Eun Jung Baek

In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

BACKGROUND: There is no appropriate alternative source of red blood cells (RBCs) to relieve the worsening shortage of blood available for transfusion. Therefore, in vitro generation of clinically available RBCs from hematopoietic stem cells could be a promising new source to supplement the blood supply. However, there have been few studies about the generation of clinical‐grade RBCs by coculture on human mesenchymal stem cells (MSCs) and various cytokine supplements, even though the production of pure RBCs requires coculture on stromal cells and proper cytokine supplements.

Direct and differential effects of stem cell factor on the neovascularization activity of endothelial progenitor cells

AIMS Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. METHODS AND RESULTS In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. CONCLUSION In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells

In vitro erythropoiesis has been studied extensively for its application in the manufacture of transfusable erythrocytes. Unfortunately, culture conditions have not been as effective as in vivo growth conditions, where bone marrow macrophages are known to be a key regulator of erythropoiesis. This study focused on the fact that some erythroblasts are detached from macrophages and only contact other erythroblasts. We hypothesized that additional factors regulate erythroblasts, likely through either physical contact or secreted factors. To further elucidate these critical factors, human erythroblasts derived from cord blood were cultured at high density to mimic marrow conditions. This growth condition resulted in a significantly increased erythroid enucleation rate and viability. We found several novel contact-related signals in erythroblasts: intercellular adhesion molecule-4 (ICAM-4) and deleted in liver cancer-1 (DLC-1). DLC-1, a Rho-GTPase-activating protein, has not previously been reported in erythroid cells, but its interaction with ICAM-4 was demonstrated here. We further confirmed the presence of a secreted form of human ICAM-4 for the first time. When soluble ICAM-4 was added to media, cell viability and enucleation increased with decreased nuclear dysplasia, suggesting that ICAM-4 is a key factor in contact between cells. These results highlight potential new mechanisms for autonomous control of erythropoiesis. The application of these procedures to erythrocyte manufacturing could enhance in vitro erythrocyte production for clinical use.

Genomic breakpoints and clinical features of MLL-TET1 rearrangement in acute leukemias

Recent rapid developments in new technology such as whole genome/exome sequencing have revealed that novel mutations in genes such as DNMT3A , IDH1 , IDH2 and TET2 contribute to the main process of leukemogenesis in patients with normal karyotype acute myeloid leukemia (AML).[1][1] Among these genes

Red Blood Cell Engineering in Stroma and Serum/Plasma-Free Conditions and Long Term Storage

In vitro generation of artificial red blood cells (RBCs) is very important to overcome insufficient and unsafe blood supply. Despite recent progresses in RBCs engineering from several stem cell sources, none of them could succeed in generation of functional RBCs in the absence of serum/plasma and feeder cells. Without the elimination of serum and plasma, human RBC engineering in a large scale is impossible, especially for the future bioreactor system. Using an appropriate combination of cost-effective and safe reagents, the present study demonstrated the terminal maturation of hematopoietic stem cells into enucleated RBCs, which were functional comparable to donated human RBCs. Surprisingly, the viability of erythroid cells was higher in our serum- and feeder-free culture condition than in the previous serum-added condition. This was possible by supplementation with vitamin C in media and hypothermic conditions. Also, our report firstly presents the storability of artificial RBCs, which possibility is essential for clinical application. In summary, our report demonstrates engineering of human applicable RBCs with a dramatically enhanced viability and shelf-life in both serum- and stroma-free conditions. This innovative culture technology could contribute to the realization of the large-scale pharming of human RBCs using bioreactor systems.

The RNA in reticulocytes is not just debris: it is necessary for the final stages of erythrocyte formation.

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

Rh D blood group conversion using transcription activator-like effector nucleases

Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

Corrigendum: Rh D blood group conversion using transcription activator-like effector nucleases

Nature Communications 6: Article number: 745110.1038/ncomms8451 (2015); Published: June162015; Updated: October222015 The original version of the Supplementary Information attached to this Article contained errors in the sequences of plasmids presented in Supplementary Note 1. The HTML has now been updated to include a corrected version of the Supplementary Information.

In‐Vitro Production of Functional RBCs from Hematopoietic Stem Cells

A great deal of research has focused on methods to produce red blood cells (RBCs) industrially in order to reduce reliance on the supply from human donations. Among the possible candidates for artificial blood, the most perfect substitute would be actual human RBCs derived from erythroid progenitor cells grown in vitro just as occurs in vivo.

The effects of plasma gelsolin on human erythroblast maturation for erythrocyte production

Gelsolin is an actin binding protein present in blood plasma and in cytoplasm of cells including macrophages. Gelsolin has important functions in cell cycle regulation, apoptotic regulation, and morphogenesis. Even though bone marrow macrophages and serum factors are critical for regulating erythropoiesis, the role of gelsolin on human erythroblasts has not been studied. Here, we investigated the effects of human recombinant plasma gelsolin (pGSN) on human immature erythroblasts. CD34+ cells isolated from cord blood were differentiated into erythroid cells in serum-free medium. When pGSN was applied to the culture medium, it accelerated basophilic and polychromatic erythroblast maturation and increased the enucleation rate with highly expressed erythropoiesis-related mRNAs. Also, pGSN was effective in reducing dysplastic changes caused by vincristine, suggesting its role in cell cycle progression at G2/M checkpoints. Also, pGSN activated caspase-3 during maturation stages in which caspase-3 functions as a non-apoptotic maturational signal or a pro-apoptotic signal depending on maturation stages. Our results suggest that pGSN has a pivotal role in maturation of erythroblasts and this factor might be one of the way how bone marrow macrophages and previously unknown serum factors work to control erythropoiesis. pGSN might be used as additive for in vitro production of erythrocytes.