1: Blood. 2005 Aug 1;106(3):860-70. Epub 2005 Apr 14. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Zambidis ET, Peault B, Park TS, Bunz F, Civin CI. Division of Immunology and Hematopoiesis, Sidney Kimmel comprehensive Cancer Center at John Hokins, The John Hopkins School of Medicine, Baltimore, MD, USA. ezambid1@jhmi.edu We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis. PMID: 15831705 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Development. 2005 Mar;132(5):1117-26. Epub 2005 Feb 2. RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells. Robert-Moreno A, Espinosa L, de la Pompa JL, Bigas A. Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain. Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been recently established, although its mechanism of action is poorly understood. Here, we show detailed analysis of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjkappa transcription factor to activate transcription, we analyzed the aorta of the para-aortic splanchnopleura/AGM in RBPjkappa mutant embryos. We found specific patterns of expression of Notch receptors, ligands and Hes genes that were lost in RBPjkappa mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. We show that in wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and demonstrate by chromatin immunoprecipitation that Notch1 specifically associated with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjkappamutants. Consistent with a role for Notch1 in regulating Gata2, we observe increased expression of this gene in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjkappa is a crucial event for the onset of definitive hematopoiesis in the embryo. PMID: 15689374 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Dev Biol. 2005 Jan 15;277(2):522-36. Zebrafish scl functions independently in hematopoietic and endothelial development. Dooley KA, Davidson AJ, Zon LI. Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston, MA 02115, USA. The SCL transcription factor is critically important for vertebrate hematopoiesis and angiogenesis, and has been postulated to induce hemangioblasts, bipotential precursors for blood and endothelial cells. To investigate the function of scl during zebrafish hematopoietic and endothelial development, we utilized site-directed, anti-sense morpholinos to inhibit scl mRNA. Knockdown of scl resulted in a loss of primitive and definitive hematopoietic cell lineages. However, the expression of early hematopoietic genes, gata2 and lmo2, was unaffected, suggesting that hematopoietic cells were present but unable to further differentiate. Using gene expression analysis and visualization of vessel formation in live animals harboring an lmo2 promoter-green fluorescent protein reporter transgene (Tg(lmo2:EGFP)), we show that angioblasts were specified normally in the absence of scl, but later defects in angiogenesis were evident. While scl was not required for angioblast specification, forced expression of exogenous scl caused an expansion of both hematopoietic and endothelial gene expression, and a loss of somitic tissue. In cloche and spadetail mutants, forced expression of scl resulted in an expansion of hematopoietic but not endothelial tissue. Surprisingly, in cloche, lmo2 was not induced in response to scl over-expression. Taken together, these findings support distinct roles for scl in hematopoietic and endothelial development, downstream of hemangioblast development. PMID: 15617691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Dev Dyn. 2005 Feb;232(2):487-97. Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells. Gerecht-Nir S, Dazard JE, Golan-Mashiach M, Osenberg S, Botvinnik A, Amariglio N, Domany E, Rechavi G, Givol D, Itskovitz-Eldor J. Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa 31096, Israel. The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified-one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left-right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFbeta3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFbetaR2, and TGFbetaR3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development. Copyright 2004 Wiley-Liss, Inc. PMID: 15614775 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Leuk Res. 2004 Nov;28(11):1227-37. Analysis of the relationship between Scl transcription factor complex protein expression patterns and the effects of LiCl on ATRA-induced differentiation in blast cells from patients with acute myeloid leukemia. Rice AM, Holtz KM, Karp J, Rollins S, Sartorelli AC. Department of Pharmacology, Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias. PMID: 15380350 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Blood. 2005 Jan 15;105(2):617-26. Epub 2004 Sep 16. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Department of Pathology and Laboratory Medicine, Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53792-8550, USA. Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells. PMID: 15374881 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Cell Death Differ. 2003 Aug;10(8):905-13. Control of erythroid cell production via caspase-mediated cleavage of transcription factor SCL/Tal-1. Zeuner A, Eramo A, Testa U, Felli N, Pelosi E, Mariani G, Srinivasula SM, Alnemri ES, Condorelli G, Peschle C, De Maria R. Department of Hematology and Oncology, Istituto Superiore di Sanita, Rome 00161, Italy. SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis. PMID: 12867998 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: EMBO J. 2002 Jun 17;21(12):3039-50. Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors. Gottgens B, Nastos A, Kinston S, Piltz S, Delabesse EC, Stanley M, Sanchez MJ, Ciau-Uitz A, Patient R, Green AR. University of Cambridge Department of Haematology, Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, UK. bg200@cam.ac.uk Stem cells are a central feature of metazoan biology. Haematopoietic stem cells (HSCs) represent the best-characterized example of this phenomenon, but the molecular mechanisms responsible for their formation remain obscure. The stem cell leukaemia (SCL) gene encodes a basic helix-loop-helix (bHLH) transcription factor with an essential role in specifying HSCs. Here we have addressed the transcriptional hierarchy responsible for HSC formation by characterizing an SCL 3' enhancer that targets expression to HSCs and endothelium and their bipotential precursors, the haemangioblast. We have identified three critical motifs, which are essential for enhancer function and bind GATA-2, Fli-1 and Elf-1 in vivo. Our results suggest that these transcription factors are key components of an enhanceosome responsible for activating SCL transcription and establishing the transcriptional programme required for HSC formation. PMID: 12065417 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10716-21. Epub 2001 Sep 4. Hematopoietic colony-forming cells derived from human embryonic stem cells. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Section of Hematology, Department of Internal Medicine, University of Wisconsin Hospital and Clinics, 600 Highland Avenue, Madison, WI 53792, USA. Human embryonic stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C166. This hematopoietic differentiation requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription factors TAL-1, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic growth factors, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34(+) cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. The in vitro differentiation of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies. PMID: 11535826 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Br J Haematol. 2001 Apr;113(1):52-7. Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia. Fujimaki S, Harigae H, Sugawara T, Takasawa N, Sasaki T, Kaku M. Department of Molecular Diagnostics, Tohoku University School of Medicine, Sendai, Japan. Aplastic anaemia is characterized by reduced haematopoiesis resulting in pancytopenia. It has been speculated that there is an injury in haematopoietic stem cells in the bone marrow; however, the precise nature of the injury has not been elucidated. In this study, the levels of expression of mRNAs for three transcription factors, GATA-2, SCL and AML1, which function in the early stages of haematopoiesis, were examined by quantitative polymerase chain reaction in patients with aplastic anaemia, idiopathic thrombocytopenic purpura (ITP) and normal subjects. Among these factors, expression of GATA-2 mRNA in purified CD34-positive cells was markedly decreased in aplastic anaemia compared with that in ITP and in normal subjects. The expression levels of SCL and AML1 mRNA in CD34-positive cells in aplastic anaemia were not different from those in normal subjects. When the expression of GATA-2 protein in CD34-positive cells was examined by immunocytochemical analysis, the percentage of GATA-2-positive cells in aplastic anaemia was lower than that in normal subjects. These findings strongly suggest that there is an aberrant expression of transcription factors in stem cells in aplastic anaemia, which may be responsible for the development of the disease. PMID: 11328281 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Exp Hematol. 2000 Jul;28(7):833-40. Sequential requirements for SCL/tal-1, GATA-2, macrophage colony-stimulating factor, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development. Yamane T, Kunisada T, Yamazaki H, Nakano T, Orkin SH, Hayashi SI. Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan. yamanet@grape.med.tottori-u.ac.jp OBJECTIVE: Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells. MATERIALS AND METHODS: We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development. RESULTS: The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors. CONCLUSION: These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units. PMID: 10907645 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Dev Dyn. 1999 Jun;215(2):139-47. Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer. Marshall CJ, Moore RL, Thorogood P, Brickell PM, Kinnon C, Thrasher AJ. Molecular Immunology Unit, Institute of Child Health, London, England. C.Marshall@ich.ucl.ac.uk The definitive long-term repopulating human hematopoietic stem cell, which seeds the adult blood system, was previously thought to derive from the extra-embryonic yolk sac. However, there is now considerable evidence that in both avian and murine systems, yolk sac hematopoietic cells are largely a transient, embryonic population and the definitive stem cell, in fact, derives from a distinct region within the embryonic mesoderm, the aorta-gonad-mesonephros region. In the human embryo, an analogous region has been found to contain a cluster of cells distinct from, but closely associated with, the ventral endothelium of the dorsal aorta, the appearance of which is restricted both spatially and temporally. We have used antibodies recognising hematopoietic regulatory factors to further characterise this region in the human embryo. These studies indicate that all factors examined, including vascular endothelial growth factor and its receptor FLK-1, Flt-3 ligand and its receptor STK-1, and stem cell leukemia transcription factor, are expressed by both hematopoietic cells in the cluster and endothelial cells. However, there is some discontinuity in cells directly underlying the cluster. Furthermore, we have identified a morphologically distinct region of densely-packed, rounded cells in the mesenchyme directly beneath the ventral wall of the dorsal aorta, and running along its entire length. In the preumbilical AGM region, directly underlying the hematopoietic cluster, but not at more rostral and caudal levels, this region of mesenchyme expresses tenascin-C, an extracellular matrix glycoprotein known to facilitate cell-cell interactions and migration. This region of cells may therefore provide the microenvironmental support for the intraembryonic development of definitive hematopoietic stem cells, a process in which tenascin-C may play a pivotal role. PMID: 10373018 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Blood. 1999 May 15;93(10):3355-68. Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. Ziegler BL, Muller R, Valtieri M, Lamping CP, Thomas CA, Gabbianelli M, Giesert C, Buhring HJ, Kanz L, Peschle C. Department of Medicine, Division of Hematology and Oncology, Eberhard-Karls-University Tubingen, Tubingen, Germany. benedikt.ziegler@uni-tuebingen.de In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development. PMID: 10233888 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Int J Hematol. 1998 Jun;67(4):369-78. Frequent expression of human Thy-1 antigen on pre-B cell acute lymphoblastic leukemia with t(9;22). Takahashi T, Mizutani M, Miwa H, Katayama N, Nishii K, Shikami M, Yamaguchi M, Shiku H, Kamada N, Kita K. Second Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan. Thy-1 (CDw90) is a phosphatidylinositol-anchored protein, and is expressed on human pluripotential hematopoietic stem cells. The expression pattern of this antigen on leukemia cells is still controversial. In this study, 72 adult patients with pre-B cell acute lymphoblastic leukemia (pre-B ALL) were examined for the expression pattern of Thy-1 by using indirect immunofluorescence and reversed transcription polymerase chain reaction (RT-PCR) methods. Twelve cases were judged positive on the basis of conventional immunophenotype criteria. Thirteen cases showed a weak clonal shift on the fluorogram, even though their positive percentages were from 6.7% to 14.9%. Thy-1 gene transcripts were detected in all of the 13 cases showing a weak clonal shift. The study of antibody binding capacity, which was calculated by the mean fluorescence intensity of the test sample on the basis of a calibration curve using standard beads, showed that cases with more than 150 sites/cell could be positive. Thy-1 positivity in pre-B ALL was not associated with the expression of B-cell differentiation antigens. Thy-1 expression was significantly higher in pre-B ALL cases with karyotypic abnormalities than in those with normal karyotype (p = 0.0071). The t(9;22) abnormality was found in 18 of the 25 Thy-1+ cases. Simultaneous expression of transcriptional factors, GATA-2 and SCL, was frequently detected in the Thy-1+ cases. bcr-abl and GATA-2 are thought to play important roles in the proliferation of immature hematopoietic cells. Indeed, cell-cycle analysis showed that the cell population in the S/G2/M phase of the present Thy-1+ cases was less than that in the Thy-1- cases (p = 0.001770). Our data suggest that Thy-1 expression indicates the proliferative status of the leukemia cells, not their phenotypic immaturity. PMID: 9695410 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Biochem Biophys Res Commun. 1998 Jun 9;247(1):65-9. Transcription factor NF-E2 is essential for the polyploidization of a human megakaryoblastic cell line, Meg-J. Kobayashi S, Teramura M, Ito K, Iwabe K, Inaba T, Mizoguchi H. Department of Hematology, Tokyo Women's Medical College, Japan. Transcription factors regulating the process of megakaryocyte development remain largely unclarified. To clarify them further, we used a human megakaryoblastic cell line, Meg-J, which showed prominant polyploidization and augmented platelet glycoprotein (GP) Ib expression after incubation with thrombopoietin (TPO, c-mpl ligand) and K252a (an indolocarbasole derivative). Under these conditions, we analyzed the expression of the transcription factors and observed that the expression of NF-E2 p45, but not those of GATA-1, GATA-2, Tal-1/SCL, Evi-1, and MafK, was increased after TPO and K252a stimulation. Gel-shift assay confirmed the enhanced binding activity to the NF-E2 site. The abolishment of NF-E2 p45 with NF-E2 antisense oligomers inhibited TPO plus K252a-induced polyploidization. These findings suggest that NF-E2 p45 is essential for the polyploidization of megakaryocytic cells. PMID: 9636655 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Hematol Oncol Clin North Am. 1997 Dec;11(6):1199-206. Stem cell transcription factors. Shivdasani RA. Dana-Farber Cancer Institute, Boston, Massachusetts, USA. The individual properties of hematopoietic stem cells, including self-renewal, maintenance of pluripotency, and asymmetric cell division, must depend at some level on the functions of specific transcription factors. Recently, valuable insights into stem cell transcription factor function have emerged from targeted gene disruption (knockout) studies in mice. Absence of transcription factors belonging to diverse protein families results in interference with expansion and differentiation of either the stem cell itself or of primitive multipotential progenitors. The findings from these and complementary experiments provide a framework for examining the transcription control of the earliest cellular events in hematopoiesis. Publication Types: Review Review, Tutorial PMID: 9443052 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Blood. 1997 Aug 15;90(4):1435-47. Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells. Elefanty AG, Robb L, Birner R, Begley CG. The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia. The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation. PMID: 9269761 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Biol Chem. 1997 Mar 28;272(13):8781-90. Distinct mechanisms direct SCL/tal-1 expression in erythroid cells and CD34 positive primitive myeloid cells. Bockamp EO, McLaughlin F, Gottgens B, Murrell AM, Elefanty AG, Green AR. University of Cambridge, Department of Haematology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early "decisions" are made during hematopoiesis. We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression. PMID: 9079714 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Blood. 1997 Feb 15;89(4):1154-64. Initiation of murine embryonic erythropoiesis: a spatial analysis. Silver L, Palis J. University of Rochester Medical Center, Department of Pediatrics and Cancer Center, NY 14642, USA. Hematopoiesis in the mouse conceptus begins in the visceral yolk (VYS), with primitive erythroblasts first evident in blood islands at the headfold stage (E8.0). VYS erythropoiesis is decreased or abrogated by targeted disruption of the hematopoietic transcription factors tal-1, rbtn2, GATA-1, and GATA-2. To better understand the potential roles of these genes, and to trace the initial temporal and spatial development of mammalian embryonic hematopoiesis, we examined their expression patterns, and that of betaH1-globin, in normal mouse conceptuses by means of in situ hybridization. Attention was focused on the 36-hour period from mid-primitive streak to early somite stages (E7.25 to E8.5), when the conceptus undergoes rapid morphologic changes with formation of the yolk sac and blood islands. Each of these genes was expressed in extraembryonic mesoderm, from which blood islands are derived. This VYS expression occurred in a defined temporal sequence: tal-1 and rbtn2 transcripts were detected earlier than the others, followed by GATA-2 and GATA-1, and then by betaH1-globin. Transcripts for all of these genes were present in VYS mesoderm cell masses at the neural plate stage (E7.5), indicating commitment of these cells to the erythroid lineage before the appearance of morphologically recognizable erythroblasts. By early somite stages (E8.5), GATA-2 mRNA expression is downregulated in VYS blood islands as terminal primitive erythroid differentiation proceeds. We conclude that primitive mammalian erythropoiesis arises during gastrulation through the ordered temporal expression of tal-1, rbtn2, GATA2, and GATA-1 in a subset of extraembryonic mesoderm cells. During the stages analyzed, tal-1 and rbtn2 expression was also present in posterior embryonic mesoderm, while GATA-1 and GATA-2 expression was evident in extraembryonic tissues of ectodermal origin. PMID: 9028937 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Blood. 1995 Oct 15;86(8):3173-80. The expression pattern of erythrocyte/megakaryocyte-related transcription factors GATA-1 and the stem cell leukemia gene correlates with hematopoietic differentiation and is associated with outcome of acute myeloid leukemia. Shimamoto T, Ohyashiki K, Ohyashiki JH, Kawakubo K, Fujimura T, Iwama H, Nakazawa S, Toyama K. First Department of Internal Medicine, Tokyo Medical College, Japan. To understand the clinical implications of transcription factors and their biologic roles during cellular differentiation in the hematopoietic system, we examined the expression of GATA-1, GATA-2, and stem cell leukemia (SCL) gene in human leukemia cell lines and various leukemia patients using the reverse transcriptase-polymerase chain reaction. Cell lines exhibiting megakaryocytic or erythrocytic phenotypes had GATA-1, GATA-2, and SCL gene transcripts, while monocytic cell lines had no detectable GATA-1, GATA-2, or SCL gene mRNA. In some myeloid cell lines, GATA-1 expression, but not SCL gene expression, was detected; GATA-1 expression in HL-60 cells was downregulated during the process of monocytic differentiation. We next examined GATA-1, GATA-2, and SCL gene expression in 110 leukemia samples obtained from 76 patients with acute myeloid leukemia (AML), 19 with acute lymphoblastic leukemia (ALL), and 15 with chronic myeloid leukemia in blast crisis (CML-BC). SCL gene expression was usually accompanied by GATA-1 expression and was preferentially detected in patients with leukemia exhibiting megakaryocytic or erythrocytic phenotypes, while patients with monocytic leukemia were clustered in the group with no detectable GATA-1 expression. None of the patients with ALL or CML-lymphoid-BC expressed SCL. De novo AML patients with SCL gene expression had a lower complete remission (CR) rate and had a significantly poorer prognosis. Among the patients with AML not expressing SCL, a high percentage of patients with CD7+ AML and CD19+ AML had detectable GATA-1, while patients with GATA-1-negative AML had the best CR rate (87.5%). Our results suggest that the expression pattern of transcription factors reflects the lineage potential of leukemia cells, and GATA-1 and SCL gene expression may have prognostic value for the outcome of patients with AML. PMID: 7579412 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Blood. 1995 Jun 15;85(12):3713-8. Ecotropic virus integration site-1 gene preferentially expressed in post-myelodysplasia acute myeloid leukemia: possible association with GATA-1, GATA-2, and stem cell leukemia gene expression. Ohyashiki JH, Ohyashiki K, Shimamoto T, Kawakubo K, Fujimura T, Nakazawa S, Toyama K. First Department of Internal Medicine, Tokyo Medical College, Japan. We investigated expression of the human ecotropic virus integration site-1 (EVI1) gene in patients with leukemia and myelodysplastic syndrome (MDS) using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The EVI1 transcripts were detected in 5 (10.0%) of 50 patients with de novo acute myeloid leukemia (AML), including two AML patients with trilineage myelodysplasia, and in 8 (34.8%) of 23 patients with post-myelodysplastic syndrome AML (post-MDS AML). EVI1 expression was also detected in 6 (35.3%) of 17 MDS patients and three of six patients with chronic myeloid leukemia (CML) in myelomegakaryoblast crisis. No EVI1 transcripts were detected in patients with acute lymphoid leukemia (n = 15) or CML in lymphoid blast crisis (n = 4). Chromosomal abnormalities at the 3q26 region, where the EVI1 gene is located, were found in one patient with MDS and two patients with CML myelomegakaryoblast crisis who had EVI1 expression. Our results showed that EVI1 expression was frequent in patients with post-MDS AML and AML with trilineage myelodysplasia, regardless of the presence or absence of 3q26 abnormalities. EVI1 expression was accompanied by expression of GATA-1 and GATA-2, and often by stem cell leukemia (SCL) gene expression. In patients with post-MDS AML, EVI1 expression was not always associated with a 3q26 abnormality, whereas EVI1 expression in CML myelomegakaryoblast crisis was often linked to a 3q26 abnormality. Our results suggest that the leukemogenic role of EVI1 expression may differ between post-MDS AML and leukemia, with EVI1 expression associated with a 3q26 abnormality. PMID: 7780155 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Oncogene. 1995 Feb 16;10(4):631-9. Regulation of lineage restricted haemopoietic transcription factors in cell hybrids. Murrell AM, Green AR. University of Cambridge, Department of Haematology, MRC Centre, UK. SCL, GATA-1, GATA-2 and GATA-3 encode lineage restricted haemopoietic transcription factors. We have previously shown that SCL, GATA-1 and GATA-2 are expressed in multipotent progenitors prior to lineage commitment, but are down-regulated during granulocyte/monocyte differentiation. The phenomenon of gene extinction in cell hybrids may reveal negative regulatory mechanisms operating during normal differentiation. We have therefore analysed the regulation of SCL, GATA-1, GATA-2 and GATA-3 in cell hybrids formed by the fusion of cell lines representing different haemopoietic lineages. Expression of GATA-3 was extinguished in both human and murine erythroid x T cell hybrids, an observation which suggests that erythroid cells contain factors capable of repressing GATA-3 expression. By contrast expression of SCL, GATA-1 and GATA-2 was not extinguished in erythroid x T or in erythroid x B cell hybrids. These data suggest that T cells and B cells do not contain trans-acting factors capable of down-regulating expression of SCL, GATA-1 or GATA-2, and therefore raise the possibility that a 'hit and run' mechanism may repress these genes during normal haemopoiesis. HpaII sites within the SCL promoter were unmethylated in erythroid cells but methylated in T cells. Erythroid x T and erythroid x B cell hybrids contained both methylated and unmethylated SCL promoters, thus implicating a heritable cis-acting mechanism in the regulation of the SCL gene in lymphoid cell lines. These results provide the first analysis of SCL and GATA gene regulation in stable cell hybrids. PMID: 7862440 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Oncogene. 1994 Sep;9(9):2623-32. GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene. Lecointe N, Bernard O, Naert K, Joulin V, Larsen CJ, Romeo PH, Mathieu-Mahul D. CNRS UMR 9942, Institut de Genetique Moleculaire de Montpellier, France. The tal-1 gene, which is frequently activated in human T cell acute leukemias (T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and potentially a transcription factor. In human and murine hematopoiesis tal-1 is expressed during the differentiation of the erythroid, megakaryocytic and mastocytic cell lineages. The expression of tal-1 appears to be comodulated with that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein may regulate the tal-1 gene activity in these hematopoietic lineages. To get further insights into the molecular mechanisms that control tal-1 expression, we have isolated 5' sequences of the murine gene and compared them to their human counterparts. The 5' flanking sequences from the two genes show several regions of high homology. The alignment of both sequences enabled us to predict that similarly, to the human, the mouse gene contains two alternative first exons (Ia and Ib). Remarkably, in both species, the proximal region of the tissue-specific exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33) and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate that GATA proteins are able to interact with both GATA-motifs in a sequence specific fashion, but with different efficiencies. Moreover transfection studies show that the GATA-1 protein directly mediates tal-1 transcription by interacting with the -122/+1 fragment, defined as a minimal promoter in erythroid cells. Mutagenesis of the promoter establishes that the -33 GATA-binding site present in this fragment is critical for tal-1 expression in erythroid cells, but by itself does not lead to full promoter activity. Indeed, further mutations show that the second -65 GATA-binding site and the binding motif for SP1 (-59) significantly contribute to the overall activity of the proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1 cooperates with the transcription factor SP1 to mediate the erythroid-specific expression of the tal-1 gene. PMID: 8058326 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: EMBO J. 1992 Nov;11(11):4073-81. The SCL gene product: a positive regulator of erythroid differentiation. Aplan PD, Nakahara K, Orkin SH, Kirsch IR. National Cancer Institute, Navy Medical Branch, Bethesda, MD 20889. The SCL (tal-1, TCL5) gene is a member of the basic domain, helix-loop-helix (bHLH) class of putative transcription factors. We found that (i) the SCL promoter for exon Ia contains a potential recognition site for GATA-binding transcription factors, (ii) SCL mRNA is expressed in all erythroid tissues and cell lines examined, and (iii) SCL mRNA increases upon induced differentiation of murine erythroleukemia (MEL) cells, and inferred that SCL may play a physiologic role in erythroid differentiation. We used gel shift and transfection assays to demonstrate that the GATA motif in the SCL promoter binds GATA-1 (and GATA-2), and also mediates transcriptional transactivation. To identify a role for SCL in erythroid differentiation, we generated stable transfectants of MEL and K562 (a human chronic myelogenous leukemia cell line that can differentiate along the erythroid pathway) cells overexpressing wild-type, antisense or mutant SCL cDNA. Increasing the level of SCL expression in two independent MEL lines (F4-6 and C19, a 745 derivative) and K562 cells increased the rate of spontaneous (i.e. in the absence of inducer) erythroid differentiation. Conversely, induced differentiation was inhibited in MEL transfectants expressing either antisense SCL cDNA or a mutant SCL lacking the basic domain. Our experiments suggest that the SCL gene can be a target for the erythroid transcription factor GATA-1 and that the SCL gene product serves as a positive regulator of erythroid differentiation. PMID: 1396592 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------