The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous ...phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.
Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate ...cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.
•A significant fraction of cell-free heme in plasma is contained in circulating erythrocyte MPs, particularly during hemolysis.•Heme-laden MPs can transfer heme to vascular endothelium and mediate oxidative stress, vascular dysfunction, and vasoocclusions.
In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the ...question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.
Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced ...pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.
We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin.
We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form.
Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.
We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the ...application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by ...establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors.
We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation.
We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice.
These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.
Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant ...neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe2+) form, indicating a His-Fe2+-His binding scheme. O2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe3+) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (kon) and a slow dissociation rate (koff) for both O2 and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O2 and CO binding is suggested to be slowin vivo. Because of this competition, the observed O2 affinity of recombinant human neuroglobin is average (1 torr at 37 °C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 °C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E′o = −129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.
The oligomeric state and kinetics of ligand binding were measured for wild‐type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The ...extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N‐ and C‐terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.
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