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      Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

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          Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

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          Most cited references 10

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          Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.

          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.
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            Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo.

            New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.
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              Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells.

              Although significant advances have been made over the last decade with respect to our understanding of stem cell biology, progress has been limited in the development of successful techniques for clinically significant ex vivo expansion of hematopoietic stem and progenitor cells. We here describe the effect of Notch ligand density on induction of Notch signaling and subsequent cell fate of human CD34+CD38- cord blood progenitors. Lower densities of Delta1(ext-IgG) enhanced the generation of CD34+ cells as well as CD14+ and CD7+ cells, consistent with early myeloid and lymphoid differentiation, respectively. However, culture with increased amounts of Delta1(ext-IgG) induced apoptosis of CD34+ precursors resulting in decreased cell numbers, without affecting generation of CD7+ cells. RNA interference studies revealed that the promotion of lymphoid differentiation was primarily mediated by Delta1 activation of Notch1. Furthermore, enhanced generation of NOD/SCID repopulating cells was seen following culture with lower but not higher densities of ligand. These studies indicate critical, quantitative aspects of Notch signaling in affecting hematopoietic precursor cell-fate outcomes and suggest that density of Notch ligands in different organ systems may be an important determinant in regulating cell-fate outcomes. Moreover, these findings contribute to the development of methodology for manipulation of hematopoietic precursors for therapeutic purposes.

                Author and article information

                [1 ]Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, United States of America
                [2 ]William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, United States of America
                [3 ]Celgene Cellular Therapeutics, Warren, New Jersey, United States of America
                [4 ]McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
                Tufts University, United States of America
                Author notes

                Competing Interests: JJC, LRM and MZ are co-authors of patents assigned to Cleveland Clinic and The Ohio State University, issued, licensed and pending that are related to certain aspects of equipment and methods developed for the purpose of this study. SA, XZ, LK and VV-B are employees of Celgene Cellular Therapeutics, Inc. (CCT) who collaborate and share materials with the rest of co-authors as supported by Defence Advanced Research Projects Agency (DARPA) funding (see Financial Disclosure). This does not alter the authors' adherence to all PLoS ONE policies on sharing data and materials.

                Conceived and designed the experiments: XZ MVK JJC MZ. Performed the experiments: XJ LK VVB RZ LRM. Analyzed the data: XJ XZ RZ MVK LRM JJC MZ. Contributed reagents/materials/analysis tools: SA MVK JJC MZ. Wrote the paper: XJ SA XZ MVK LRM JJC MZ.

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                27 August 2012
                : 7
                : 8

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Pages: 10
                The work was supported by funding from Defence Advanced Research Agency (DARPA) Projects, BAA07-21 “Continuous RBC Production” to SA; the National Health Institutes (NIH), CA062349 to MZ; and the National Science Foundation (NSF), BES-0124897 to JJC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Cell Mechanics
                Biomedical Engineering
                Medical Devices
                Tissue Engineering
                Physical Chemistry
                Chemical Properties
                Physicochemical Properties
                Biomedical Engineering
                Medical Devices
                Chemical Engineering
                Fluid Mechanics
                Anatomy and Physiology
                Cell Physiology
                Iron Deficiency Anemia
                Bone Marrow and Stem Cell Transplantation
                Heme Synthesis
                Red Cells
                Transfusion Medicine
                Cell Mechanics



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