Stromal Expression of Jagged 1 Promotes Colony Formation by Fetal Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1505-1511 ◽  
Author(s):  
Philip Jones ◽  
Gill May ◽  
Lyn Healy ◽  
John Brown ◽  
Gerald Hoyne ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Culture Conditions ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract The Notch signaling system regulates proliferation and differentiation in many tissues. Notch is a transmembrane receptor activated by ligands expressed on adjacent cells. Hematopoietic stem cells and early progenitors express Notch, making the stromal cells which form cell-cell contacts with progenitor cells candidate ligand-presenting cells in the hematopoietic microenvironment. Therefore, we examined primary stromal cell cultures for expression of Notch ligands. Using reverse transcription-polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting, we demonstrate expression of Jagged 1 in primary stromal cultures. To investigate if the stromal expression of Jagged 1 has functional effects on hematopoietic progenitors, we cultured CD34+, c-kit+ hematopoietic progenitor cells derived from the aorto gonadal mesonephros region of day 11 mouse embryos on the Jagged 1− stromal cell line S17 and on S17 cells engineered to express Jagged 1. The presence of Jagged 1 increased the number of colonies formed in subsequent methylcellulose culture fourfold. Larger increases in colony numbers were observed under the same culture conditions with CD34+, c-kit+ hematopoietic progenitor cells derived from d11 fetal liver. These results obtained in vitro table Jagged 1 as a candidate regulator of stem cell fate in the context of stromal microenvironments in vivo. © 1998 by The American Society of Hematology.

Stromal Expression of Jagged 1 Promotes Colony Formation by Fetal Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1505-1511 ◽  
Author(s):  
Philip Jones ◽  
Gill May ◽  
Lyn Healy ◽  
John Brown ◽  
Gerald Hoyne ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Culture Conditions ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

The Notch signaling system regulates proliferation and differentiation in many tissues. Notch is a transmembrane receptor activated by ligands expressed on adjacent cells. Hematopoietic stem cells and early progenitors express Notch, making the stromal cells which form cell-cell contacts with progenitor cells candidate ligand-presenting cells in the hematopoietic microenvironment. Therefore, we examined primary stromal cell cultures for expression of Notch ligands. Using reverse transcription-polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting, we demonstrate expression of Jagged 1 in primary stromal cultures. To investigate if the stromal expression of Jagged 1 has functional effects on hematopoietic progenitors, we cultured CD34+, c-kit+ hematopoietic progenitor cells derived from the aorto gonadal mesonephros region of day 11 mouse embryos on the Jagged 1− stromal cell line S17 and on S17 cells engineered to express Jagged 1. The presence of Jagged 1 increased the number of colonies formed in subsequent methylcellulose culture fourfold. Larger increases in colony numbers were observed under the same culture conditions with CD34+, c-kit+ hematopoietic progenitor cells derived from d11 fetal liver. These results obtained in vitro table Jagged 1 as a candidate regulator of stem cell fate in the context of stromal microenvironments in vivo. © 1998 by The American Society of Hematology.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

scholarly journals Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture

2009 ◽  
Vol 18 (8) ◽  
pp. 887-897 ◽  
Author(s):  
Xiaosong Huang ◽  
L. Jeanne Pierce ◽  
Paul A. Cobine ◽  
Dennis R. Winge ◽  
Gerald J. Spangrude
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Copper Chelation

Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2 treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2 decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.

scholarly journals Essential and Instructive Roles of GATA Factors in Eosinophil Development

2002 ◽  
Vol 195 (11) ◽  
pp. 1379-1386 ◽  
Author(s):  
Ryutaro Hirasawa ◽  
Ritsuko Shimizu ◽  
Satoru Takahashi ◽  
Mitsujiro Osawa ◽  
Shu Takayanagi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Fetal Liver ◽  
The Other ◽  
Th2 Cells ◽  
Functional Domain ◽  
Hematopoietic Stem ◽  
Gata Factors

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

C/EBPα deficiency results in hyperproliferation of hematopoietic progenitor cells and disrupts macrophage development in vitro and in vivo

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1639-1647 ◽  
Author(s):  
Victoria Heath ◽  
Hyung Chan Suh ◽  
Matthew Holman ◽  
Katie Renn ◽  
John M. Gooya ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Fetal Liver ◽  
Cell Types ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Progenitor ◽  
Differentiation Potential

Abstract CCAAT enhancer binding protein-α (C/EBPα) inhibits proliferation in multiple cell types; therefore, we evaluated whether C/EBPα-deficient hematopoietic progenitor cells (HPCs) have an increased proliferative potential in vitro and in vivo. In this study we demonstrate that C/EBPα-/- fetal liver (FL) progenitors are hyperproliferative, show decreased differentiation potential, and show increased self-renewal capacity in response to hematopoietic growth factors (HGFs). There are fewer committed bipotential progenitors in C/EBPα-/- FL, whereas multipotential progenitors are unaffected. HGF-dependent progenitor cell lines can be derived by directly culturing C/EBPα-/- FL cells in vitro Hyperproliferative spleen colonies and myelodysplastic syndrome (MDS) are observed in mice reconstituted with C/EBPα-/- FL cells, indicating progenitor hyperproliferation in vitro and in vivo. C/EBPα-/- FL lacked macrophage progenitors in vitro and had impaired ability to generate macrophages in vivo. These findings show that C/EBPα deficiency results in hyperproliferation of HPCs and a block in the ability of multipotential progenitors to differentiate into bipotential granulocyte/macrophage progenitors and their progeny. (Blood. 2004; 104:1639-1647)

Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2286-2292 ◽  
Author(s):  
Christian Buske ◽  
Michaela Feuring-Buske ◽  
Jennifer Antonchuk ◽  
Patricia Rosten ◽  
Donna E. Hogge ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Aberrant Expression ◽  
Hematopoietic Development ◽  
The Impact

Abstract Several studies point to multiple members of the Hox transcription factor family as playing key roles in normal hematopoietic development, and they link the imbalanced expression of these transcription factors, in particular of the Abd-like A cluster HOXgenes HOXA9 and HOXA10, to leukemogenesis. To test directly the hypothesis that HOXA10is involved in human hematopoietic development, the gene was retrovirally overexpressed in human highly purified CD34+/GFP+ hematopoietic progenitor cells derived from cord blood or fetal liver sources, and the impact of aberrant gene expression was analyzed on differentiation and proliferation in vitro and in vivo. HOXA10 misexpression profoundly impaired myeloid differentiation with a higher yield of blast cells in liquid culture and a greater than 100-fold increased generation of blast colonies after in vitro expansion or after replating of primary colonies first plated in methylcellulose directly after transduction (P < .01). Furthermore, aberrantHOXA10 expression almost completely blocked erythroid differentiation in methylcellulose (P < .02).HOXA10 deregulation also severely perturbed the differentiation of human progenitors in vivo, reducing B-cell development by 70% in repopulated NOD/SCID mice and enhancing myelopoiesis in the transduced compartment. The data provide evidence that the balanced expression of HOXA10 is pivotal for normal human hematopoietic development and that aberrant expression of the gene contributes to impaired differentiation and increased proliferation of human hematopoietic progenitor cells. These results also provide a framework to initiate more detailed analyses ofHOX regulatory domains and HOX cofactors in the human system in vitro and in vivo.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Lineage Instructive Function of C/EBPα in Multipotent Hematopoietic Progenitor Cells Revealed in a Novel Cebpa-Cre Knock-in Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2458-2458
Author(s):  
Albert Wolfler ◽  
Astrid A Danen-van Oorschot ◽  
Jurgen Haanstra ◽  
Marijke Valkhof ◽  
Paulette van Strien ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Cell Development ◽  
Hematopoietic Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Multipotent Progenitors ◽  
Reported Data

Abstract Transcription factors control the lineage specification and differentiation of hematopoietic progenitor cells. They are expressed in a cell type-restricted pattern and activate lineage specific genetic programs. Recent studies have demonstrated that expression of GATA-1 or PU.1 in multipotent lin−Sca-1+c-Kit+ (LSK) cells specifies them to develop into myeloerythroid progenitors or lymphomyeloid progenitors, respectively. In contrast, C/EBPα, a transcription factor indispensable for the production of granulocytes and macrophages, is thought to predominantly act at a later stage of hematopoietic commitment, by governing the transition from common myeloid progenitors (CMPs) into granulocytic/monocytic progenitors (GMPs). To study whether C/EBPα may already exert a lineage instructive function at an earlier stage of hematopoietic cell development, i.e., at the level of multipotent LSK cells, we generated a knock-in mouse model expressing Cre recombinase under the regulation of the cebpa promoter and crossed C/EBPαcre/+ mice with R26 YFP reporter mice. This model faithfully demonstrates high levels of C/EBPα expression in myeloid cells and enabled us to trace cebpa-driven Cre/YFP expression in single LSK cells and their progeny by flow cytometry and colony cultures. On average cebpa-driven YFP expression was found in 17% (range 10–25%) of the total LSK fraction (n=12 mice). Within the CD150+CD48− CD34− subset of LSK cells, which contains the most primitive hematopoietic stem cells (HSC), 3–8% of the cells expressed YFP, indicating that cebpa is lowly expressed in bona fide HSC. This low level of expression appears insufficient for lineage determination, since the same levels of YFP expression (1–10%) were found in peripheral T and B cells. Within the CD34+ fraction of LSK cells, a population enriched for multipotent progenitors, 19% (range 14%–28%) of the cells expressed YFP. Identical distributions of YFP+ cells among the different LSK subsets were found in fetal livers of day 14.5 embryos, suggesting a comparable regulation of cebpa expression in fetal and adult cells. Similar to the reported data for GATA-1 and PU.1, cebpa-expressing LSK cells were predominantly found in the Sca-1low fraction. When cultured in a multilineage cytokine cocktail, YFP+ LSK cells gave predominantly rise to GM colonies (73% of all colonies; range 65–85%), whereas YFP− cells formed multiple types of colonies including mixed, megakaryocytic and erythroid colonies. The predominant outgrowth of YFP+ LSK cells to GM lineages was further supported in GM-CSF-supplemented colony assays, which gave rise to cloning efficiencies of 26% for YFP+ and 4% for YFP− LSK cells, respectively. In conclusion, our results show that C/EBPα starts to exert its instructive function towards GM cell development already within the LSK population, at the level of the multipotent progenitors. This has important ramifications for our understanding of the role of C/EBPα in early hematopoietic cell fate decisions.

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