BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3613-3613
Author(s):  
Claudia Lengerke ◽  
Yuan Wang ◽  
Frank Yates ◽  
Leila Maouche-Chretien ◽  
George Q. Daley
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Murine Embryonic Stem Cells

Abstract Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.

scholarly journals Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 268-278 ◽  
Author(s):  
Shannon L. McKinney-Freeman ◽  
Olaia Naveiras ◽  
Frank Yates ◽  
Sabine Loewer ◽  
Marsha Philitas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Murine Embryonic Stem Cells ◽  
Isolation And Characterization

Abstract Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.

Derivation of Hematopoietic Stem Cells from Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 223-223 ◽  
Author(s):  
Yuan Wang ◽  
Frank Yates ◽  
Eugenia Dikovskaia ◽  
Patricia Ernst ◽  
Alan J. Davidson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Lymphoid Cells ◽  
Time Interval ◽  
Hematopoietic Stem

Abstract Despite the significant in vitro blood-forming potential of murine embryonic stem cells (ESCs), deriving hematopoietic stem cells (HSCs) that can reconstitute irradiated mice has proven to be challenging. Previously, we successfully engrafted lethally irradiated adult mice with ESCs engineered to ectopically express the homeodomain gene hoxB4. In engrafted animals, blood reconstitution showed a myeloid predominance, likely due to an inability to fully pattern the adult HSC from these embryonic populations. Recently, we have investigated cdx4, a caudal-related homeobox gene whose function has been linked to blood development in the zebrafish. During in vitro differentiation of murine ESCs, cdx4 is expressed during a very narrow time interval on day 3, coincident with the specification of hematopoietic mesoderm. To further characterize the function of cdx4 in mouse hematopoiesis, we have established a tetracycline-inducible murine embryonic stem cell line. When cdx4 expression is conditionally induced over a protracted period from day 2 and 6, we observe a marked enhancement of hemangioblast formation as well as significant increases in primitive and definitive hematopoietic colonies. Cdx4 acts to induce a broad array of hox genes, including a modest elevation in hoxb4. Co-expression of cdx4 and hoxb4 promotes robust expansion of hematopoietic blasts on supportive OP9 stromal cultures. When injected intravenously into lethally-irradiated mice, these cell populations provide robust radio-protection, and reconstitute high-level lymphoid-myeloid donor chimerism. Marrow from engrafted primary animals can be transplanted into irradiated secondary mice. B220+ splenic lymphoid cells and Mac-1/Gr-1+ marrow myeloid cells purified from primary and secondary mice show multiple common sites of retroviral integration, thereby proving the derivation of long-term hematopoietic stem cells from embryonic stem cells in vitro. Our data support a central role for the cdx4-hox gene pathway in specifying murine HSC development, and establish a robust system for hematopoietic reconstitution from ESCs. We have coupled techniques for generating ESCs by nuclear transfer with these methods for blood reconstitution to model the treatment of genetic disorders of the bone marrow.

Comprehensive Analysis Of HOX Gene Expression and DNA Methylation From 189 Primary AMLs Demonstrates Canonical Patterns Associated With Hematopoietic Stem/Progenitors and Recurrent AML Mutations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2496-2496 ◽  
Author(s):  
David H Spencer ◽  
Margaret A. Young ◽  
Jeffery M. Klco ◽  
Timothy J. Ley
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Cd34 Cells ◽  
Methylation Patterns

Abstract HOX genes encode a family of homeodomain transcription factors with important roles in hematopoiesis. Expression of HOX genes is also a common feature of acute myeloid leukemia (AML), and functional studies have suggested that HOX-dependent pathways may contribute to leukemogenesis. Although HOX expression is known to correlate with specific AML mutations, the patterns of expression of all 39 HOX genes in primary AML samples, and their relationships with recurrent AML mutations, are incompletely understood. In addition, little is known about the influence of AML mutations on DNA methylation at the HOX loci, and the relationship between HOX gene expression and methylation in AML. In this study, we carried out a combined analysis of gene expression data from microarray and RNA-sequencing platforms and genome-wide DNA array-based methylation from 189 primary AML samples that have been previously characterized by either whole-genome or whole exome sequencing. We also measured expression and methylation using the same platforms from normal bone marrow subsets, including CD34+ cells, promyelocytes, monocytes, neutrophils and lymphocytes, and obtained expression data from CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells. Our analysis confirmed previous work on the general patterns of HOX expression in AML. The HOXA and HOXB genes showed variation both within each cluster and across the AMLs, although high level expression was restricted to a subset of these genes, including HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXB2-HOXB4, and HOXB6, as well as HOX cofactor MEIS1; HOXC and HOXD genes were minimally expressed in all of the samples. These observations were orthogonally validated by RNA-seq, and with a targeted Nanostring expression platform. Consistent with previous studies, MLL-positive AML samples (n=11) expressed only HOXA genes and MEIS1. AML samples with CBFB-MYH11 rearrangements (n=12) showed expression of only MEIS1, and HOXB2-HOXB4 at moderate levels; RUNX1-RUNX1T1 (n=7) and PML-RARA (n=19) samples did not detectably express any HOX genes. In AMLs with a normal karyotype (n=85), we observed two distinct patterns; one pattern displayed little or no HOX gene expression (7/85; 8%), and another displayed canonical expression of a specific subset of the HOXA and HOXB genes and MEIS1 (78/85; 92%) with similar relative HOX gene expression levels in all cases. Comparison of this pattern with normal bone marrow revealed the same HOX expression pattern in normal CD34+ cells; additional analysis showed that this pattern was confined to hematopoietic stem/progenitor cells, but was not seen in more mature cells, including other CD34+ subsets, promyelocytes, monocytes and neutrophils. We also measured HOX gene expression in CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells, which revealed expression of only MEIS1 and the canonical HOXB genes, suggesting that activation of these genes may represent the earliest events in the HOX pathway of hematopoietic development. Correlation of HOX expression with recurrent AML mutations by gene set enrichment analysis demonstrated a significant association with NPM1 (P<10-4) and DNMT3A (P<10-2) mutations, but not with other recurrent somatic mutations, including FLT3,IDH1/IDH2, and TET2. Methylation at the HOX loci demonstrated patterns that correlated with HOX expression, including hypomethylation at HOX promoters in samples with high expression. However, additional mutation-specific patterns were apparent. For example, NPM1-mutant AMLs demonstrated a distinct methylation pattern that included hypomethylation at the HOXB3 promoter, which was not shared with CBFB-MYH11 cases or other AMLs with HOXB3 expression. In summary, our comprehensive analysis demonstrates canonical expression and methylation patterns at the HOX loci in AML. These patterns correspond to specific recurrent AML mutations, and the dominant pattern in most normal karyotype AMLs mimics the signature of hematopoietic stem cells. This supports previous observations of developmental regulation of HOX genes in hematopoiesis, and implies that this normal stem cell signature is “captured” in the majority of AMLs with normal karyotype. In addition, distinct methylation patterns at HOX loci suggest that multiple regulatory mechanisms are involved in HOX expression in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Delayed Mesoderm and Erythroid Differentiation of Murine Embryonic Stem Cells in the Absence of the Transcriptional Regulator FUBP1

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Josephine Wesely ◽  
Marlene Steiner ◽  
Frank Schnütgen ◽  
Manuel Kaulich ◽  
Michael A. Rieger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Early Time ◽  
Transcriptional Regulator ◽  
Erythroid Differentiation ◽  
Embryoid Bodies ◽  
Hematopoietic Stem ◽  
Murine Embryonic Stem Cells ◽  
Delayed Differentiation

The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.

scholarly journals Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Selami Demirci ◽  
Juan J. Haro-Mora ◽  
Alexis Leonard ◽  
Claire Drysdale ◽  
Daniela Malide ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Close Association ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Embryoid Bodies ◽  
Confocal Imaging ◽  
Hematopoietic Stem ◽  
Tcr Repertoire

Abstract Background Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders. Methods To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions. Results Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p < 0.01) and lowest number of primitive (CD34− CD235a+, 1.55%, p < 0.01) precursor cells along with the highest colony-forming units (149.8 ± 11.6, p < 0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38− CD45RA− CD49f+ CD90+) was 7.6–8.9% after 10 days of hematopoietic differentiation with 14.5% adult β-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5 ± 65.7, p < 0.001), while the CD34− CD43+ fraction produced only a small number of colonies (21.6 ± 11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center. Conclusions In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.

scholarly journals Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (3) ◽  
pp. e4820 ◽  
Author(s):  
Kenji Matsumoto ◽  
Takayuki Isagawa ◽  
Toshinobu Nishimura ◽  
Takunori Ogaeri ◽  
Koji Eto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Stepwise Development
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