Ann. N.Y. Acad. Sci. 1044: 51–59 (2005). © 2005 New York Academy of Sciences.
Genetic and Functional Characterization of
Isolated Stromal Cell Lines from the
KATJA C. WEISELa AND MALCOLM A. S. MOOREb
aUniversity of Tübingen, Medical Center,
Department of Hematology, Oncology, and Immunology, Tübingen, Germany
bLaboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center,
New York, New York, USA
ABSTRACT: The hematopoietic system interacts with a supportive stromal
environment allowing maintenance and differentiation of hematopoietic stem
cells (HSCs). The aorta-gonado-mesonephros (AGM) region serves as a unique
embryonic microenvironment, generating the first adult repopulating HSCs in
the mouse embryo. To eludicate factors involved in hematopoietic support and
induction of hematopoietic differentiation, we isolated more than 100 stromal
cell clones derived from the AGM region of embryonic day (E) 10.5 mouse
embryos for functional and genetic analysis. Selected isolated AGM stromal
cell lines are highly efficient in supporting maintenance and expansion of
mouse and human hematopoietic stem and progenitor cells. In addition, we can
demonstrate for the first time that AGM stromal cell lines are also potent
inducers of hematopoietic differentiation of murine embryonic stem cells.
Stromal gene array analysis has identified genes that could play a role in
KEYWORDS: aorta-gonado-mesonephros region; stromal coculture; murine ES
cells; ex vivo expansion
During hematopoietic development, hematopoiesis first begins in the yolk sac
(YS) at day 7.5 postgestation (E7.5) before shifting into the fetal liver, spleen, and
bone marrow.1,2 Nondefinitive progenitors first appear in YS blood islands at E8.5.3
Definitive murine hematopoietic stem cells (HSCs) emerge from the intraembryonic
aorta-gonado-mesonephros (AGM) region, appearing initially on the floor of the
dorsal aorta.4,5 These hematopoietic cells, capable of reconstituting definitive, adult
hematopoiesis, first appear at day 10.5 after gestation.
Address for correspondence: Katja C. Weisel, M.D., University of Tübingen, Department of
Hematology, Oncology, and Immunology, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany.
Voice: +49-7071-2982726; fax: +49-7071-293671.
52 ANNALS NEW YORK ACADEMY OF SCIENCES
Stromal cells are known to be essential in the regulation of hematopoiesis. In non-
cytokine conditions, hematopoiesis is not maintained when stromal cell growth is
impeded.6 Further studies have demonstrated that bone marrow (BM) and fetal liver
stromal cells support long-term proliferation of HSCs, as measured by late second-
ary progenitor colony formation and cobblestone area formation beneath the
stroma.7–9 A number of immortalized stromal cell lines from adult murine BM and
fetal liver have been developed for long-term support of both murine and human
HSCs.8–10 More recently, the presumptively unique characteristics of the embryonic
hematopoietic microenvironment have been investigated further, and a limited num-
ber of stromal cell lines from the YS and the AGM region have been developed that
support murine and human hematopoiesis.11–16 However, until now, only two cell
lines derived from the AGM region and the surrounding urogenital ridge have been
reported that are able to support human hematopoiesis in a cytokine-independent
manner. More recently, BM-derived stromal cell lines have been reported that can
induce hematopoietic differentiation when cocultured with totipotent embryonic
stem (ES) cells. For hematopoietic differentiation of murine17 and human18 ES cells,
so far the OP9 stromal cell line derived from the BM of the op−/− mouse is well
established as a potent inducer of hematopoiesis.
Murine mesenchymal stromal lines supporting growth and maintenance of
hematopoietic stem and progenitor cells derived from marrow, spleen, and fetal liver
represent several stages along the vascular smooth muscle cell (VSMC) differenti-
ation pathway.19,20 However, until now, mechanisms of hematopoietic differentia-
tion and hematopoietic stem cell maintenance or expansion have not been fully
Here, we describe the isolation and characterization of more than 100 cloned stro-
mal cell lines derived from the AGM region of E10.5 mouse embryos. When cocul-
tured with the stromal cells, hematopoietic cells, especially primitive hematopoietic
stem/progenitor cells in adult mouse bone marrow and human cord blood, prolifer-
ated significantly without additional cytokines. Moreover, one of these AGM strom-
al cell lines induced significant hematopoietic differentiation of murine ES cell lines
comparable to that reported in OP9 stromal coculture. Genetic characterization of
supporting lines, including microarray analysis, revealed a mesenchymal and vascu-
lar smooth muscle cell phenotype.
Functional Characterization of Isolated AGM Stromal Cell Lines
Effect of AGM Stromal Cell Lines on Mouse BM Progenitor Cells
A total of 106 stromal cell lines (AGM-S1 to AGM-S106) were obtained from
E10.5 mouse embryos by dissecting the AGM region and culturing outgrowing stro-
mal cells as described.13 It has previously been shown that AGM-derived stromal
cell lines are capable of supporting murine adult hematopoiesis.13,14 As a first
screening to determine whether the generated clones could support growth and
maintenance of HSCs, we examined the ability of the 106 established stromal cell
lines to support hematopoiesis following coculture with mouse bone marrow mono-
53 WEISEL & MOORE: GENETIC AND FUNCTIONAL CHARACTERIZATION
nuclear cells (MNCs) from C57BL/6 mice. BM MNCs were cocultured with AGM
stromal cells for three weeks and evaluated for stem and progenitor cell function
(cell growth, colony, and cobblestone-area formation).21 Results were compared
with those obtained coculturing BM MNCs under the same conditions with the MS-
5 BM stromal cell line, which is well established in its ability to support mouse and
human HSCs. After three weeks of coculture, hematopoiesis was maintained with all
of the 106 stromal cell lines, with a minimum expansion of 0.8-fold; only 4 lines
failed to support colony-forming cells (CFCs) and cobblestone area–forming cells
(CAFCs). In comparison, Xu et al. reported that only 3 of 17 of their stromal lines
supported hematopoiesis. Oostendorp et al. showed that between 4% and 38% of
isolated AGM lines supported hematopoiesis; this was dependent on their origin
from different AGM subregions.
Colony-forming assays of week-3 suspension cells showed secondary colony for-
mation in cocultures with 102 of 106 AGM cell lines with a minimum of 2 and a
maximum of 2,000 colonies per flask with 100,000 BM MNCs input at day 0. Twenty-
nine stromal lines were superior to the MS-5 stromal line in supporting secondary
CFC production. Stromal lines that supported the generation of >50 CFCs at week
3 also supported production of a spectrum of progenitor types, including the eryth-
ropoietic burst formation unit (BFU-E) and the mixture colony-forming unit (CFU-
Mix), in addition to the predominant granulocyte-macrophage colony-forming unit
(CFU-Gm). Primary cobblestone formation occurred in a wide range of 1 to 200
CAFCs per flask. High CAFC counts were, in general, correlated with high CFC
counts, demonstrating that the cobblestone-forming earlier progenitor cells release
the committed progenitor population in the supernatant. However, in some cocul-
tures, despite a low number of cobblestones, a high CFC count was detected. This
might be the result of lower expression of cell adhesion molecules or chemokines
such as SDF-1 in the stromal cell population, without affecting progenitor cell
expansion in the supernatant.
AGM Stromal Cell Line Coculture with Human Cord Blood CD34+ Cells
For evaluation of the functional support of human HSCs and progenitors, 28 of
the best supportive stromal cell clones were selected. It was previously shown that
AGM-derived stromal cells have the ability to expand human HSCs.16 Kusadasi
et al. demonstrated that 19 of 100 cloned stromal cell clones supported long-term
proliferation of human CD34+ cord blood cells in vitro. However, only one cell line
was able to maintain HSCs in the absence of exogenous cytokines. We investigated
the coculture of human cord blood CD34+ cells with selected AGM stromal cell
clones in comparison with the reference cell lines MS-5 and OP9, under serum-con-
taining conditions in medium without cytokines, or in the presence of continuously
secreted thrombopoietin (TPO) following transduction of the stromal cells with a
TPO-expressing adenovirus (Ad-TPO). We have shown previously that the OP9/Ad-
TPO system is highly effective for extensive amplification of human cord blood
CD34+ cells, sustaining stem and progenitor cell activity for over five months with-
out significant cell senescence, as indicated by constant telomere length.22 Cocul-
ture of cord blood CD34+ cells on selected AGM stromal lines revealed early
cobblestone formation under most of the stromal cells, with release of nonadherent
hematopoietic cells into the suspension. Twenty-seven of 28 cell lines supported ex-
54ANNALS NEW YORK ACADEMY OF SCIENCES
pansion or maintenance of cord blood cells. Expansion rates of 50- to 100-fold were
observed after 4 weeks of coculture. Progenitor cell assays of four selected lines
demonstrated expansion of early and committed progenitors in this period, compa-
rable to expansion on OP9/TPO or MS-5. Generation of CFU-Mix, the immature
multipotential progenitors, was sustained until week 5 of coculture. In addition, we
assayed secondary cobblestone formation at week 5 of coculture. The production of
week-5 CAFC in human CD34+ ex vivo expansion cultures has been shown to be in-
dicative of the presence and quantity of in vivo repopulating HSCs, as measured us-
ing the quantitative nonobese diabetic/severe combined immunodeficiency (NOD/
SCID) mouse xenotransplant model.23,24 Cobblestone formation was present in the
four stromal line cocultures, comparable to the reference stromas MS-5 and OP9.
For one of the supporting cell lines, AGM-S2, we also investigated the effect on
human reconstituting hematopoietic stem cells before and after the coculture in the
NOD/SCID transplantation assay. When 2 × 104 cord blood CD34+ cells cocultured
with AGM-S2 cells for 4 weeks were transplanted into irradiated adult NOD/SCID
mice, human CD45+ cells were found (in 29%) in the bone marrow MNC fraction of
the recipient mice, as determined by flow cytometry.
Effect of AGM Stromal Cell Lines on Hematopoietic Differentiation of Murine
Embryonic Stem Cells
Totipotent undifferentiated murine ES cells can be differentiated into hematopoi-
etic cells using a sequential stromal coculture system based on the macrophage col-
ony-stimulating factor (M-CSF)–deficient OP9 stromal cell line17 With human ES
cells, the OP9 cell line and the BM stromal cell line S17 can also induce production
of CD45+ hematopoietic cells.18,25 We evaluated 10 AGM stromal cell lines for their
capacity to induce hematopoietic differentiation of murine ES cells with or without
additional TPO, using a sequential OP9 coculture. After 10 d of sequential coculture,
hematopoietic cells were found in the supernatant and cobblestone areas formed
beneath the stroma. After transferring the supernatant onto fresh AGM stroma,
expansion of hematopoietic cells as well as secondary cobblestone formation was
achieved. Immunophenotyping of day 18 cultures revealed a marked production of
CD45+ hematopoietic cells. In addition, early B cell differentiation with a B220-
positive fraction was detected.
Genetic Characterization of Supporting Lines
Surface Epitope Expression on AGM Stromal Cells
In an effort to elucidate the phenotype of supporting cell lines, we investigated
surface marker expression on three representative stromal clones that were efficient
in supporting human cord blood CD34+ cells. The three cell lines showed a nearly
identical phenotype, with expression of Sca-1, CD13, VCAM-1, and CXCR-4, but
were negative for PECAM-1, c-kit, CD49d, and CD34. This is comparable to the
data reported by Xu et al. on their AGM stromal lines. The constitutive expression
of VCAM-1 by marrow stromal cells is well known.26 Our present work supports the
finding that VCAM-1 also is expressed by stroma of the AGM region, as previously
shown,13,20 and suggests that hematopoietic stem and progenitor cells adhere to stro-
mal cells through the VCAM-1/VLA-4 pathway. Finally, this expression profile is
55WEISEL & MOORE: GENETIC AND FUNCTIONAL CHARACTERIZATION
consistent with the postulated VSMC phenotype of stromal cells that can support
hematopoiesis,20 because VCAM-1 is expressed by VSMCs early in development.
Cytokine Expression by AGM Stromal Cells
We compared cytokine and chemokine expression on the three selected lines by
RT-PCR. All three lines showed expression of SCF, M-CSF, and SDF-1, but unde-
tectable levels of IL-3, IL-6, and Flt3 ligand. Our data and previous reports13,27
show that cytokine expression of the tested AGM stromal cell lines is similar to that
in MS-5 cells.
Gene Expression Profiles of AGM Stromal Cell Lines
To analyze the transcriptosome of stromal lines that most efficiently support
hematopoietic stem cell activity, we performed microarray analysis comparing gene
expression profiles of a supporting and a nonsupporting cell line (AGM-S26 vs.
AGM-80). The results are shown in TABLE 1. Five hundred two genes were differen-
tially expressed in AGM-S26; 321 were upregulated, and 181 were downregulated.
The genes that were upregulated in the supporting line included many that are asso-
ciated with the VSMC phenotype, such as Eda, VCAM-1, osteoglycin, and smooth
muscle actin.20 Furthermore, mesenchymal genes were found, such as laminin al-
pha-2, laminin gamma-1, laminin B1, and thrombospondin-1, as well as many cy-
toskeleton genes, such as myosin heavy chain IX, myosin light polypeptide kinase,
myosin regulatory light chain interacting protein, myosin X, transgelin, and tro-
pomyosin 1. Reflecting the early developmental status of AGM cells and hematopoi-
etic development at this time, mesodermal genes such as bone-morphogenetic
protein 4 (BMP-4) and osteomodulin were highly expressed. BMP-4 is known to act
early in hematopoietic differentiation processes and can promote hematopoietic dif-
ferentiation in vitro.28,29 In addition to the above-described genes, upregulation of
early B cell differentiation genes was detected, with the member of the TNF receptor
superfamily, osteoprotegerin, which was expressed at a nearly 40-fold higher level
than in the nonsupporting line, as well as early B cell factor 1. In fact, early B cell
differentiation, with a small but significant progenitor population, is described in the
AGM region.30,31 Ostoprotegerin, which is known to be a crucial regulator of bone
metabolism, also regulates B cell function.32 In an opg−/− mouse model it was shown
that these mice revealed perturbations in central and peripheral B cell compartments,
including an accumulation of type 1 transitional B cells. Although these mice
seemed not to show a defect in the hematopoietic stem cell compartment, a potential
role for osteoprotegerin in growth and maintenance of hematopoietic stem and pro-
genitor cells seems to be possible and will be evaluated further. In addition, we de-
tected increased expression of Igf-2, which has already been described to be
expressed in hematopoietic stromal cell lines,33 and which is overexpressed in poly-
cythemia vera, a rare myeloproliferative disease with enhanced stem cell prolifera-
tion.34 This could indicate a role for this gene in stem cell expansion in early
development. In addition to genes involved in hematopoietic support and differenti-
ation, gene expression analysis also revealed genes involved in kidney differentia-
tion and renal cell function, such as angiotensinogen, angiotensin II receptor, and
renin binding protein (TABLE 1). This demonstrates the close ontogenic relationship
between these organ systems in the AGM region.
56 ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE 1. Differential gene expression in the AGM-S26 (supporting) stromal cell
line compared with the AGM-S80 (nonsupporting) stromal cell linea
Gene Gene symbolFold change
Tumor necrosis factor receptor superfamily, member 11b
Interferon-activated gene 205
Interferon, alpha-inducible protein
Interferon-activated gene 203
Interferon-γ induced GTPase
Angiotensin II receptor, type 2
Insulin-like growth factor 2
Mast cell protease 8
Interferon-inducible protein 1
Interferon-regulatory factor 7
Matrix gamma-carboxyglutamate (gla) protein
Laminin, alpha 2
Bone morphogenetic protein 4
Laminin, gamma 1
Natriuretic peptide receptor 3
Transforming growth factor, beta 3
Actin, gamma 2, smooth muscle, enteric
Erythroid differentiation regulator 1
Myeloid/lymphoid or mixed-lineage leukemia
Myosin heavy chain IX
Myosin, light polypeptide kinase
Tropomyosin 1, alpha
Laminin B1 subunit 1
Vascular cell adhesion molecule 1
Renin binding protein
57 WEISEL & MOORE: GENETIC AND FUNCTIONAL CHARACTERIZATION
In hematopoiesis, the stromal microenvironment plays an important role in sup-
port and regulation of self-renewal and differentiation of hematopoietic stem cells
capable of a lifetime of continuous expansion of all hematopoietic lineages. Several
cell lines have been established from fetal and adult mouse hematopoietic tissues
that can support the long-term survival of murine long-term repopulating hematopoi-
etic stem cells. Here, we describe the efficient isolation of more than 100 stromal cell
clones derived from the AGM region of E10.5 mouse embryos. Using coculture sys-
tems with selected AGM stromal cell lines and murine and human hematopoietic
stem cells, we demonstrate that AGM stromal cell lines are highly effective in sup-
porting adult murine and human stem/progenitor cell proliferation without exoge-
nous cytokine support. A significant number of these cell lines were superior to the
MS-5 BM stromal cell line in hematopoietic support. Regarding the embryonic
microenvironment that is represented by the AGM stromal cell clones, we show that
AGM-S62 is comparable to OP9 in its capacity to induce hematopoietic differentia-
tion of murine ES cells. The hematopoietic support and the induction of hematopoi-
etic differentiation seem to be associated with genes expressed in cells of a
mesenchymal and VSMC phenotype, with characteristic genes expressed including
VCAM-1, laminins, and smooth muscle actin. With a growing interest in therapeutic
application of differentiated cells for regenerative and transplantation medicine,
understanding key features that distinguish factors essential for hematopoietic
support may have future clinical significance.
Early B cell factor 1
Growth hormone receptor
Interferon-activated gene 204
Interferon gamma receptor
Myosin regulatory light chain interacting protein
Procollagen, type IV, alpha 2
Procollagen, type XVI, alpha 1
WNT1 inducible signaling pathway protein 2
aTotal RNA was isolated and used to hybridize gene arrays.
NOTE: A change in gene expression was considered significant only when fold change was
>1.87, with P<.05 and signal value >200.
TABLE 1. (continued) Differential gene expression in the AGM-S26 (supporting)
stromal cell line compared with the AGM-S80 (nonsupporting) stromal cell linea
Gene Gene symbolFold change
58ANNALS NEW YORK ACADEMY OF SCIENCES
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