Cell ◽

Cell Disease ◽

Acute Crisis

Abstract BACKGROUND: Mature circulating red blood cells, though devoid of a nucleus, have been shown to contain an abundance of miRNAs. Further, it has been shown that sickle cell patient-derived RBCs have a dramatic difference in miRNA content than normal RBCs. Given that a range of miRNAs are involved in the regulation of immunity, including the release of inflammatory mediators, we hypothesize that miRNAs enriched in circulating red blood cells function to prolong the inflammatory state in sickle cell disease. Further, we hypothesize that these miRNAs can be used as biomarkers for use in the clinic to predict crisis and differentiate acute versus chronic pain. Exploring this miRNA enrichment in circulating red blood cells in sickle cell patients will provide practical insight for the inflammation state and will inform characteristics of patients who may need greater care in the clinic. METHODS: Twenty steady state patients were recruited and categorized according to their chronic pain status and crisis frequency per year. Whole blood was drawn during routine visits to the OSU Wexner Medical Center Hematology Clinic. Additionally, whole blood was drawn from five patients either in acute pain crisis (recruited prior to crisis) or within a few days of crisis. Samples were subject to double gradient centrifugation and red cells were resuspended in Trizol and cryopreserved. MiRNAs were isolated from red cell Trizol suspensions using a commercial isolation kit (QIAGEN Cat#217004). Isolated miRNAs were then subject to a NanoString Human miR (v3) expression assay. Differential expression analysis was conducted to compare miRNAs with at least 1.5 fold difference (p = 0.05) between steady state and acute crisis. Target prediction and GO ontology analysis was performed for statistically significant miRNAs using DIANA Tools mirPath v3. Follow-up qPCRs were performed using TaqMan Advanced miRNA cDNA Synthesis Kit (Cat#A28007) and TaqMan Advanced miRNA Assays (Cat#A25576) to validate the decreased expression of miRNAs. Additional qPCRs were performed using TaqMan Gene Expression Assays (Cat#4331182) to investigate mRNA regulatory effects of significant miRNAs in the total red cell population. Western blots were also performed to investigate regulatory effects of these miRNAs at the protein level. RESULTS & CONCLUSION: Comparison of RBC miRNA profiles from patients during acute crisis to those in steady state shows several significantly decreased (>1.5 fold) miRNAs in crisis. Among these miRs we have found previously uncharacterized miRNAs, hsa-miR-2116-5p and hsa-miR-302d-3p. DIANA tools miRNA analysis software predicts these miRNAs to be involved in regulation of cell-to-cell adhesion pathways through gene transcripts such as Protocadherin Beta 6 (PCDHB6) and Neural Cell Adhesion Molecule 2 (NCAM2). Interestingly, inspection of miRNA predicted targets that fall under significant GO terms also predicts several individual miRNAs to regulate inflammatory response and nociceptive signaling gene transcripts like A20 (TNFAIP3) and Cathepsin S (CTSS). Validation of these miRNAs was performed via qPCR for 5 out of the 6 significantly decreased miRNAs. Of the 5 miRNAs tested, hsa-miR-2116-5p, hsa-miR-302d-3p, and hsa-miR-1246 were validated as having decreased expression in acute crisis patients compared to steady state. qPCRs were then performed to probe for miRNA based regulation of top predicted target mRNA transcripts. Both CTSS and TNFAIP3 showed increased expression of mRNA transcripts in acute crisis patient red cells as compared to steady state. Next, western blot analysis was performed on red cell protein lysate. Interestingly, this analysis revealed a pattern in activated CTSS expression that was independent of acute crisis. Steady state patients reporting chronic pain showed increased activated CTSS compared to those without chronic pain. Activated CTSS was not found in red cell lysates from three normal, non-SCD donors. Taken together, these results suggest that red blood cells may play a larger role in inflammation and pain responses in sickle cell disease than previously thought. Further these results suggest activated CTSS as a potential biomarker for differentiating chronic pain in patients. Follow-up studies are underway to further stratify and investigate these findings. Disclosures Desai: University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Committee; NIH: Research Funding; FDA: Research Funding; Selexy/Novartis: Research Funding; Pfizer: Research Funding.

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GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro

Hematology Reports ◽

10.4081/hr.2018.7419 ◽

2018 ◽

Vol 10 (2) ◽

Cited By ~ 4

Author(s):

Kobina Dufu ◽

Donna Oksenberg

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Oxygen Affinity ◽

Microvascular Blood Flow ◽

Cell Disease ◽

Hypoxic Conditions

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

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ML-0207/ASP8731: A Novel BACH1 Inhibitor That Induces Fetal Hemoglobin in Treatment of Sickle Cell Disease

Blood ◽

10.1182/blood-2021-151606 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 854-854

Author(s):

Selva Nataraja ◽

Maneet Singh ◽

Shilpa Demes ◽

Lyndsay Olson ◽

Jeff Stanwix ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Heme Oxygenase ◽

Sickle Cell ◽

Blood Cells ◽

Research Funding ◽

Heme Oxygenase 1 ◽

Cell Disease ◽

Nrf2 Pathway ◽

F Cells

Abstract Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the β-globin subunit resulting in hemoglobin S (HbS). Following deoxygenation of red blood cells, HbS forms polymers that can promote hemolysis and the release of free heme that cause pro-oxidative and pro-inflammatory stress, vaso-occlusive pain crises, and ischemia-reperfusion pathophysiology. Heme also functions as an intracellular activator of antioxidant and globin gene expression. Heme binds to the transcriptional repressor BTB and CNC homolog 1 (BACH1), which relieves BACH1's repression of gene transcription. The release of BACH1 repression increases the binding of nuclear factor erythroid 2-related factor 2 (NRF2) to antioxidant response elements (ARE) and the cell-specific transcription of antioxidant genes such as heme oxygenase-1 (HMOX1), glutathione reductase (GR), solute carrier family 7 member 11 (SLC7A11), and NAD(P)H dehydrogenase [quinone] 1 (NQO1). We have previously shown that pharmacologic activation of the NRF2 pathway in SCD mice provides protection against heme-induced vascular occlusion, is anti-inflammatory, and decreases hepatic necrosis. NRF2 activation also promotes erythroid expression of the A-gamma (HBG1) and G-gamma (HBG2) globins, which are subunits of hemoglobin F (HbF) that replace β S-globins and thus increase HbF and decrease HbS in red blood cells. Thus, BACH1 inhibitors have the potential to increase expression of antioxidant and HbF genes and prevent or reduce SCD-related pathophysiology, resulting in reduction of hemolysis, inflammation, and vaso-occlusive pain crises. Mitobridge is currently developing ML-0207/ASP8731, a highly potent, selective small molecule inhibitor of BACH1 capable of activating the Nrf2 pathway in human and murine models and investigated the ability of ML-0207 to modulate antioxidant and anti-inflammatory genes and induce HbF in human translational cellular models and a preclinical murine model of SCD. ML-0207 induced mRNA expression of Nrf2 target genes HGB1, HBG2, HMOX1, SLC7A11, GCLM, and NQO1 in human bone marrow-derived CD34+ cells differentiated to erythrocytes. We observed 2-fold increases in both the percentage and number of CD71+/HbF+ erythrocytes by FACS using 1 µM ML-0207 and 10 μM HU compared to DMSO control (Figure 1A). The combination of ML-0207 and HU induced significantly more HbF+ erythrocytes compared to each drug alone (Figure 1B). In a single healthy CD34+ donor non-responsive to 10 µM HU, we observed ML-0207 was able to significantly induce CD71+/HbF+ cells at 1 & 3 µM (Figure 1C). In Townes SCD mice, there were significant increases in heme oxygenase 1 and decreases in VCAM-1, ICAM-1, and decreases in phospho-p65 NF-ĸB protein. Furthermore, we observed a significant reduction in hemin-induced vaso-occlusion and an increase in the percentage of F-cells. The increases in F-cells were accompanied by increases in blood A-gamma globin and erythrocytes and decreases in leukocytes. Taken together, these data support BACH1 inhibitors as potential novel and effective treatments for SCD patients. Figure 1 Figure 1. Disclosures Nataraja: Mitobridge: Current Employment. Singh: Mitobridge: Current Employment. Demes: Astellas: Current Employment. Olson: Mitobridge: Current Employment. Stanwix: Mitobridge: Current Employment. Biddle: Rheos Medicine: Current Employment. Vercellotti: Mitobridge, an Astellas Company: Consultancy, Research Funding; CSL Behring: Research Funding. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding.

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Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650577 ◽

1996 ◽

Vol 76 (03) ◽

pp. 322-327 ◽

Cited By ~ 73

Author(s):

Dominique Helley ◽

Amiram Eldor ◽

Robert Girot ◽

Rolande Ducrocq ◽

Marie-Claude Guillin ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Annexin V ◽

Mean Value ◽

Procoagulant Activity ◽

Dependent Manner ◽

Cell Disease ◽

Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

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Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽

10.1039/d0lc01133a ◽

2021 ◽

Author(s):

Yuncheng Man ◽

Debnath Maji ◽

Ran An ◽

Sanjay Ahuja ◽

Jane A Little ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Red Blood Cells ◽

Sickle Cell ◽

Red Blood Cell ◽

Blood Cells ◽

Electrical Impedance ◽

Cell Disease ◽

Blood Disorders

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contributes to vaso-occlusion and disease pathophysiology. However, there are few...

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Role of Adhesion Molecules and Vascular Endothelium in the Pathogenesis of Sickle Cell Disease

Hematology ◽

10.1182/asheducation-2007.1.84 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 84-90 ◽

Cited By ~ 27

Author(s):

Marilyn J. Telen

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Adhesion Molecules ◽

Sickle Cell ◽

Vascular Endothelium ◽

Blood Cells ◽

Cell Disease ◽

Adhesive Interactions ◽

Lines Of Evidence

AbstractA number of lines of evidence now support the hypothesis that vaso-occlusion and several of the sequelae of sickle cell disease (SCD) arise, at least in part, from adhesive interactions of sickle red blood cells, leukocytes, and the endothelium. Both experimental and genetic evidence provide support for the importance of these interactions. It is likely that future therapies for SCD might target one or more of these interactions.

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Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽

10.1182/blood-2020-141603 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Yuanbin Song ◽

Rana Gbyli ◽

Liang Shan ◽

Wei Liu ◽

Yimeng Gao ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Red Cell ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

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