Erythroid Development in the Absence of Hemoglobin.

Abstract The mammalian erythrocyte is a highly specialized blood cell that differentiates via an orderly series of committed progenitors in the bone marrow in a process termed erythropoiesis. Homeostasis of the erythron is carefully maintained by balancing the proliferation and destruction of early and late erythroid progenitors. In mature red blood cells over ninety-five percent of the protein is hemoglobin (Hb). What happens to committed erythroid cells in the absence of hemoglobin? To answer this question we have derived a novel line of embryonic stem (ES) cells from mouse embryos that have all eight adult alpha and beta globin genes knocked out. These “Null” Hb ES cells were injected into wild-type blastocysts to examine their in vivo potential to contribute to the tissues of developing chimeric mice. Examination of the peripheral blood and bone marrow of these chimeras by flow cytometry revealed that the “Null” Hb ES cells were able to produce normal levels of each type of white blood cell analyzed. However, “Null” erythrocytes were absent from the circulation and only early committed progenitors were found in the bone marrow. Very few “Null” erythroid cells matured beyond the proerythoblast to the basophilic erythroblast stage (Ter119low, CD71hi). To study this maturational block in more detail, an erythroid culture system was established by in vitro differentiation of the “Null” Hb ES cells. These pure erythroid progenitor (EP) cultures support and amplify the proerythroblast stage of development. Interestingly, EP cells could be derived from “Null” Hb ES cells demonstrating that Hb is not required for the development of proerythroblasts. “Null” derived EP cells express erythroid lineage markers (EKLF, GATA1, GypA, EpoR, Tal1), but express no adult globins or markers of other hematopoietic lineages (Mpl, GATA3, IL7R, PAX5, CEBPα, CD41b). Upon terminal differentiation most “Null” derived EP cells undergo apoptosis by 48 hours (7AAD−, Annexin V+) and are dead (7AAD+) by 72 hours. These “Null” Hb ES cells provide a novel experimental system to elucidate the role of hemoglobin during erythroid differentiation, maturation, and homeostasis.

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Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1642 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1642-1651 ◽

Cited By ~ 240

Author(s):

M J Weiss ◽

C Yu ◽

S H Orkin

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Zinc Finger ◽

Es Cells ◽

Embryonic Stem ◽

Erythroid Cell ◽

Transactivation Domain ◽

Stage Of Development ◽

Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

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Development of hematopoietic cells lacking transcription factor GATA-1

Development ◽

10.1242/dev.121.1.163 ◽

1995 ◽

Vol 121 (1) ◽

pp. 163-172 ◽

Cited By ~ 4

Author(s):

L. Pevny ◽

C.S. Lin ◽

V. D'Agati ◽

M.C. Simon ◽

S.H. Orkin ◽

...

Keyword(s):

Transcription Factor ◽

Mast Cell ◽

Cell Differentiation ◽

Mast Cells ◽

Blood Cell ◽

Embryonic Stem ◽

Sequence Motif ◽

Erythroid Cells ◽

Gata Factor

GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-1 in hematopoietic cell differentiation, we have tested the ability of embryonic stem cells, carrying a targeted mutation in the X-linked GATA-1 gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-1- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this factor at some point in the erythroid lineage (L. Pevny et al., (1991) Nature 349, 257–260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-1 in other lineages. We found that the development of erythroid progenitors in embryonic yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation.

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 54

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors

Blood ◽

10.1182/blood.v89.4.1207 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1207-1213 ◽

Cited By ~ 21

Author(s):

Takumi Era ◽

Tomomi Takahashi ◽

Katsuya Sakai ◽

Kazuo Kawamura ◽

Toru Nakano

Keyword(s):

Colony Formation ◽

Es Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

In Vitro Differentiation ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Proliferation And Differentiation ◽

Differentiation Status

Abstract Thrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.

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Selective Expression of the Receptor Tyrosine Kinase, HTK, on Human Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v89.8.2757 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2757-2765 ◽

Cited By ~ 32

Author(s):

Tomohisa Inada ◽

Atsushi Iwama ◽

Seiji Sakano ◽

Mitsuharu Ohno ◽

Ken-ichi Sawada ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Bone Marrow Cells ◽

Regulatory System ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Marrow Cells

Abstract HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34low, and glycophorin A−/low. Assays of progenitors showed that HTK+c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK−c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

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Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.

Blood ◽

10.1182/blood.v106.11.4255.4255 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4255-4255

Author(s):

Ewa Carrier ◽

Shermila Kausal ◽

Anand S. Srivastava

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Growth Factors ◽

Es Cells ◽

Embryonic Stem ◽

Globin Genes ◽

Rt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.

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

Blood ◽

10.1182/blood.v124.21.3846.3846 ◽

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.

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108.423k29_4108_4118 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 4

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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