Generation Of Functional Erythrocytes From Human Embryonic Stem Cell-Derived Definitive Hematopoiesis

Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2008; 105(35):13087-92. DOI: 10.1073/pnas.0802220105
Source: PubMed

ABSTRACT A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic epsilon-globin-expressing erythroid cells from individual clones decreased, whereas adult-type beta-globin-expressing cells increased to approximately 100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34(+) progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.

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Available from: Hiromi Sakai, Jun 11, 2014
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    • "It was once expected that CD34 + cells from cord blood (CB) or bone marrow would someday provide a means for ex vivo expansion or in vitro generation of erythrocytes (red blood cells; RBCs) for transfusion (Giarratana et al., 2011), but the inability to produce sufficient numbers of CD34 + cells remains a bottleneck in this process . It was also thought that the advent of pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), would eliminate the need for blood donation, but a series of differentiation trials to create various blood cells from human ESCs or iPSCs has highlighted the difficulty of obtaining blood cells in quantities sufficient for use in transfusion using this method (Lu et al., 2007, 2011; Takayama et al., 2008, 2010). "
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    ABSTRACT: The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.
    Stem Cell Reports 12/2013; 1(6):499-508. DOI:10.1016/j.stemcr.2013.10.010
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    • "In addition to shedding light on the developmental biology of erythroid cells, the investigation of HESC-derived erythroblasts (ESERs) and/or HiPSC-derived erythroid cells may be clinically relevant. ESERs and HiPSC-derived erythroblasts reportedly exhibit characteristics of cord blood (CB) in terms of their oxygen dissociation curve, G6PD activities , CO rebinding kinetics, and response to 2,3-DPG depletion [7] [8] [9]. Cell enucleation can also occur under appropriate culture conditions [5]. "
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    ABSTRACT: To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs-ESERs, ESERs-FLERs, and FLERs-PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell-cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.
    Genomics 10/2013; 102(5-6). DOI:10.1016/j.ygeno.2013.09.005 · 2.79 Impact Factor
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    • "One group has cultured whole human PSC colonies on immortalized human fetal liver cells until CD34 þ cells arose, then treated the CD34 þ cells with hematopoietic differentiation cytokines and cocultured with mouse stromal cells (Olivier et al. 2006; Qiu et al. 2008). A second group cultured whole human PSC colonies on mouse fetal liver stromal cells with hematopoietic differentiation cytokines (Ma et al. 2008). A third group differentiated human PSCs as EBs, which were then directly plated on Matrigel with hematopoietic differentiation cytokines (Chang et al. 2006). "
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    ABSTRACT: Pluripotent stem cells (PSCs) hold great promise for research and treatment of hemoglobinopathies. In principle, patient-specific induced pluripotent stem cells could be derived from a blood sample, genetically corrected to repair the disease-causing mutation, differentiated into hematopoietic stem cells (HSCs), and returned to the patient to provide a cure through autologous gene and cell therapy. However, there are many challenges at each step of this complex treatment paradigm. Gene repair is currently inefficient in stem cells, but use of zinc finger nucleases and transcription activator-like effector nucleases appear to be a major advance. To date, no successful protocol exists for differentiating PSCs into definitive HSCs. PSCs can be directly differentiated into primitive red blood cells, but not yet in sufficient numbers to enable treating patients, and the cost of clinical scale differentiation is prohibitively expensive with current differentiation methods and efficiencies. Here we review the progress, promise, and remaining hurdles in realizing the potential of PSCs for cell therapy.
    Cold Spring Harbor Perspectives in Medicine 04/2012; 2(4):a011841. DOI:10.1101/cshperspect.a011841 · 7.56 Impact Factor
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