Biomechanical properties of native and cultured red blood cells-Interplay of shape, structure and biomechanics.

Modern medicine increases the demand for safe blood products. Ex vivo cultured red blood cells (cRBC) are eagerly awaited as a standardized, safe source of RBC. Established culture models still lack the terminal cytoskeletal remodeling from reticulocyte to erythrocyte with changes in the biomechanical properties and interacts with membrane stiffness, viscosity of the cytoplasm and the cytoskeletal network. Comprehensive data on the biomechanical properties of cRBC are needed to take the last step towards translation into clinical use in transfusion medicine. Aim of the study was the comparative analysis of topographical and biomechanical properties of cRBC, generated from human CD34+ adult hematopoietic stem/progenitor cells, with native reticulocytes (nRET) and erythrocytes (nRBC) using cell biological and biomechanical technologies. To gain the desired all-encompassing information, a single method was unsatisfactory and only the combination of different methods could lead to the goal. Topographical information was matched with biomechanical data from optical tweezers (OT), atomic force microscopy (AFM) and digital holographic microscopy (DHM). Underlying structures were investigated in detail. Imaging, deformability and recovery time showed a high similarity between cRBC and nRBC. Young's modulus and plasticity index also confirmed this similarity. No significant differences in membrane and cytoskeletal proteins were found, while lipid deficiency resulted in spherical, vesiculated cells with impaired biomechanical functionality. The combination of techniques has proven successful and experiments underscore a close relationship between lipid content, shape and biomechanical functionality of RBC.

Generation of the human erythroblast-derived iPSC line UBTi001-A.

We performed reprogramming of human erythroblasts derived from CD34+ hematopoietic stem / progenitor cells of a healthy donor. CD34+ cells were differentiated in-vitro into a pure population of CD36+ erythroblasts and nucleofected with four episomal plasmids expressing SOX2, OCT3/4, KLF4, LIN28, L-MYC and TP53-shRNA. The established iPSC line showed normal karyotype. Pluripotency was confirmed by expression of pluripotency markers and in-vitro differentiation into tissues of all three germ layers. The UBTi001-A iPSC line might provide an attractive source for developmental research on human hematopoiesis and erythropoiesis.

Membrane Properties of Human Induced Pluripotent Stem Cell-Derived Cultured Red Blood Cells.

Cultured red blood cells from human induced pluripotent stem cells (cRBC_iPSCs) are a promising source for future concepts in transfusion medicine. Before cRBC_iPSCs will have entrance into clinical or laboratory use, their functional properties and safety have to be carefully validated. Due to the limitations of established culture systems, such studies are still missing. Improved erythropoiesis in a recently established culture system, closer simulating the physiological niche, enabled us to conduct functional characterization of enucleated cRBC_iPSCs with a focus on membrane properties. Morphology and maturation stage of cRBC_iPSCs were closer to native reticulocytes (nRETs) than to native red blood cells (nRBCs). Whereas osmotic resistance of cRBC_iPSCs was similar to nRETs, their deformability was slightly impaired. Since no obvious alterations in membrane morphology, lipid composition, and major membrane associated protein patterns were observed, reduced deformability might be caused by a more primitive nature of cRBC_iPSCs comparable to human embryonic- or fetal liver erythropoiesis. Blood group phenotyping of cRBC_iPSCs further confirmed the potency of cRBC_iPSCs as a prospective device in pre-transfusional routine diagnostics. Therefore, RBC membrane analyses obtained in this study underscore the overall prospects of cRBC_iPSCs for their future application in the field of transfusion medicine.

Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy.

Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBCPlasma with plasma and cRBCHPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBCHPL, and different characteristics for cRBCPlasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes.

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a "per cell" basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.

Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43+ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34+/CD45+ progenitors with high erythroid colony-forming potential and CD36+ erythroid precursors. A total of 1.5 × 107 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA+ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

Cholesterol Deficiency Causes Impaired Osmotic Stability of Cultured Red Blood Cells.

Ex vivo generation of red blood cells (cRBCs) is an attractive tool in basic research and for replacing blood components donated by volunteers. As a prerequisite for the survival of cRBCs during storage as well as in the circulation, the quality of the membrane is of utmost importance. Besides the cytoskeleton and embedded proteins, the lipid bilayer is critical for membrane integrity. Although cRBCs suffer from increased fragility, studies investigating the lipid content of their membrane are still lacking. We investigated the membrane lipid profile of cRBCs from CD34+ human stem and progenitor cells compared to native red blood cells (nRBCs) and native reticulocytes (nRETs). Ex vivo erythropoiesis was performed in a well-established liquid assay. cRBCs showed a maturation grade between nRETs and nRBCs. High-resolution mass spectrometry analysis for cholesterol and the major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lysophosphatidylcholin, demonstrated severe cholesterol deficiency in cRBCs. Although cRBCs showed normal deformability capacity, they suffered from increased hemolysis due to minimal changes in the osmotic conditions. After additional lipid supplementation, especially cholesterol during culturing, the cholesterol content of cRBCs increased to a subnormal amount. Concurrently, the osmotic resistance recovered completely and became comparable to that of nRETs. Minor differences in the amount of phospholipids in cRBCs compared to native cells could mainly be attributed to the ongoing membrane remodeling process from the reticulocyte to the erythrocyte stage. Obtained results demonstrate severe cholesterol deficiency as a reason for enhanced fragility of cRBCs. Therefore, the supplementation of lipids, especially cholesterol during ex vivo erythropoiesis may overcome this limitation and strengthens the survival of cRBCs ex vivo and in vivo.

Emergence of CD43-Expressing Hematopoietic Progenitors from Human Induced Pluripotent Stem Cells.

BACKGROUND: The ex vivo generation of human hematopoietic stem cells (HSCs) with long-term repopulating capacity and multi-lineage differentiation potential represents the holy grail of hematopoiesis research. In principle, human induced pluripotent stem cells (hiPSCs) provide the tool for both studying molecular mechanisms of hematopoietic development and the ex vivo production of 'true' HSCs for transplantation purposes and lineage-specific cells, e.g. red blood cells, for transfusion purposes. CD43-expressing cells have been reported as the first hematopoietic cells during development, but whether or not these possess multilineage differentiation and long-term engraftment potential is incompletely understood. METHODS: We performed ex vivo generation of hematopoietic cells from hiPSCs using an embryoid body(EB)-based, xeno-product-free differentiation protocol. We investigated the multilineage differentiation potential of different FACS-sorted CD43-expressing cell subsets by colony-forming assays in semisolid media. Further, erythroid differentiation was investigated in more detail using established protocols. RESULTS: By using CD43, we were able to measure hematopoietic induction efficiency during hiPSC-derived EB differentiation. Further, we determined CD43+ cells as the cell population of origin for in vitro erythropoiesis. Furthermore, colony formation demonstrates that the multipotent hematopoietic stem and progenitor cell fraction is particulary enriched in the CD43hi CD45+ population.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

Prolonged anemia in an intrauterine-transfused neonate with Rh-hemolytic disease: no evidence for anti-D-related suppression of erythropoiesis in vitro.

BACKGROUND: Rh-hemolytic disease may be complicated in some cases by a prolonged postnatal anemia with an extended need for postnatal red blood cell (RBC) transfusion. Besides ongoing hemolysis, marrow suppression and erythropoietin (EPO) deficiency are discussed as underlying mechanisms of this so-called "late hyporegenerative anemia." CASE REPORT: We present a case of a newborn with Rh-hemolytic disease caused by anti-D who received several intrauterine RBC transfusions. After birth, no reticulocytes or D+ RBCs were detectable in peripheral blood of the infant; thus further RBC transfusions were necessary. Administration of intravenous immunoglobulins had no obvious effect. Reticulocytes first became detectable 15 weeks after birth, when anti-D titer had decreased to 16. A few days later, hemoglobin started to increase and no further treatment was necessary. To investigate whether anti-D is able to cause maturation arrest of erythroid progenitors, maternal serum was added to an in vitro assay of erythropoiesis, induced from human CD34+ cells. RESULTS: In this case, no variables of hemolysis (e.g., elevated bilirubin) were observed. The EPO level was normal and a marrow sample showed increased erythropoiesis. The in vitro erythropoiesis assay revealed no influence of anti-D on RBC proliferation and differentiation. CONCLUSION: Anemia in our patient seemed to be mainly caused by ongoing intramedullar hemolysis due to persistent high anti-D titers. In such cases, variables for hemolysis are not necessarily found. Release of patient's own RBCs into the circulation may become sufficient when anti-D has declined to a very low level of approximately 16.

Acquired von Willebrand syndrome in a 10-year-old girl with acute lymphoblastic leukaemia.

UNLABELLED: Following diagnosis of acute lymphoblastic leukaemia (ALL) in a 10-year-old girl, routine coagulation screening including von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and factor VIIIC (FVIII:C) detected no pathological findings. After the first HR2' element of the high-risk group of the ALL-BFM 2000 protocol, the patient demonstrated extensive bleeding symptoms and acquired von Willebrand syndrome was diagnosed. VWF:Ag (13%), VWF:RCo (13%) and FVIII:C (27%) were decreased. Multimer analysis showed a loss of large multimers and a loss in triplet structures. The observed pattern was thought to be typical for monoclonal IgG gammopathy; however, in this case, unexpectedly, biclonal IgM gammopathy (κ and λ) was detected. After treatment with intravenous immunoglobulin over 5 days, coagulation factors increased to normal levels. Although this effect was assumed to be at best only temporary, especially in a case of IgM gammopathy, no further bleeding symptoms have been observed. TRIAL REGISTRATION NUMBER: M208.

In vitro proliferation and differentiation of human CD34+ cells from peripheral blood into mature red blood cells with two different cell culture systems.

BACKGROUND: An in vitro erythropoiesis assay is a powerful tool for investigating red blood cell (RBC) development and diseases of the erythroid lineage. Most assays, however, failed in either proliferation or terminal differentiation. Here two liquid cultures (LCs) for in vitro generation of RBCs from peripheral blood CD34+ cells were compared. STUDY DESIGN AND METHODS: Granulocyte-colony-stimulating factor-mobilized CD34+ cells were cultured for 16 days in a two-phase LC (2P-LC; Days 1-8, stem cell factor [SCF], erythropoietin [EPO], insulinlike growth factor [IGF]-1, and steroids; Days 9-16, EPO and insulin) and for 21 days in a three-phase LC (3P-LC; Days 1-7, SCF, thrombopoetin, and Flt3-ligand; Days 8-14, SCF, EPO, and IGF-1; Days 15-21, EPO and IGF-1). Maturation was analyzed by flow cytometry (CD36, CD71, glycophorin A [GPA]) and microscopy. RESULTS: In the 2P-LC, cell numbers increased from 0.5 x 10(6) to 25.7 x 10(6) +/- 15.1 x 10(6) cells per mL. More than 95 percent were GPA+ and showed morphologic characteristics of normoblasts (52 +/- 15%) and enucleated reticulocytes (43 +/- 18%). In the 3P-LC, a higher overall proliferation to 55.7 x 10(6) +/- 37.7 x 10(6) cells per mL was achieved (p < 0.05). This was also accompanied by a high degree of normoblasts (36 +/- 16%) and reticulocytes (48 +/- 24%). The amount of GPA+ cells was slightly lower (88.4 +/- 16.4%), associated with a significantly higher contamination by nonerythroid cells (15.8 +/- 19.3% vs. 3.9 +/- 2.9%, p < 0.05). CONCLUSION: Both LCs were able to generate fully matured RBCs and represent powerful tools for fundamental research in erythroid development and diseases targeting the erythroid lineage. A slightly higher proliferation was achieved in the 3P-LC. This was associated with a limited homogeneity due to more nonerythroid cells, however. Therefore the 2P-LC is favored, also saving additional culture days and growth factors.

Interferon-alpha stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection.

BACKGROUND/AIMS: RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore plays a central role in the hepatitis-delta-virus (HDV) life-cycle. Editing is catalyzed by the enzyme Adenosine-deaminase-acting-on-RNA1 (ADAR1) of which two different forms, ADAR1-L and ADAR1-S, exist. As ADAR1-L is induced by interferon (IFN)-alpha, we examined the influence of IFN-alpha-stimulation of host cells on HDV-RNA editing. METHODS: Editing was studied in Huh-7-cells transfected with HDV-RNA on days 7, 14, 21 and 28 after transfection. ADAR1-L mRNA was measured by RT-PCR. RESULTS: IFN-alpha-treatment led to a 5-fold higher expression of ADAR1-L and to an increase in editing from 14+/-2% (SD) in unstimulated controls to 27+/-4% (SD) on day 7 after transfection. Editing further increases over time to the same maximum level of 35% in IFN-alpha-treated as well as untreated cells. CONCLUSIONS: By IFN-alpha-stimulation both ADAR1-L expression and editing are increased in Huh-7-cells at day 7, and the maximum level of edited antigenomes is reached earlier with IFN-alpha-treatment as compared to untreated cells. Thus, ADAR1-L appears to be able to increase editing, but the HDV genome apparently has an intrinsic negative feed-back regulation mechanism that limits editing to roughly a third of the genomes.

Recommendations for optimized settings of the Amicus Crescendo cell separator for the collection of CD34+ progenitor cells.

BACKGROUND: CD34+ PBPCs for autologous transplantation purposes are collected by leukapheresis procedures on automated cell separators. In this study, the influence of different parameters on collection efficiency (CE) of the Amicus Crescendo cell separator (Baxter) was investigated. STUDY DESIGN AND METHODS: A total of 146 PBPC collections with Amicus cell separators were performed in 56 patients with either settings recommended by the manufacturer or modified settings to identify variables that have a significant and important impact on CE. RESULTS: By use of a standard setting with a cycle volume of 1400 mL, CE significantly decreases when patients' preapheresis peripheral blood WBC counts are between 25,000 and 35,000 per micro L. CE can be improved if cycle volume is reduced to 1000 mL. If WBC concentrations exceed 55,000 per micro L before apheresis, CE also significantly decreases despite of reduced cycle volume. Additionally, high flow rates greater than 60 mL per minute significantly reduce CE. CONCLUSION: Parameters influencing the outcome of CD34+ PBPC collections were identified, such as patients' WBC count, cycle volume, and whole blood flow rate. An optimized adjustment of these variables will further increase the CE of the device.

Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry.

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix, was used. By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG+ cells can be observed, in Crohn's disease also a higher percentage of IgM+ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2+ cells, patients with ulcerative colitis an increase in IgG1+ and IgG3+ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.