The Transcription Factor Repertoire of Flt3+ ...

Published online: January 4, 2008

Cells Tissues Organs 2008;188:103–115 DOI: 10.1159/000112836

The Transcription Factor Repertoire of Flt3+ Hematopoietic Stem Cells Thomas Hieronymus a David Ruau a Julia Ober-Blöbaum a Jea-Hyun Baek a Alexandra Rolletschek b Stefan Rose-John c Anna M. Wobus b Albrecht M. Müller d Martin Zenke a a

Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Medical School, and Helmholtz Institute for Biomedical Engineering, RWTH Aachen, Aachen, b In Vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, and c Department of Biochemistry, Christian Albrechts University, Kiel, d Institute of Medical Radiation and Cell Research (MSZ), University of Würzburg, Würzburg, Germany

Key Words Stem cells ⴢ Expression profiling ⴢ Transcription factors ⴢ Mouse

Abstract Hematopoietic stem cells maintain the development of all mature blood cells throughout life due to their sustained self-renewal capacity and multilineage differentiation potential. During development into specific cell lineages, the options of stem cells and multipotent progenitor cells be-

come increasingly restricted concomitant with a successive decline in self-renewal potential. Here we describe an Flt3+CD11b+ multipotent progenitor that can be amplified in vitro with a specific combination of cytokines to yield homogeneous populations in high cell numbers. By employing gene expression profiling with DNA microarrays, we studied the transcription factor repertoire of Flt3+CD11b+ progenitors and related it to the transcription factor repertoire of hematopoietic stem cells and embryonic stem cells. We report here on overlapping and nonoverlapping expression patterns of transcription factors in these cells and thus pro-

Abbreviations used in this paper

DC ES Ets FCS Flt3 Flt3L GM-CSF HMG

basic helix-loop-helix basic leucine zipper classical cystein and histidine containing zinc finger domain dendritic cells embryonic stem erythroblast transformation-specific fetal calf serum fms-like tyrosine kinase 3 Flt3 ligand granulocyte macrophage colony-stimulating factor high mobility group

© 2008 S. Karger AG, Basel 1422–6405/08/1882–0103$24.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cto

HSC hyper-IL-6 IGF-1 IL6st KRAB LSK MPP PCA PHD SCF SDF-1 TF

hematopoietic stem cell IL-6/soluble IL-6 receptor fusion protein insulin-like growth factor 1 IL-6 signal transducer Krüppel associated box Lin–Sca-1+c-kit+ multipotent progenitor principal component analysis plant homeodomain stem cell factor stromal cell-derived factor 1 transcription factor

Prof. Dr. Martin Zenke RWTH Aachen University Medical School Institute for Biomedical Engineering, Department of Cell Biology Pauwelsstrasse 30, DE–52074 Aachen (Germany) Tel. +49 241 80 80760, Fax +49 241 80 82008, E-Mail [email protected]

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bHLH bZip C2H2

Introduction

Stem cell commitment and differentiation entails the successive loss of self-renewal and multilineage differentiation potential that results in the final restriction to a terminally differentiated and fully functional mature cell type. All mature blood cells develop from a population of hematopoietic stem cells (HSC), which due to their sustained self-renewal capacity and multilineage differentiation potential maintain hematopoiesis throughout life. Blood cell differentiation has been particularly well studied and multiple developmental intermediates have been identified. In mammals, the HSC compartment contains (1) long-term reconstituting HSC, which generate all hematopoietic lineages for life after transplantation into lethally irradiated mice, and (2) short-term HSC and their multipotent progenitor (MPP) progeny, which give rise to all hematopoietic lineages but with diminished selfrenewal capacity [Orkin, 2000; Weissman, 2000; Graf, 2002]. HSC and MPP cells exhibit a broad gene expression repertoire and promiscuously express genes of several lineages, but at low levels [Orkin, 2000; Weissman, 2000; Graf, 2002; Miyamato et al., 2002]. During development of the different hematopoietic lineages, the gene expression repertoire and the options of HSC and MPP become increasingly restricted, leading to the establishment of a specific lineage from the choice of several. Among these more restricted progenitor cells are the well-studied common lymphoid progenitors and common myeloid progenitors [Orkin, 2000; Weissman, 2000; Graf, 2002], which produce mature lymphoid and myeloid cells, respectively. Stem/progenitor cell renewal and cell fate choice in hematopoiesis have been proposed to be regulated by instructive signals provided by the microenvironment, including cell-cell contacts, Wnt and Notch factors and hematopoietic cytokines. The fms-like tyrosine kinase-3 ligand (Flt3L) is such a nonredundant cytokine that has recently emerged as a potent growth factor for stem/progenitor cells expressing Flt3 receptor. Flt3, also termed Flk-2 [Matthews et al., 104

Cells Tissues Organs 2008;188:103–115

1991] and STK-1 [Small et al., 1994], is a receptor tyrosine kinase with homology to c-kit (the receptor for stem cell factor, SCF) and c-fms (the receptor for macrophage colony-stimulating factor), and bone marrow from Flt3–/– mice display reduced reconstitution activity in vivo [Mackarehtschian et al., 1995]. Flt3 expression is detected in various early progenitors at different rates including MPP, common lymphoid progenitors and common myeloid progenitors [Adolfsson et al., 2001; D’Amico and Wu, 2003; Karsunky et al., 2003; Sitnicka et al., 2003]. In the primitive Lin–Sca-1+c-kit+ (LSK) HSC compartment approximately 60% of cells express Flt3 and those cells exhibit short-term reconstituting activity, whereas LSK HSC that lack Flt3 expression comprise long-term HSC activity. These findings associate Flt3 expression in the HSC compartment with loss of sustained self-renewal capacity [Adolfsson et al., 2001; Christensen and Weissman, 2001]. The observation that highly enriched populations of HSC do not respond to single cytokines, but require the presence of two or more cytokines before initiating a mitogenic response, supports the idea that self-renewal requires the coordinate activities of multiple signaling pathways. However, all extrinsically activated signaling pathways within stem/progenitor cells will amalgamate in orchestrated gene expression programs driven by specific transcriptional regulators and modulate the gene activity of lineage-determining transcription factors (TF). TF were demonstrated to play a central role in lineage commitment and differentiation, and factors that control the development of red blood cells, macrophages, B cells and T cells have been particularly well studied [Sieweke and Graf, 1998; Orkin, 2000; Graf, 2002]. In contrast, our understanding of how maintenance of self-renewal and multipotentiality of HSC is regulated at the genetic level is scarce. A variety of cytokines and growth factors have been reported to efficiently expand hematopoietic progenitor cells in vitro, such as Flt3L, SCF, insulin-like growth factor 1 (IGF-1), interleukin (IL)-3, IL-6 and soluble IL-6 receptor fusion protein (hyper-IL-6) and others, and also various combinations thereof. Given the importance of Flt3 for early HSC/progenitor cell expansion in vivo, Flt3L has been frequently used in combination with other hematopoietic cytokines for HSC/progenitor cell expansion in vitro [Lyman and Jacobsen, 1998; Hacker et al., 2003; Ju et al., 2003; Hieronymus et al., 2005]. We recently described in vitro culture systems to efficiently amplify Flt3+ progenitors from mouse bone marrow or human cord blood using specific growth facHieronymus et al.


vide novel insights into the dynamic networks of transcriptional regulators in embryonic and adult stem cells. Additionally, the results obtained open the perspective for elucidating lineage and ‘stemness’ determinants in hematopoiesis. Copyright © 2008 S. Karger AG, Basel

Materials and Methods Cells and Cell Culture Mouse ES cell line R1 was maintained in DMEM (Gibco BRL, Eggenstein, Germany) supplemented with 10 ng/ml recombinant human leukemia inhibitory factor, 15% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1% nonessential amino acids (all from Gibco BRL) and 50 ␮ M ␤-mercaptoethanol (Serva, Heidelberg, Germany) on a layer of primary mouse embryonic fibroblasts [Rolletschek et al., 2001].

Transcription Factor Repertoire of Flt3+ Hematopoietic Stem Cells

HSC from bone marrow of 8- to 12-week-old NRMI mice were isolated by depletion of lineage-positive cells using immunomagnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and monoclonal antibodies against B220 (RA3-6B2), CD11b/Mac-1 (M1/70), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8 (53-6.72) and TER119 (all antibodies from BD Biosciences, San Jose, Calif., USA). Lineage-negative cells were then sorted with a FACSVantage device (BD Biosciences) for c-kit (ACK45) and Sca-1 (E13-161.7) into Lin–Sca-1–c-kit+ and LSK populations, and LSK HSC were used for further analysis. To generate Flt3+CD11b+ progenitor cells, bone marrow suspensions were prepared from C57BL/6 mice (Charles River, Sulzfeld, Germany). Cells were seeded at 2 ! 106 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (all from Gibco BRL) and 50 ␮ M ␤-mercaptoethanol containing recombinant murine SCF (100 ng/ml), 25 ng/ml Flt3L (PeproTech, London, UK), 40 ng/ml recombinant long-range IGF-1 (Sigma-Aldrich, Taufkirchen, Germany), 5 ng/ ml recombinant IL-6/soluble IL-6 receptor fusion protein (hyperIL-6) [Fischer et al., 1997], 20 U/ml recombinant mouse GM-CSF and 10 –6 M dexamethasone. After 3 days in culture, cells were subjected to Ficoll density gradient centrifugation (density 1.077 g/ml; PAA, Cölbe, Germany) to remove residual erythrocytes, dead cells and debris. Medium with growth factors was replenished every 2 days and cells were maintained at 2 ! 106 cells/ml cell density. Cell numbers were determined with an electronic cell counter device (CASY1; Schärfe System, Reutlingen, Germany). Cell Proliferation Assay A total of 5 ! 104 cells were incubated in 200 ␮l medium in a 96-well flat-bottom microtiter plate for 72 h at 37 ° C with the indicated growth factors and combinations thereof. Samples were then pulsed for 2 h with 0.75 ␮Ci/well [3H]thymidine (29 Ci/ mmol; Amersham Biosciences, Braunschweig, Germany) and harvested onto glass fiber filters. Radioactivity was measured by liquid scintillation counting in a Microbeta counter (Wallac, Turku, Finland). Flow Cytometry Flow cytometry analysis for surface antigen expression was performed as previously described [Kurz et al., 2002]. The following antibodies were used: FITC-conjugated anti-MHC class II (IA/I-E; clone 2G9), CD11b-FITC (M1/70), CD93-FITC (AA4.1), CD11c-PE (HL3), CD24-PE (M1/69), CD135/Flt3-PE (A2F10.1), CD3␧-bio (145-2C11), Gr-1 (RB6-8C5), CD14 (rmC5-3), CD29 (9EG7), CD49f (GoH3), CD71 (C2) and the respective isotype controls were all purchased from BD Biosciences. Isotypematched FITC or PE-labeled secondary antibodies, streptavidinFITC and streptavidin-Tri-Color were from Caltag Laboratories (Invitrogen, Karlsruhe, Germany). Anti-mouse CD133/prominin antibody (clone AC133) was obtained from Miltenyi Biotec. Antimouse CXCR4/CD184 antibody was a kind gift from R. Foerster (Hannover Medical School, Hannover, Germany). Cells were analyzed with a FACSCalibur flow cytometer device using CELLQuest software (BD Biosciences). RNA Isolation and DNA Microarray Analysis RNA isolation, cDNA and cRNA synthesis, DNA microarray hybridization, and analysis for ES cells and Flt3+CD11b+ progenitor cells were carried out essentially as previously described


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tor/cytokine combinations that include Flt3L [Hacker et al., 2003; Ju et al., 2003; Hieronymus et al., 2005]. These culture systems yield homogenous populations of stem/ progenitor cells that readily differentiate into dendritic cells (DC) in vitro with differentiation factors, such as granulocyte macrophage colony-stimulating factor (GM-CSF). We employed such two-step culture systems for transcriptional profiling by DNA microarrays to study the changes in gene expression during DC development [Hacker et al., 2003; Ju et al., 2003; Hieronymus et al., 2005]. Additionally, we used bone marrow transplantation to corroborate the multilineage reconstitution potential of sorted mouse Flt3+CD11b+ progenitors and demonstrated the expression of several stem cell antigens, such as CD93 (Ly68/AA4.1), CD133 (AC133/ prominin), integrin ␣6 (CD49f) and stromal cellderived factor-1␣ (SDF-1␣) receptor (CXCR4/CD184) [Hieronymus et al., 2005]. Furthermore, the surface marker make-up of Flt3+CD11b+ MPP was compared to that of highly purified LSK HSC and revealed a broad overlap of the surface antigen repertoire of Flt3+CD11b+ MPP and HSC [Hieronymus et al., 2005]. However, the LSK HSC compartment also contains cells with longterm reconstituting multilineage differentiation potential that are Flt3– and therefore express genes that are exclusively associated with their unlimited self-renewal capacity. In this study, we further extended our analysis on the phenotypic and functional properties of mouse Flt3+ CD11b+ progenitors and report on a comprehensive analysis of their TF repertoire determined by gene expression profiling. The goal of the study was to compare the TF expression of Flt3+CD11b+ MPP, highly purified LSK HSC and embryonic stem (ES) cells, and to define overlapping and nonoverlapping TF networks. This study provides novel information on the TF repertoire in hematopoietic stem and progenitor cells, and can be expected to allow delineating lineage and ‘stemness’ determining transcriptional regulators in hematopoiesis.

Table 1. Growth factor receptor expression in Flt3+CD11b+ progenitor cells and LSK HSC

Ligand

Receptor

Flt3+CD11b+ progenitor cells

SCF Flt3L Hyper-IL-6 GM-CSF

c-kit/CD117 Flt3/CD135 IL6st/CD130 Csf2ra/CD116 Csf2rb1/CD131 Csf2rb2 Igf1r/CD221

135832 5168265 56828 3888158 3548283 308869 6358153

IGF-1

P P P P P P P

LSK HSC 135836 1,3588719 11181,4 82861 41816 162820 8178474

P P P P A P P

Gene expression in Flt3+CD11b+ progenitor cells and LSK HSC was assessed by Affymetrix MG-U74Av2 GeneChip arrays. Results shown are mean intensity values 8 SD of normalized data from 5 independent experiments for Flt3+CD11b+ progenitor cells and 2 independent replicate results of LSK HSC hybridization data. All microarray data are available in the GEO database (series accession No. GSE692 and GSE693). P and A indicate the presence or absence, respectively, of the respective transcript in each cell population as indicated by the Affymetrix Suite 5.0 software algorithm.

Bioinformatics Scanned GeneChip.DAT files were processed with Affymetrix Microarray Suite 5.0 analysis software. Data were imported into GeneSpring software (Agilent Technologies, Santa Clara, Calif., USA) and raw gene expression values were normalized per chip to the 50th percentile and per gene to the median. Classification of genes encoding for mouse TF and transcriptional regulators was done using information from various databases including Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), the Pfam Protein Families Database (http://www. sanger.ac.uk/Software/Pfam/) [Bateman et al., 2004] and the PANTHER Classification System (http://www.pantherdb.org/) [Thomas et al., 2003]. TF with present call in this and 2 previously published studies [Ivanova et al., 2002; Ramalho-Santos et al., 2002] were considered for further analysis. Hierarchical clustering [Eisen et al., 1998] was done in GeneSpring software using the Pearson correlation with a separation ratio of 0.5 and a minimum distance of 0.001. All microarray data are available at the GEO database (http://www.ncbi.nlm.nih.gov/geo/; series accession No. GSE692, GSE693 and GSE2375).

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Results and Discussion

To determine the gene expression repertoire of HSC we sorted lineage-negative cells from mouse bone marrow into Lin–Sca-1–c-kit+ and LSK populations. Total RNA was isolated and subjected to DNA microarray analysis using Affymetrix MG-U74Av2 GeneChip arrays (GEO accession No. GSE693) [Hieronymus et al., 2005]. Data were first analyzed for growth factor and cytokine receptor expression (table 1) and second for cell surface antigen expression (table 2). Based on the observation that LSK HSC express transcripts for c-kit, Flt3, gp130/ IL6st, IGF-1 receptor and the GM-CSF receptor ␣- and ␤2-chains, the respective ligands were chosen and tested in various combinations for their mitogenic potential on unseparated bone marrow cells by measuring DNA synthesis (fig. 1). An optimized combination of SCF, Flt3L, hyper-IL-6, IGF-1 and GM-CSF in the presence of dexamethasone was used to cultivate bone marrow cells that yield a homogeneous population of Flt3-expressing progenitor cells after about 7 days in culture (fig. 2). The synthetic glucocorticoid hormone dexamethasone was used as an alternative for hydrocortisone initially introduced by Dexter et al. [1984] to establish long-term culture of bone marrow cells. Dexamethasone significantly reduced the rate of spontaneous differentiation [Hieronymus and Zenke, unpubl. data] as observed in other cell culture systems [Panzenbock et al., 1998; von Lindern et al., 1999]. The activity of SCF, Flt3L, hyper-IL-6, IGF-1 and GMCSF on growth of established Flt3+ progenitor cells (day 7 of culture) was quantitatively assessed using Hieronymus et al.


[Hacker et al., 2003; Ju et al., 2003]. In brief, total RNA was isolated with RNeasy kits including DNAse digestion (Qiagen, Hilden, Germany). Five micrograms of total RNA was used to generate cDNA according to the Expression Analysis Technical Manual (Affymetrix, Santa Clara, Calif., USA). cRNA was generated with the BioArray High-Yield Transcript Labeling kit (Enzo, Farmingdale, N.Y., USA). Total cellular RNA from a total of 75,000 LSK HSC per experiment was isolated with an RNeasy Mini kit (Qiagen). In vitro transcription-based RNA amplification was performed essentially as described [Van Gelder et al., 1990], employing the Ambion Megascript T7 kit (Applied Biosystems, Darmstadt, Germany) for 2 rounds of in vitro transcription reaction. Finally, 10 ␮g cRNA per sample was hybridized to Affymetrix mouse MG-U74Av2 GeneChip arrays at 45 ° C for 16 h. DNA chips were stained, washed and scanned according to the manufacturer’s protocol.

Table 2. Gene expression of surface antigens in Flt3+CD11b+ progenitor cells and LSK HSC

Gene

Entrez Gene ID

CD3␦ CD3␧ CD3␥ CD11b CD11c CD14 CD24 CD29 CD49f CD71 CD93/AA4.1 CD133/prominin CD135/Flt3 CD184/CXCR4 Gr-1/Ly6c Gr-1/Ly6g MHC II/H2-Aa MHC II/H2-Ab1

12500 12501 12502 16409 16411 12475 12484 16412 16403 22042 17064 19126 14255 12767 17067 17072 14960 14961

Flt3+CD11b+ progenitor cells 39827 18810 984 3608200 68832 401866 3,8328982 3,98081,172 194860 6608223 1,4858316 194860 5168265 530888 10,87983,940 197873 42840 23813

A A A P A P P P P P P P P P P P A A

LSK HSC 17820 3283 29825 1587 14784 6608395 1,090852 13,43484,461 106830 1,4398613 1,1078350 46842 1,3588719 113827 4,57983,441 71824 28817 74850

A A A A A P P P A P P A P P P A A A

[3H]thymidine incorporation assay and compared to their response on bone marrow cells (fig. 1). When single factors were applied, SCF, Flt3L and GMCSF were found to be most effective (fig. 1). The activities of hyper-IL-6 and IGF-1 were less pronounced individually, yet these factors exhibited additive or even synergistic effects when applied in various combinations with SCF, Flt3L and GM-CSF. Flt3L in combination with SCF, hyper-IL-6 and/or IGF-1 clearly enhanced proliferation rates, especially in established Flt3+ progenitor cells. A different effect was seen for GM-CSF that highly increased proliferation rates of bone marrow cells in all factor combinations, but decreased proliferation rates in established Flt3+ cell cultures, presumably because GM-CSF downregulates Flt3, gp130 and IGF-1 receptors (table 1). GM-CSF is widely used to generate bone marrowderived DC in in vitro culture [Inaba et al., 1992; Lutz et al., 1999; Kurz et al., 2002], and in previous studies we used the Flt3+ cell culture system to generate DC in high cell numbers by replacing the growth-promoting factors with a high dose of GM-CSF only [Hacker et al., 2003; Hieronymus et al., 2005; Himmelreich et al., 2006]. The initial phenotypic characterization of Flt3+ cells as a precursor for DC revealed expression of the lineage marker CD11b along with the stem cell antigens CD93 (Ly68/

AA4.1), CD133 (AC133/prominin) and integrin ␣6 (CD49f), hence we refer to these cells as Flt3+CD11b+ progenitors [Hieronymus et al., 2005]. Hematopoietic cells are frequently characterized by their surface antigen expression. Therefore, we extended our initial studies on the surface marker make-up of Flt3+CD11b+ progenitors and additionally compared these to microarray data of the respective surface antigens (table 2). RNA was isolated from Flt3+CD11b+ progenitors and subjected to DNA microarray analysis using Affymetrix MG-U74Av2 GeneChip arrays. All microarray data sets of Flt3+CD11b+ progenitors are available in the GEO database under series accession No. GSE692 [Hieronymus et al., 2005]. Surface antigen expression of Flt3+CD11b+ progenitors was analyzed by flow cytometry and we found a good correlation for the expression on the transcript and protein levels of the tested surface antigens (fig. 2; table 2). There was no surface expression of CD3, CD11c and MHC class II on Flt3+CD11b+ progenitor cells and no expression of the transcripts for these genes both in Flt3+CD11b+ progenitors and LSK HSC. Higher transcript expression levels in LSK HSC than in Flt3+CD11b+ progenitors were found for CD14, CD29 (integrin ␤1), CD71 (transferrin receptor) and Flt3. The high expression levels for Flt3 in LSK HSC might reflect the high frequency of Flt3+ cells within the population of LSK HSC. Abundant but lower expres-



107


Gene expression in Flt3+CD11b+ progenitor cells and LSK HSC was assessed by Affymetrix MG-U74Av2 GeneChip arrays as described in table 1.

Medium

Bone marrow

SCF 100 ng/ml

Flt3+CD11b+ progenitor

Flt3L 25 ng/ml Hyper-IL-6 5 ng/ml IGF 40 ng/ml GM-CSF 10 U/ml SCF+Flt3L SCF+Hyper-IL-6 SCF+IGF SCF+GM-CSF Flt3L+Hyper-IL-6 Flt3L+IGF Flt3L+GM-CSF Hyper-IL-6+IGF Hyper-IL-6+GM-CSF IGF+GM-CSF SCF+Flt3L+IL-6

*

SCF+Flt3L+IGF

*

SCF+Flt3L+GM-CSF SCF+Hyper-IL-6+IGF SCF+Hyper-IL-6+GM-CSF SCF+IGF+GM-CSF Flt3L+Hyper-IL-6+IGF Flt3L+Hyper-IL-6+GM-CSF Flt3L+IGF+GM-CSF SCF+Flt3L+Hyper-IL-6+IGF

*

SCF+Flt3L+Hyper-IL-6+GM-CSF SCF+Flt3L+IGF+GM-CSF SCF+Hyper-IL-6+IGF+GM-CSF Flt3L+Hyper-IL-6+IGF+GM-CSF SCF+Flt3L+Hyper-IL-6+IGF+GM-CSF

0

2

4

6

8

10 12 14 16

[3H]thymidine incorporation (×103 cpm)

Fig. 1. Growth factor response of bone marrow cells and Flt3+CD11b+ progenitor cells. Growth factors were applied to bone marrow cells and established Flt3+CD11b+ progenitor cells at day 7 of culture as indicated and [3H]thymidine incorporation was determined 72 h later. Results (mean triplicate values 8 SD) from 1 representative of 3 individual experiments are shown. Asterisks indicate conditions of augmented growth response of Flt3+CD11b+ progenitor cells to Flt3L.

Fig. 2. Surface phenotype of Flt3+CD11b+ progenitor cells. Flow

sion in LSK HSC than in Flt3+CD11b+ progenitors was also found for the transcripts of Ly6c, CD24 (HSA), CD93 (AA4.1) and CD184 (CXCR4/SDF-1 receptor). We found no transcripts of CD11b, Ly6g, CD49f and CD133 (prominin) in LSK HSC that were expressed both on the transcript as well as on the protein level in Flt3+CD11b+ progenitor cells (table 2; fig. 2).

Gene Expression Profiling of TF in LSK HSC and Flt3+CD11b+ Progenitor Cells Recent transcriptional profiling studies on the expression pattern of a total of 206 surface antigen genes in Flt3+CD11b+ progenitors revealed a considerable overlap with LSK HSC [Hieronymus et al., 2005]. Here we extended our analysis of the microarray data to gain new


Hieronymus et al.


108

cytometry analysis of surface antigen expression on Flt3+CD11b+ progenitor cells was performed after 7 days in culture. For detection of Flt3/CD135, cells were starved for Flt3L for 16 h prior to staining. Open areas represent staining with isotype control antibody. A representative of 5–8 experiments is shown.

Table 3. Expression of transcriptional regulators in Flt3+CD11b+ progenitor cells and LSK HSC

Probe sets

Genes

Present in Flt3+CD11b+

Present in LSK HSC

Overlap

% of HSC

31 66 94 52 28 33 46 187 60

21 59 82 40 21 28 30 156 44

15 35 21 22 6 3 11 15 3

17 28 21 20 3 1 12 19 6

14 27 18 18 2 1 9 12 0

82 96 86 90 67 100 75 63 0

66 83 21 50 177 999

51 66 15 37 123 773

11 26 10 22 53 253

13 23 10 17 45 235

6 17 10 13 40 187

46 74 100 76 87 80

267 110 59 436

189 83 38 310

113 50 32 195

82 41 27 150

73 40 27 140

88 98 100 93

1,435

1,083

448

385

327

85

TF Basal TF TF cofactors bHLH domain bZip domain Ets domain Forkhead domain HMG box domain Homeobox domain Nuclear hormone receptor ZF KRAB box domain Other C2H2 domain PHD domain Other domains Other TF Subtotal Non-TF Non-TF DNA binding Unclassified RNA binding factor Subtotal Total

insights into the TF repertoire of Flt3+CD11b+ progenitors and LSK HSC. First, we generated a list of putative TF represented on the Affymetrix MG-U74Av2 GeneChip array based on the gene ontology terms ‘nucleus’, ‘DNA binding’, ‘TF activity’, ‘transcription regulator activity’, ‘transcription’ and ‘regulation of transcription‘ within the 3 organizing principles of gene ontology ‘cellular component’, ‘biological process’ and ‘molecular function’. Histone genes and expressed sequence tags were excluded. The remaining 1,435 probe sets, representing 1,083 single genes, were further annotated and classified into various TF and non-TF families (table 3) using public databases such as the Pfam Protein Families Database (http://www.sanger.ac.uk/Software/Pfam/) [Bateman et al., 2004] and the PANTHER Classification System (http://www.pantherdb.org/) [Thomas et al., 2003]. NonTF were separated into DNA and RNA binding factors and yet unclassified gene products. However, some of

these genes might be of interest as regulators of transcription by epigenetic or other mechanisms, such as histone deacetylases, DNA methyltransferases, DNA repair factors and other chromatin-remodeling components. We specified TF cofactors and different TF families according to defined TF DNA binding domains and listed those where 15 or more genes were identified on the microarray (table 3). Probe sets and genes that encode multiple DNA binding domains are listed and counted in 1 family only for clarity. The largest single class of TF (169 genes, not including nuclear hormone receptors) was the zinc finger (ZF) family that was further subdivided into KRAB, C2H2, PHD and other domains containing ZF TF. We identified 156 genes that encode for homeobox domain TF, 82 basic helix-loop-helix (bHLH) TF and 44 nuclear hormone receptors. We further discriminated basal TF, bZip, Ets, HMG and forkhead domain TF. The class of other TF compiled TF-encoding genes with less than 15



109


Putative transcriptional regulators present on the Affymetrix MG-U74Av2 GeneChip array were classified as TF and TF cofactors based on TF DNA binding domains or as non-TF binding factors. Some genes are represented by more than one probe set. Genes encoding multiple DNA binding domains are classified and counted in one family for clarity. Expression of nonredundant genes in Flt3+CD11b+ progenitor cells and LSK HSC was analyzed using the Affymetrix Suite 5.0 software.

100 Normalized intensity

Transcriptional regulators

577

10 1 0.1 0.01

327

3

2

1

3

5

4

0.8 PCA component 2

progenitor cells and LSK HSC. Summary of expressed genes encoding transcriptional regulators in Flt3+CD11b+ progenitor cells and LSK HSC as described in table 3.

LSK HSC

0.6


0.4 ES cells

0.2 0 –0.2 –0.4 –0.6 LSK HSC –0.8 0

–0.2

family members, such as NF-␬B factors, interferon regulatory factors and STAT factors. We found a total number of 448 of 1,083 genes (41%) to be expressed in Flt3+CD11b+ progenitors, while LSK HSC expressed 385 genes (36%). The majority of genes (327) were found in both LSK HSC and Flt3+CD11b+ progenitor cells, while only 58 and 121 were solely found in stem cells and progenitor cells, respectively (fig. 3; table 3). The resulting overlap of expressed genes in Flt3+CD11b+ progenitors to genes expressed in HSC (85%) is highly consistent with the overlap of the 206 CD molecule-encoding genes (83%) found in our previous study [Hieronymus et al., 2005]. However, within the various classes of TF and non-TF, substantial differences were observed. First, the majority of factors that contribute to the basal transcriptional machinery, and are therefore preferentially listed in the classes of basal TF, TF cofactors and RNA binding factors, are expressed in both Flt3+CD11b+ progenitors and LSK HSC. In contrast, factors that belong to the classes of forkhead domain, homeobox domain and nuclear hormone receptor TF, are underrepresented in Flt3+CD11b+ progenitors and LSK HSC, as only less than 10% of those factors were found to be expressed. Second, for most factor classes we found the overlap of expressed genes in Flt3+CD11b+ progenitors to genes expressed in LSK HSC to be more than 70%. However, Ets domain, homeobox Cells Tissues Organs 2008;188:103–115


1.0

Flt3+CD11b+ progenitors

Fig. 3. Expression of transcriptional regulators in Flt3+CD11b+

110

2

1

ES cells

a

LSK HSC

2

1

121

0.2

b

1

c

0.4

0.6

0.8

1.0

3

4

PCA component 1

3 ES cells

2

1

2

LSK HSC

1

5

2


Fig. 4. Microarray analysis of ES cells, LSK HSC and Flt3+CD11b+

progenitor cells. Gene expression in ES cells, LSK HSC and Flt3+CD11b+ progenitor cells was assessed using Affymetrix mouse MG-U74Av2 GeneChip arrays. Bioinformatics analysis was performed by GeneSpring software. a The raw data of individual hybridization experiments were normalized and signal log intensities for all genes are shown. b Principal component analysis (PCA) was performed on the entire list of genes for all data sets. Two principal components separate ES cells, LSK HSC and Flt3+CD11b+ progenitor cells, with replicate samples of the same cell type forming individual clusters. PCA component 1 most efficiently separates hematopoietic cells from ES cells, while PCA component 2 separates in vitro cultured cells from ex vivo isolated LSK HSC. c Gene array data were subjected to conditional tree cluster analysis using the entire gene list. The condition tree dendrogram shows the relationship of individual cell types; replicate experiments of the same cell type cluster together.

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Gene Expression Profiling of TF in Hematopoietic Cells and ES Cells LSK HSC represent a heterogeneous population of stem cells containing both Flt3+ cells showing only shortterm reconstituting activity and Flt3– cells that include those responsible for long-term HSC activity [Adolfsson et al., 2001; Christensen and Weissman, 2001]. Likewise, the Flt3+CD11b+ progenitors described here exhibit a multipotent differentiation potential and a short-term reconstitution capacity [Hieronymus et al., 2005; Hieronymus et al., unpubl. results]. To date, no robust protocol for maintaining or even expanding and manipulating transplantable long-term reconstituting HSC ex vivo has been achieved. Since ES cells are unique in their capacity to maintain both pluripotency and self-renewal in vitro, the differentiation of long-term reconstituting HSC from ES cells has been considered an alternative approach [Kyba et al., 2002; Nakano, 2003; Martin and Kaufman, 2005; Olsen et al., 2006]. Hence, a number of protocols have been established for generating various hematopoietic cell types from ES cells, including HSC, and thus providing new means for investigating mechanisms of HSC selfrenewal [Nakano et al., 1994; Nishikawa et al., 1998; Pinto-do et al., 1998; Perlingeiro et al., 2001; Kyba et al., 2002; Schroeder et al., 2006]. We therefore extended our analysis of the TF repertoire to undifferentiated ES cells and compared it to that of LSK HSC and Flt3+CD11b+ progenitor cells. Image analysis of chip hybridizations was done for ES cells, LSK HSC and Flt3+CD11b+ progenitor cells using Affymetrix’s Microarray Suite software applying global scaling. The raw data were then normalized in GeneSpring software per chip to the 50th percentile and per gene to the median, resulting in normalized signal intensities that are comparable among each hybridization experiment (fig. 4a). Next, we analyzed the gene array data by principal component analysis (PCA) that clusters data sets according to their degree of correlation (fig. 4b). PCA demonstrates that samples of each cell population cluster together and that hematopoietic cells are separated from ES cells (PCA component 1). LSK HSC cluster separately from Flt3+CD11b+ progenitor cells and ES cells (PCA Transcription Factor Repertoire of Flt3+ Hematopoietic Stem Cells

component 2), probably because LSK HSC were not obtained by in vitro culture and samples required 2 rounds of RNA amplification before hybridization. We next proceeded to analyze the array data sets by hierarchical clustering to describe the relationships among experiments and between genes in more detail. First, we performed conditional clustering of all samples on the total gene list (fig. 4c). The condition tree shows clustering of the samples in an ordered fashion according to their degree of correlation. Moreover, samples of each cell population cluster together to constitute subtrees, thereby obtaining essentially the same result as by PCA. Second, we performed gene tree clustering on the established gene list of transcriptional regulators with abundant expression in either LSK HSC or Flt3+CD11b+ progenitor cells comprising 506 of the total number of 1,083 genes, thereby excluding all TF that are only expressed in ES cells. This method revealed specific patterns of differentially expressed genes in the individual cell populations (fig. 5a). Genes with the highest expression in ES cell, LSK HSC or Flt3+CD11b+ progenitor cell populations, respectively, are indicated with dashed boxes (clusters I–III). The solid framed box indicates genes that are highly expressed in both ES cells and LSK HSC but not (or at lower levels) in Flt3+CD11b+ progenitor cells (cluster IV). This cluster is shown in larger scale in figure 5b with the mean intensity values of all samples of each individual cell population together with the detailed information on the compiled genes. Given the fact that ES cells and LSK HSC have an exceptionally high self-renewal capacity, those genes can be considered to be particularly important for self-renewal. This cluster lists 27 genes from almost all classes of TF and non-TF DNA binding factors except for RNA binding factors, forkhead domain TF or those compiled as ‘other TF’. The ZF motif containing factor Cxxc5 is the only yet unclassified factor. Numerous genes are involved in DNA/ chromatin remodeling, such as histone deacetylase 7a (Hdac7a), nucleosome assembly protein (Nap1l4) or the telomere length-controlling factor telomerase reverse transcriptase (Tert) that has already been implicated to play a role in self-renewal [Bodnar et al., 1998]. ZF domain TF represent the most abundant class of TF, including a total of 7 genes (Egr1, Prdm1/Blimp1, Zfp1, Zfp90, Zfp239, Zfpm1, Zfx). Recently, Zfx was described to control selfrenewal of both HSC and ES cells, suggesting a common genetic mechanism of self-renewal in stem cells [GalanCaridad et al., 2007]. Some of the other ZF TF have been investigated for their role in hematopoietic cell differentiation, but whether these factors are involved in mainCells Tissues Organs 2008;188:103–115

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domain, KRAB box domain and nuclear hormone receptor TF revealed a significantly more divergent expression pattern in Flt3+CD11b+ progenitors compared to LSK HSC. This finding might be an indication for a rather cell type-specific role of these TF in the respective populations.

Flt3+CD11b+ progenitors

LSK HSC

1

I

II

III

IV

Relative expression 0.4

1.0

6.0

a

3

Gene

Gene ID

Probe set ID

Tert

21752

94104_at

Nr5a1

26423

101665_at

Zfpm1

22761

97974_at

Hoxd8

15437

160460_at

Hdac7a

56233

97550_at

Dntt

21673

161409_f_at

Polr3d

67065

160650_at

Sox4

20677

160109_at

Zfx

22764

101919_at

Nap1l4

17955

97860_at

Taf1c

21341

92456_at

Tcf7

21414

97995_at

Pola2

18969

98006_at

Nmyc1

18109

103048_at

Cbfa2t2h

12396

97406_at

Hes1

15205

160887_at

Prdm1

12142

92904_at

Cxxc5

67393

95701_at

Fosb

14282

103990_at

Nr4a1

15370

102371_at

Zfp239

22685

102411_at

Polr2c

20021

160146_r_at

Tgif2

228839

93621_at

Egr1

13653

98579_at

Zfp90

22751

92934_at

Nr4a2

18227

92249_g_at

Zfp1

22640

92443_i_at

b

Fig. 5. Hierarchical cluster analysis of transcriptional regulators.

Gene expression in ES cells, LSK HSC and Flt3+CD11b+ progenitor cells was assessed by Affymetrix mouse MG-U74Av2 GeneChip arrays. Hierarchical cluster analysis of transcriptional regulators was performed with GeneSpring software. The list of 1,435 probe sets representing 1,083 TF and non-TF transcriptional regulators were filtered for genes with abundant expression in LSK HSC or Flt3+CD11b+ progenitor cells. a Columns represent data sets that were grouped according to the condition tree clustering (fig. 4b). Each of the 506 selected genes is depicted by a single row of colored boxes. The color of the respective box in one row represents the expression value of the gene transcript in one sample compared to the median expression level of the gene’s

112

2


transcript for all samples shown. Blue = Transcript levels below median; yellow = transcript levels equal to median; red = transcript levels higher than median. Dashed boxes indicate the genes with the highest expression in the respective cell population (clusters I–III). Cluster IV (solid box) indicates genes expressed in ES cell and LSK HSC but not, or at lowest level, expressed in Flt3+CD11b+ progenitor cells. b Enlarged representation of genes compiled in cluster IV with mean intensity values of all samples of the respective cell population. Color code as in a. 1 = ES cells; 2 = LSK HSC; 3 = Flt3+CD11b+ progenitor cells. Gene name, Entrez Gene ID and Affymetrix probe set ID are shown. All microarray data sets are deposited with series accession No. GSE692, GSE693 and GSE2375 in the GEO database.

Hieronymus et al.


ES cells

Conclusions

The culture system described in this paper employs SCF, Flt3L, IGF-1, GM-CSF, hyper-IL-6 and the glucocorticoid dexamethasone to generate Flt3+CD11b+ MPP from mouse bone marrow. These factors were chosen based on microarray studies of LSK HSC that demonstrated expression of their cognate receptors [Ivanova et al., 2002; RaTranscription Factor Repertoire of Flt3+ Hematopoietic Stem Cells

malho-Santos et al., 2002; Hieronymus et al., 2005]. The established cytokine combination yields an effective outgrowth and amplification of an Flt3+CD11b+ MPP that overcomes limitations in number, purity and homogenicity for such progenitors and therefore is most suitable for further cellular, biochemical and molecular studies. Gene expression profiling by high-density oligonucleotide microarrays provides a powerful means of exploring transcriptional patterns on a genome-wide scale. By employing microarray analysis, we recently identified the bHLH TF Id2 as a major determinant for DC development [Hacker et al., 2003]. Therefore, microarray data of Flt3+CD11b+ progenitors were further evaluated for their expression pattern of transcriptional regulators. These data were compared to those of LSK HSC and ES cells [Rolletschek et al., 2001; Ivanova et al., 2002; Ramalho-Santos et al., 2002] and it was found that Flt3+CD11b+ progenitors exhibit a considerable overlap with HSC. ES cells show the unique property of maintaining pluripotency and self-renewal in culture and this has emerged as an important means to study blood cell development. Differentiation of multipotent hematopoietic progenitors from ES cells has been achieved via embryoid body formation or by coculturing with stromal cell lines such as OP9. However, irrespective of the ES cell differentiation protocols used, the ES cell-derived blood progenitors exhibit merely a limited repopulation capacity. A potential reason might be an inadequate recapitulation of the appropriate microenvironment for optimal survival of HSC in vitro that resembles the HSC niche in vivo. The insights into the dynamic regulatory networks of transcriptional activators and repressors in embryonic and adult stem cells will (1) help to understand the instructive signaling of the microenvironment composed of soluble and cell-cell contact-dependent factors and (2) thereby elucidate the requirements to reproduce such stem cell-like niches in vitro to facilitate cellular-based therapies.

Acknowledgments We would like to thank R. Foerster for anti-mouse CXCR4 antibody. We are grateful to R. Ensenat-Waser and X. Ding for critical reading of the manuscript and P. Podlatis and A. Offergeld for expert assistance on manuscript editing. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Priority Program 1109, Ze432/1 and Ze432/2, SFB542) and by funds from the Interdisciplinary Center for Clinical Research BIOMAT within the Faculty of Medicine at the RWTH Aachen University (VV B110-d and VV B112-a). T.H. received a grant from the START program of the Faculty of Medicine at the RWTH Aachen.


113


taining stem cell properties has not yet been addressed. Other interesting candidate genes belong to the families of nuclear hormone receptors (Nr5a1/SF-1, Nr4a1/nur77, Nr4a2/Nurr1), HMG box factors (Sox4, Tcf7), homeobox domain TF (Hoxd8, Tgif2) and bHLH TF (Nmyc1, Hes1). Several members of these TF families have been shown to be critically involved in self-renewal mechanisms. This has been demonstrated, for example, for the HMG box factor Sox2 [Avilion et al., 2003] and nuclear hormone receptor Nr3b2/Esrrb [Ivanova et al., 2006] in ES cells. Interestingly, the orphan nuclear hormone receptor Nr5a1/ SF-1 identified in our study has recently been described as a transcriptional activator of human Oct4 through direct interaction with an SF-1 binding element in the human Oct4 proximal promoter [Yang et al., 2007]. Several members of the Hox family of homeobox domain TF have been reported to be involved in hematopoiesis, such as HoxB4 that enhances the in vivo repopulating activity of HSC [Sauvageau et al., 1995; Schmittwolf et al., 2005]. Moreover, HoxB4 expression transformed ES cell-derived hematopoietic progenitors into long-term repopulating HSC [Kyba et al., 2002]. Finally, we and others have shown that bHLH TF are critically involved in cell fate decisions in a wide array of developmental processes including the hematopoietic system [Hacker et al., 2003; for review, see Massari and Murre, 2000; Murre, 2005], and this study identified, for example, Hes1 as a candidate gene involved in self-renewal. Noteworthy, Kunisato et al. [2003] demonstrated that Hes1 expression in HSC maintained the long-term reconstituting activity of these cells in culture. Hes1 functions downstream of the Notch receptor and retroviral transduction of LSK HSC with a constitutively active form of Notch1 resulted in immortalized, cytokine-dependent pluripotent HSC lines [Varnum-Finney et al., 2000]. However, whether Notch signaling plays an essential role for HSC self-renewal is controversially discussed, as Notch signaling in ES cell-derived HSC was found to accelerate myeloid differentiation rather than maintaining HSC properties [Schroeder et al., 2006].

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