Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2534-2534
Author(s):  
Akira Niwa ◽  
Tomoki Fukatsu ◽  
Katsutsugu Umeda ◽  
Itaru Kato ◽  
Hiromi Sakai ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Oxygen Carriers ◽  
Developmental Pathway ◽  
Ips Cell

Abstract Abstract 2534 Poster Board II-511 Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production. Disclosures: No relevant conflicts of interest to declare.

Generation of Blood Cells from Human Ips Cells in Vitro through the Hematopoietic Progenitors Concentrated within the Unique Structures, Ips-Sac.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1992-1992 ◽  
Author(s):  
Naoya Takayama ◽  
Koji Eto ◽  
Hiromitsu Nakauchi ◽  
Shinya Yamanaka
Keyword(s):  
Cell Lines ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Dermal Fibroblasts ◽  
Hematopoietic Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell ◽  
Human Ips Cells

Abstract Human embryonic stem cells (hESCs) are proposed as an alternative source for transfusion therapy or studies of hematopoiesis. We have recently established an in vitro culture system whereby hESCs can be differentiated into hematopoietic progenitors within the ‘unique sac-like structures’ (ES-sacs), that are able to produce megakaryocytes and platelets (Takayama et al., Blood, 111, 5298–306, 2008). However there is a little concern that repetitive transfusion with same human ESC-derived platelets may induce immunological rejection against transfused platelets expressing allogenic HLA. Meanwhile, induced pluripotent stem (iPS) cells established from donor with identical HLA are well known as a potential and given source on platelet transfusion devoid of rejection. To examine if human iPS cells could generate platelets as well as from hESCs, we utilized 3 different human iPS cell lines; two were induced by transduction of 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) in adult dermal fibroblasts, and one was by 3 genes without c-Myc. Sac-like structures (iPS-sac), inducible from 3 iPS cell lines, concentrated hematopoietic progenitors that expressed early hemato-endothelial markers, such as CD34, CD31, CD41a (integrin αIIb) and CD45. These progenitors were able to form hematopoietic colonies in semi-solid culture and differentiate into several blood cells including leukocytes, erythrocytes or platelets. Of these, obtained platelets responded to agonist stimulation, in which the function was as much as human ESC-derived platelets, as evidenced by PAC-1 binding with activated αIIbβ3 integrin or full spreading onto fibrinogen. These results collectively indicated that human dermal fibroblasts could generate functional and mature hematopoietic cells through the reprogramming process and this method may be useful for basic studies of hematopoietic disorders and clinical therapy in the future.

scholarly journals Adamts13-Cultured Red Blood Cells to Treat Thrombotic Thrombocytopenic Purpura

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 89-89
Author(s):  
Kai Wang ◽  
Khulan Batbayar ◽  
Karl Roberts ◽  
Emmanuel Olivier ◽  
Eric E. Bouhassira
Keyword(s):  
Red Blood Cells ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Blood Cells ◽  
Vascular System ◽  
Conflicts Of Interest ◽  
Direct Injection ◽  
Culture Method ◽  
Thrombocytopenic Purpura ◽  
Membrane Bound

Red Blood Cells (RBCs) have long been considered a potentially useful means of delivering drugs to the circulation because delivery through therapeutic RBCs as compared to direct injection in the plasma can lengthen the half-life of the therapeutic agent in the circulation, spatially restrict the drugs to the lumen of the cardio-vascular system, and shield the drug from the immune system. Despite some progress, loading the cells with therapeutically useful cargo remains technically challenging. We have recently developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated cultured RBCs. This provides an ideal method to produce therapeutic RBCs since iPSCs can be genetically manipulated with powerful CRISPR-based technologies. ADAMTS13, whose deficiency is responsible for congenital and acquire Thrombotic Thrombocytopenic Purpura (TTP) is a good target as a therapeutic that could be delivered through drug-carrying RBCs because large amounts of plasma concentrate, or more recently recombinant proteins, are necessary to treat TTP. We report here we have produced engineered erythroid cells that contains globin-LCR driven ADAMTS13 fusion transgenes inserted at safe harbor AAVS1, and that these cells express a membrane bound version of an inhibitor-resistant version of ADAMTS13. We show using flow cytometry that the fusion protein is express at high levels, and using a FRET assay that detect cleavage of the von Willebrand cognate site, that the membrane bound ADAMTS13 is enzymatically active. Comparison of enzymatic activity with plasma concentrate suggests that about 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate. This suggests that a transfusion of about 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP. The number of cRBCs necessary to treat even a few patients is very large. This has been considered a major obstacle to the development of treatment based on in vitro produced RBCs because of the volume of culture that is necessary to produce the cells. We also report that we have developed a culture method based on holo-fiber bioreactors that allows the production of cRBCs at a density of 5.108 cell/mL which is sufficient to produce enough cells to performed small clinical trials. Disclosures No relevant conflicts of interest to declare.

Identification of secreted proteins that can promote erythroid progenitor expansion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4864-4864
Author(s):  
Bing Xu ◽  
Xiangmeng Wang ◽  
Peng Li ◽  
Yiren Xiao ◽  
Huijuan Dong ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Secreted Proteins ◽  
Erythroid Progenitor ◽  
In Vitro Expansion ◽  
Stromal Cell Line ◽  
Expansion System

Abstract Erythroid blasts are progenitors that can differentiate to mature red blood cells (RBC). Identification of these growth factors and understanding their downstream pathways can help us to optimize RBC production ex vivo. We derived a stromal cell line (PL16) from mouse embryonic fetal livers that promotes proliferation of mouse and human erythroid blasts in vitro. Among specifically highly expressed secreted proteins in PL16, we identified a new growth factor (FA) that is capable of stimulating human erythroid blast expansion in vitro significantly. Furthermore, erythroid blasts from our in vitro expansion system can differentiate into functional mature red blood cells. Disclosures: No relevant conflicts of interest to declare.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3846-3846
Author(s):  
Ji-Yoon Noh ◽  
Shilpa Gandre-Babbe ◽  
Yuhuan Wang ◽  
Vincent Hayes ◽  
Yu Yao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell

Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

MODELING GENETIC Diseases through Reprogramming of HUMAN Amniotic Liquid Derived CELLS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4789-4789
Author(s):  
Noufissa Oudrhiri ◽  
Frank Yates ◽  
Olivier Feraud ◽  
Emilie Gobbo ◽  
Cecile BAS ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Sickle Cell Anemia ◽  
Genetic Diseases ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Ips Cell

Abstract Abstract 4789 Pluripotency and self-renewal, two key characteristics of induced pluripotent stem cells (IPS), make these cells ideally suited for modeling diseases in vitro and generating biological resources usable for drug screening and cell therapy. However, the reprogramming efficiency of somatic cells greatly varies according to the cell type, to the in vitro proliferation index, the number of passages and the age of the donor. Human amniotic liquid-derived cells (hALDC), collected during amniocentesis for the prenatal diagnosis of genetic diseases, represent an abundant source of primary cells. In preliminary experiments we have shown that hALDC expressed endogenous Oct4 and Sox2 proteins suggesting that could be readily amenable to reprogramming. To this end, we have used two strategies using either hALDC or neonatal fibroblasts: (1) lentivirus mediated gene transfer of OCT4, SOX2, LIN28, NANOG, (2) retroviruses mediated gene transfer of OCT4, SOX2, CMYC, KLF4 and (3) lentiviral transfer of OCT4, SOX2. hALDC transduced by these viruses were placed on MEF and b-FGF (10 ng/ml) with daily medium changes. One to three weeks after infection, typical human ES-like colonies could be picked up for expansion before being characterized. HALDC show an increased reprogramming potential with the [OCT4, SOX2, LIN28, NANOG] and [OCT4, SOX2] cocktails, when compared to reprogramming of neonatal fibroblasts. Twelve hALDC-derived-IPS cells were obtained from 12 different samples of amniotic fluid. All hALDC-IPS cell lines maintained a normal karyotype in culture and displayed the morphology and characteristics of human embryonic stem cells, including the surface expression of Tra-160, SSEA-3, SSEA-4, HESCA-1 and alkaline phosphatase, and formed multi-lineaged teratomas upon injection to NOD-SCID mice. Gene expression profiles of the IPS cell lines reveal a high correlation coefficient between hALDC-iPS cells and human embryonic stem cells, and a low correlation between hALDC-iPS and hALDC. When compared to hES cells H1, H9 and Cl01, these cell lines generated hematopoiesis with a variable efficiency in vitro. Amongst the hALDC-IPS cell lines generated by our laboratory (http://www.hescreg.eu/) four lines carry an inherited trisomy of chromosome 21, and three lines carry the homozygous “S” mutation in the beta-globin gene of sickle-cell anemia. All hALDC-IPS cell are currently banked at the Human Pluripotent Stem Cell Core Facility, France. In conclusion, hALDC can be rapidly and efficiently reprogrammed to pluripotency with a limited number of transgenes. Moreover, hALDC-IPS cell lines derived from patients can be used to modelize in vitro the phenotypic features of monogenic diseases such as sickle cell anemia or more complex, multifactorial disorders such as Down's syndrom. The ability to generate hematopoietic differentiation from these cell lines will facilitate the modelling of these hematopoietic disorders. Disclosures: No relevant conflicts of interest to declare.

Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4334-4334
Author(s):  
Anand S Srivastava ◽  
Babak Esmaeli Azad ◽  
Rosalia De Necochea Campion ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4334 It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold. We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture. The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use. Disclosures: Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.

scholarly journals Biologic properties and enucleation of red blood cells from human embryonic stem cells

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4475-4484 ◽  
Author(s):  
Shi-Jiang Lu ◽  
Qiang Feng ◽  
Jennifer S. Park ◽  
Loyda Vida ◽  
Bao-Shiang Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Embryonic Stem ◽  
Globin Chain ◽  
Nuclear Condensation

Abstract Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (1010-1011 cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult β-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of β-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 μm. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.

scholarly journals An Essential Role for the RNA Editor-Exonuclease Axis in Terminal Erythroid Differentiation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Areum Han ◽  
Alena V. Yermalovich ◽  
Vanessa Lundin ◽  
Daniel S. Pearson ◽  
Mariam Hachimi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Rna Degradation ◽  
Erythropoietin Receptor ◽  
Conditional Knockout ◽  
Erythroid Progenitors

Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Research Progress and Application Prospect of IPS Cells

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Ceng Yiwu
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Ips Cells ◽  
New Drugs ◽  
Theoretical Research ◽  
Research Progress ◽  
Ips Cell

Differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPS cells) by introducingspecific transcription factors. This technique avoids immune rejection and ethical problems in stem cell research.A great revolution in the fi eld of science. As with embryonic stem cells (ES cells), iPS cells are able to self-renewand maintain undiff erentiated state. In vitro, iPS cells can be induced to diff erentiate into a variety of mature cells,therefore, iPS cells in theoretical research and clinical applications are extremely valuable. IPS cell diff erentiationand transplantation in the treatment of blood diseases have a great use, iPS cells can treat nervous system diseases,to provide in vitro disease model, to study the mechanism of disease formation, screening new drugs and thedevelopment of new to provide a new treatment The The use of iPS cells as a nuclear donor cell, with the appropriatereceptor cells after fusion can be directly obtained transgenic animals. Not only can improve the genetic nature ofanimals, but also can break the boundaries of species and get the new animal traits that cannot achieve by usingtraditional mating methods. The research of iPS cells has been widely concerned, and it is the research hotspot in cellbiology and molecular biology. In this paper, the defi nition of iPS cells, the acquisition of iPS cells, the history ofdevelopment, the signifi cance of research, the progress of research, the application of iPS cells, and the problems ofiPS cells were reviewed.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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