1 Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive,
Thousand Oaks, CA 91320, USA
2 Department of Pathology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA
91320, USA
3 Department of Flow Cytometry Laboratory, Amgen Inc., One Amgen Center Drive,
Thousand Oaks, CA 91320, USA
* Author for correspondence (e-mail: naoki.nakayama{at}amgen.com)
Accepted 7 February 2003
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Summary |
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Key words: Embryonic stem cells, Cartilage, BMP, TGFß, PDGF
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Introduction |
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Upon differentiation of ES cells in a conventional serum-containing medium,
some cells starts to express the vascular endothelial growth factor (VEGF)
receptor-2 (VEGFR-2 or flk-1) protein, whose function is essential for
embryonic hematopoiesis and vasculogenesis in mice
(Shalaby et al., 1997;
Shalaby et al., 1995
), and
some begin to express the platelet-derived growth factor receptor
(PDGFR
) protein (Kabrun et al.,
1997
; Nishikawa et al.,
1998a
). The ES-cell-derived flk-1+ cells contain the
hemangioblast, the common progenitor cell for both the hematopoietic and
endothelial cell lineages (Choi et al.,
1998
; Faloon et al.,
2000
; Nishikawa et al.,
1998a
; Ogawa et al.,
1999
). Embryo-derived cells with similar cell-surface
characteristics also show hemogenic and angiogenic activities
(Nishikawa et al., 1998b
;
Ogawa et al., 1999
).
Consistently, both the extraembryonic mesoderm, the origin of the primitive
endothelial cells and hematopoietic cells, and the intraembryonic mesoderm,
which includes the presumptive endocardium and the proximal lateral mesoderm,
express flk-1 (Dumont et al.,
1995
; Kataoka et al.,
1997
; Takakura et al.,
1997
; Yamaguchi et al.,
1993
). Therefore, flk-1+ cells are considered to
represent the hemoangiogenic lateral plate mesoderm, that is, the
splanchnopleuric mesoderm. Another part of the lateral plate mesoderm, the
somatopleuric mesoderm, participates in the formation of limb bone. However,
the potential of flk-1+ cells to generate cartilage and bone has
not been reported yet.
During early mouse embryogenesis, the PDGFR gene is
expressed in most of mesodermal cells
(Orr-Urtreger et al., 1992
;
Schatteman et al., 1992
), and
PDGFR
signaling is essential for normal development of many mesodermal
tissues, including the axial skeleton
(Soriano, 1997
).
Interestingly, protein expression appeared to be limited to the paraxial
mesoderm, and later to the somite
(Takakura et al., 1997
). The
strong expression of the PDGFR
gene in the somitic block is
later restricted in the dermatome and sclerotome, but not in the myotome.
PDGFR
is also expressed in the lateral mesoderm, the proximal
limb mesenchyme, and the perichondrium. Thus, the ES-cell-derived
PDGFR
+ cells may form the paraxial mesoderm, somite cells or
the chondrogenic mesenchymal cells, including the sclerotome. However, the
chondrogenic potential of the ES-cell-derived PDGFR
+ cells
has not yet been demonstrated.
Chondrogenesis from undifferentiated mesenchymal cells can be divided into
several stages (Cancedda et al.,
1995; Hall and Miyake,
2000
). The first stage is precartilage condensation, in which the
mesenchymal cells are closely packed and start to differentiate into
chondroblasts and chondrocytes. Chondroblasts/chondrocytes proliferate and
secrete increasing amounts of cartilage matrix macromolecules, until each
single cell is completely surrounded by a matrix. Cells at the periphery of
condensation are relatively undifferentiated and later form a sheath of
spindle-shaped cells, which becomes the perichondrium, around the cartilage
rudiment. Each stage requires a different and/or an overlapping set of
extracellular factors. For example, transforming growth factor (TGF)ß is
a potent factor for driving the differentiation of mesenchymal cells toward
chondrocytes in vitro (Kulyk et al.,
1989
). It seems to be necessary for the initial mesenchymal
condensation stage (Leonard et al.,
1991
). In addition, TGFß also plays an inhibitory role in
chondrocyte maturation (Ballock et al.,
1993
; Kato et al.,
1988
; Serra et al.,
1997
; Yang et al.,
2001
), for which the importance of the perichondrium has been
demonstrated (Alvarez et al.,
2001
). On the other hand, bone morphogenetic protein (BMP)
function is also implicated in several steps in cartilage formation. Like
TGFß, BMP has early positive roles, that is, proliferation, maintenance
and maturation of chondrocytes, and a negative role at a later stage, that is,
delay of terminal hypertrophic differentiation in the growth plate
(Capdevila and Johnson, 1998
;
Duprez et al., 1996
;
Enomoto-Iwamoto et al., 1998
;
Minina et al., 2001
;
Pathi et al., 1999
;
Pizette and Niswander, 2000
;
Zou et al., 1997
). Different
BMPs are detected around the mature chondrocytes in the mid to deep zone of
the articular cartilage and the hypertrophic/calcifying zone of the growth
plate (Anderson et al.,
2000
).
BMP4, required for mesoderm formation in mice
(Winnier et al., 1995) and for
hematopoietic cell genesis during the early embryogenesis of vertebrates, is a
strong hematopoietic-cell-inducer for ES cells in a serum-free environment
(Johansson and Wiles, 1995
;
Nakayama et al., 2000
). We
have previously demonstrated that treatments with BMP4 followed by VEGF, a
flk-1 ligand, synergistically stimulate the generation of lymphohematopoietic
progenitor cells from ES cells in a serum-free medium, which supports the idea
that BMP4-induced mesoderm produces hematopoietic cell progenitors in a
flk-1-dependent manner like it does in vivo
(Nakayama et al., 2000
). Here,
we have demonstrated that two types of mesodermal cells, flk-1+
cells and PDGFR
+ cells, are induced by BMP4 in serum-free
medium, from which macroscopic cartilage particles are generated in vitro.
Hematopoietic progenitor cells were generated predominantly from the
flk-1+PDGFR
cells. However, under the
optimal conditions established with TGFß3, BMP4 and PDGF-BB, the
flk-1PDGFR
+ cells, the
flk-1+PDGFR
+ cells and the
flk-1+PDGFR
cells produced hyaline
cartilage particles, which were further mineralized. Therefore, the
ES-cell-derived mesodermal cells seem to possess the full developmental
potential to form mature cartilage.
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Materials and Methods |
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Induction of differentiation in a serum-free medium by the embryoid
body formation method
Differentiation was performed as described previously
(Nakayama et al., 2000).
Briefly, E14 ES cells were cultured on a fibronectin-coated 60 mm plate
(Becton Dickinson) for 2 days in the KnockOut-SR (Gibco)-based serum-free
medium containing 10 ng/ml human leukemia inhibitory factor. The cells were
then differentiated at 4500 cells/ml in the serum-free medium containing 0.9%
methylcellulose (Stem Cell Technology) in the presence of 1.9 ng/ml BMP4 under
5% CO2, 5% O2. On days 3.6 and 4.6, embryoid bodies
(EBs) were collected, combined and treated with 0.5 mM EDTA, and single cell
suspensions were obtained by passing the EBs through a 22-gauge needle. The
cells were then stained with 2 µg/ml PDGFR
-bio and 4 µg/ml
Flk-1-PE or with 2 µg/ml SA-APC. The stained samples were analyzed and
sorted using a Vantage SE cell sorter (Becton Dickinson).
Serum-free erythro-myeloid colony-forming cell analysis
Each sorted EB cell fraction was mixed with a serum-free colony-forming
cell (CFC) medium, containing 1% methylcellulose and supplemented with a
mixture of hematopoietic cytokines, and was distributed into two to four 35 mm
bacterial-grade dishes at 2.5x104 cells/plate. The serum-free
CFC medium was as described previously
(Heyworth and Spooncer, 1993),
except that linoleic acid and soybean lipids were replaced with 2% chemically
defined lipids (Gibco). The cytokines included were the same as described
before for the FACS-purified EB cells, except that 10 ng/ml TPO was added
(Nakayama et al., 2000
). On
days 10 to 12, burst-forming-unit erythrocyte, colony-forming unit (CFU)
macrophage/monocyte, CFU neutrophil, CFU mast cell and CFU mix were
counted.
Co-culture method for developing erythro-myeloid CFCs,
lymphokine-activated killer cells and pre-B cells from sorted EB cells
The OP9 cell co-culture method for generating the erythro-myeloid CFC
activity and the pre-B and/or lymphokine-activated killer (LAK) lymphocytes
was as described previously (Nakayama et
al., 1998). For the former, sorted EB cells were plated at
5x104 cells/well onto a confluent layer of OP9 cells within a
six-well plate and were cultured for 3 days without interleukin (IL)-2 and
IL-7. Both non-adherent cells and loosely attached cells were then
mechanically harvested and subjected to CFC analysis. Lymphoid potential was
detected with the same culture with IL-2 and IL-7, except that the sorted
cells were plated at 1x104 cells/well. On days 10 to 12, both
non-adherent cells and loosely attached cells were mechanically harvested,
followed by FACS phenotyping (FACScan, Becton Dickinson) using the Sca-1-FITC
and B220-PE antibodies to confirm the generation of B220+
lymphocytes: pre-B cells (Sca-1) and LAK cells
(Sca-1+), as described previously
(Nakayama et al., 1998
).
Serum-free micromass cultures for chondrogenesis
The FACS-purified EB cells were re-suspended at 2x107
cells/ml in a serum-free chondrogenesis medium. The medium was a modification
of that reported by Mackay et al. (Mackay
et al., 1998), in that Dulbecco's modified Eagle's medium (DMEM)
was replaced with DMEM (high glucose): Ham's F12=1:1 (Gibco), 0.3% glucose
(Sigma), 50 µM monothioglycerol (MTG, Sigma) and 50 ng/ml IGF1 were added.
Then, 7.5 µl (1.5x105 cells)/well of the cell suspensions
were spotted onto a fibronectin-coated 24-well plate (Becton Dickinson),
incubated for 1 hour, and cultured in 1 ml/well of the same medium under 5%
CO2, 5% O2 for 7 to 8 days. The culture was washed twice
with PBS and the total protein was extracted with LDS gel-loading buffer
(Invitrogen) including the protease-inhibitor mix (Boehringer Mannheim) for
western blot analysis. For the COL2 analysis, 1 µg/ml 6B3 was used. To
detect the sulfated glycosaminoglycans, the culture was fixed with 10%
formalin, stained with 1% Alcian blue at pH 1 (American Master Tech
Scientific) and destained with acetic acid, according to Tallquist et al.
(Tallquist et al., 2000
).
The pellet culture was performed as described previously
(Mackay et al., 1998) except
that the serum-free chondrogenesis medium included 50 µM MTG. The
3-4x105 FACS-purified EB cells were first re-suspended in 0.5
ml of the chondrogenesis medium supplemented with or without 10 ng/ml
TGFß3 and/or other factors indicated. Cells were then centrifuged and
cultured as a pellet. On days 14 to 20, each cartilage particle was fixed with
10% buffered zinc formalin (Anatech) or Gendre's fluid
(Bedossa et al., 1987
) for less
than 24 hours at room temperature, paraffin-embedded, sectioned through the
center part of each particle and stained with 0.1% Toluidine blue (Sigma)
(Sheehan and Hrapchak, 1987
).
Two additional sections were also made through different parts of each
particle, to confirm reproducibility. Some sections were immunostained with 4
µg/ml 2B1.5 for COL2 or with 2 µg/ml AB765P for COL1, according to the
manufacturer's recommendation, and were counterstained with Gill 2 Hematoxylin
(Shandon) (Sheehan and Hrapchak,
1987
).
Mineralization was induced in the modified hypertrophic differentiation
medium described by Mackay et al. (Mackay
et al., 1998). We added 50 µM MTG and replaced 50 ng/ml
thyroxine with 10 nM T3 (Sigma), as described previously
(Alini et al., 1996
). After 5
days of culture, the particle was fixed with 10% buffered zinc formalin,
paraffin-embedded, sectioned, stained with von Kossa and counterstained with
Nuclear FastRed (Sheehan and Hrapchak,
1987
). Some sections were immunostained with 1:20 diluted X53 for
COL10, according to the manufacturer's recommendations.
Cartilage-specific gene expression by the reverse
transcriptase-polymerase chain reaction method
Two to five aggregates were harvested at each designated time point and
were disrupted immediately in the guanidine isothiocyanate solution provided
with the RNeasy Kit (Qiagen). Total RNAs were purified using the protocol
recommended by the manufacturer, including the DNase I treatment step.
Essentially the same materials and protocols were used for reverse
transcription (RT) and nested polymerase chain reaction (PCR) as previously
described (Nakayama et al.,
1998). Modifications were (1) approximately 0.1 µg cDNA per
reaction was used, (2) the annealing temperature was set at 68°C and (3)
the cycle number was 21 with an outside primer set, followed by another 21
cycles with the corresponding inside primer set. The primers for
cartilagespecific genes, including parathyroid hormone-related protein
(PTHrP), aggrecan, cartilage oligomeric matrix protein (COMP), COL2, COL10 and
Chordin-like 1 (CHL1), are shown in Table
1.
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Results |
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After FACS isolation, single positive cells
(flk-1PDGFR+ and
flk-1+PDGFR
), double positive (DP) cells
(flk-1+PDGFR
+) and double negative (DN) cells
(flk-1PDGFR
) were individually
examined for their erythro-myeloid CFC activities and lymphoid potentials.
Prior to the OP9 culture, CFCs were hardly detected in any of these cell
fractions. However, the CFC activities were strongly induced from the
flk-1+PDGFR
cells and, to a lesser degree,
from the DP cells in 3 days on OP9 (Fig.
1B). In contrast, the
flk-1PDGFR
+ and DN cell fractions were
virtually devoid of such a CFC-generating activity. Similarly, the
flk-1+PDGFR
cells and, to a lesser degree,
the DP cells, eventually generated B220+ lymphocytes during the 10
to 12 day culture on OP9 in the presence of IL-2 and IL-7
(Fig. 1C). The
flk-1PDGFR
+ and DN cell fractions never
generated B220+ pre-B or LAK cells. Endothelium-like cell clusters
were also observed during the culture of
flk-1+PDGFR
cells and DP cells on OP9,
whereas flk-1PDGFR
+ cells never generated
such cells (data not shown).
These results were consistent with the previous observations that the
FACS-purified flk-1+ cells have CFC-generating (hemogenic) activity
and angiogenic activity. Thus, the mesodermal progenitor cells expressing
flk-1 and/or PDGFR generated with BMP4 in the serum-free medium are
likely to be equivalent to those made in the serum-containing medium described
before.
In vitro cartilage formation with the EB cells in the presence of
TGFß
Both the paraxial mesoderm and the lateral plate mesoderm form cartilage in
vivo. Therefore, we addressed whether the ES-cell-derived mesodermal cells
were also capable of forming cartilage. The sorted EB cell fractions were
subjected to the serum-free pellet culture with 10 ng/ml TGFß3, as
described for mesenchymal stem cells
(Mackay et al., 1998). 24
hours later, the cells pelleted in a tube at the beginning of the culture were
detached from the bottom to form a round cell aggregate. By day 14, areas that
stained light- to dark-blue with Alcian blue or light- to dark-red/purple with
Toluidine blue (metachromatic staining) became noticeable within the cell
particle, which is indicative of cartilage-like extracellular matrix
accumulation.
At frequencies of 96%, 87% and 65%
(Table 2), the
flk-1PDGFR+ cells,
flk-1+PDGFR
cells and DP cells,
respectively, formed a particle containing one to three cartilage nodules that
consisted of well separated, round cells and large intercellular spaces that
stained metachromatically with Toluidine blue
(Fig. 2, Fig. 3A). The other areas
contained loosely attached, spindle-shaped cells and showed weak to no
metachromatic staining with Toluidine blue. For further confirmation, the
contiguous sections were stained with an anti-COL1 antibody as well as an
anti-COL2 antibody (Fig. 3A).
Most of the areas that stained weakly with Toluidine blue expressed both COL1
and COL2, whereas some cartilage nodules that strongly stained with Toluidine
blue consisted predominantly of COL2 (Fig.
3A), suggesting that the particles were heterogeneous, in that
they consisted of hyaline cartilage nodules, fibro cartilage areas and
non-cartilaginous areas. On the other hand, the DN cells formed a large
particle, consisting mostly of a non-cartilaginous area that was not
metachromatically stained (Fig.
2G). The DN cell population was expected to be heterogeneous,
because APA5 is known to recognize part of the PDGFR
mRNA-expressing
mesoderm (Takakura et al.,
1997
).
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In conclusion, the pellet culture in the presence of TGFß3 allowed
fractions of ES-cell-derived mesodermal cells, such as
flk-1PDGFR+ cells,
flk-1+PDGFR
cells and DP cells, to form a
cartilage-nodule-containing particles.
Cartilage-specific gene expression during the pellet culture of the
ES-cell-derived mesodermal cells
For further validation, we investigated whether the in-vitro-formed
cartilage expressed cartilage-specific genes by nested RT-PCR
(Fig. 3B). Prior to the
induction of chondrogenesis (day 0), transcripts for aggrecan COMP, COL10 and
PTHrP were hardly detected in the
flk-1PDGFR+ and
flk-1+PDGFR
cell fractions, whereas the
COL2 transcript was readily detectable. Upon differentiation, all of them were
upregulated and became detectable by days 7 to 16. Interestingly, the CHL1
transcript, which is preferentially expressed in condensing mesenchymes as
well as mature chondrocytes of the developing skeleton
(Nakayama et al., 2001
), and
the PTHrP transcript were induced earlier (from day 3). Consistent with the
previous histological observations, these results supported the idea that the
ES-cell-derived mesodermal cells were capable of generating
cartilage-containing particles in vitro.
TGFß-dependent and TGFß-independent chondrogenic
activities
Without TGFß3, the efficiency of the initial aggregate formation was
significantly reduced (data not shown), and the size of the particles formed
became smaller (Fig. 2A,C,E).
Furthermore, in the case of flk-1+PDGFR
cells and DP cells, no area was stained metachromatically with Toluidine blue
within the particles (Fig.
2C,E). In contrast, in the case of the
flk-1PDGFR
+ cells, seven out of 10 small
particles formed contained one to two small cartilaginous nodules that stained
strongly with Toluidine blue (Fig.
2A and Table
2).
To quantify the TGFß-independent cartilage-forming activity, three
chondrogenic EB cells, flk-1PDGFR+ cells,
flk-1+PDGFR
cells and DP cells, were
individually subjected to a serum-free micromass culture. In the presence of
50 ng/ml PDGF-BB, the cells survived or slowly proliferated during the first 8
days, as observed with the somite cells
(Tallquist et al., 2000
).
Consistent with the results shown in Fig.
2, the addition of TGFß3 stimulated the accumulation of
sulfated glycosaminoglycan in all three cultures
(Fig. 4A). Furthermore, the
addition of TGFß3 induced COL2 accumulation during the culture of the
flk-1+PDGFR
cells and the DP cells
(Fig. 4Ca). The
flk-1PDGFR
+ cell culture that accumulated
low levels of COL2 without TGFß3 also displayed an approximately
three-fold increase in the COL2 level with TGFß3.
|
Without TGFß, however, approximately 79 small cartilage nodules that
stained positively with Alcian blue were detected per 2x105
flk-1PDGFR+ cells initially seeded
(Fig. 4B). Smaller cartilage
nodules were detected in the DP cell fraction at a lower frequency
(approximately 14 per 2x105 cells). No cartilage nodule was
found with the flk-1+PDGFR
cells
(Fig. 4B). Therefore, in
contrast to the lymphohematopoietic potential, the TGFß-independent
chondrogenic activity was highly enriched in the
flk-1PDGFR
+ EB cell fraction and was
absent from the flk-1+PDGFR
cell
fraction.
Thus, there seem to be two modes of chondrogenesis. In one type, exogenous
TGFß is absolutely essential (for
flk-1+PDGFR as well as DP mesodermal
cells) and in the other, chondrogenesis occurs as small, distinct nodules
without the exogenous TGFß, and the addition of TGFß expanded the
chondrogenic area (for flk-1PDGFR
+
cells).
PDGF-BB augments TGFß-induced chondrogenesis
TGFß alone was not sufficient to form a particle filled with the
cartilage matrix macromolecules (Figs
2,
3). Since PDGF-BB sustained the
viability of the ES-cell-derived mesodermal cells during the serum-free
micromass culture, we further investigated the effect of PDGF. The addition of
20 to 50 ng/ml PDGF-BB significantly elevated the COL2 accumulation during the
micromass culture of all three cell types tested in the presence of TGFß3
(Fig. 4Cb). Twenty to 100 ng/ml
of PDGF-AA did not significantly elevate the COL2 level.
On the other hand, the addition of 50 ng/ml PDGF-BB to the pellet culture
of flk-1PDGFR+ cells
(Fig. 5A),
flk-1+PDGFR
cells
(Fig. 5B) or DP cells (data not
shown) in the presence of TGFß3 markedly enlarged the particle volume as
well as the areas staining with the anti-COL2 antibody and metachromatically
with Toluidine blue, so that, except for a thin surface cell layer, the
cartilage matrix was distributed throughout the particle. We refer to such
particles as `full' cartilage in this report. The COL2+ interior of
the large `full' cartilage particle formed in the presence of TGFß3 and
PDGF-BB (Fig. 5Ad-f, 5Bj-l) was
weakly stained with the anti-COL1 antibody. However, a layer of cells on the
surface of the particle showed relatively high levels of COL1, and COL2 was
also detected at a level similar to that in the interior COL2+
area. Thus, even in the presence of PDGF-BB, the `full' cartilage was still
fibro cartilage.
In contrast, PDGF-BB treatment alone resulted in small particles with
either flk-1+PDGFR cells
(Fig. 5Bg-i) or DP cells (data
not shown) that were devoid of cartilage matrix. The TGFß-independent
chondrogenesis of the flk-1PDGFR
+ cells
was still observed in the presence of PDGF-BB alone
(Fig. 5Ba-c). However, a large
`full' cartilage particle was formed only with TGFß3
(Table 2).
Thus, PDGF-BB seems to be a synergistic factor for the TGFß-induced
chondrogenesis of the ES-cell-derived mesodermal cells, leading to `full'
cartilage particle formation. Further analyses were performed, mostly with two
interesting cell types that also efficiently formed `full' cartilage
(Table 2): the
hemo-chondrogenic flk-1+PDGFR cells (75%)
and the chondrogenic flk-1PDGFR
+ cells
(83%).
Inhibitory effect of noggin
The BMP family is also implicated in chondrogenesis
(Cancedda et al., 1995). To
determine whether TGFß-induced or TGFß+PDGF-induced chondrogenesis
involved BMP activity, we added a pan-BMP-binding inhibitor, noggin, to the
pellet culture. Noggin interacts with BMP2, BMP4 and BMP7, but does not bind
to Activin, TGFß1 (Zimmerman et al.,
1996
) and TGFß3 (data not shown).
When added at the beginning of the culture, noggin-Fc inhibited the TGFß-induced cartilage formation in a dose-dependent manner (Fig. 6; Table 2). One µg/ml of noggin-Fc was sufficient for complete inhibition of the formation of areas stained metachromatically with Toluidine blue, and 0.1 µg/ml produced weaker inhibitory effects (Fig. 6A). A similar inhibitory effect on the TGFß+PDGF-induced `full' cartilage formation was observed (Fig. 6B), in that the area immunostained with the anti-COL2 antibody was also abolished. Moreover, the noggin-Fc at 1 µg/ml significantly retarded the growth of particles formed either with TGFß3 alone or with TGFß3 and PDGF-BB, resulting in a small particle. At 0.1 µg/ml, no effect on the particular volume was observed.
When TGFß3 was removed on days 10 to 12, which otherwise would have resulted in a cartilage-nodule-containing particle, the addition of noggin-Fc at 1 µg/ml at this point prevented the formation of cartilaginous areas (0-20% of particles contained areas showing metachromatic staining) during the following 7 to 8 days (Fig. 7C; Table 3). An inhibitory effect on the particle growth was also noted.
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Thus, the BMP family of proteins seems to be involved in the chondrogenesis of the ES-cell-derived mesodermal cells, probably downstream of TGFß or TGFß+PDGF.
Synergistic enhancement of the TGFß-induced cartilage formation
by BMP4
The roles of BMP were further investigated. First, 50 ng/ml BMP4, added in
place of TGFß3 at the beginning of the culture, resulted in a small
particle without cartilage nodules, although it supported the initial
aggregate formation. Rather, BMP4 prevented the TGFß-independent
formation of the small cartilage nodules in the
flk-1PDGFR+-cell-derived particle
(Fig. 7Ab;
Table 2).
By contrast, the addition of 20 to 50 ng/ml BMP4 to the TGFß-containing culture markedly enhanced the depositions of cartilage matrix (Fig. 7B) and COL2 (data not shown), leading to `full' cartilage formation. The enhancement was dose dependent, and 5 ng/ml of BMP4 were not effective (Fig. 7Bb). The frequency of `full' cartilage formation reached 100% in the presence of TGFß3 and 50 ng/ml BMP4 (Table 2). A similar synergistic effect was observed even when TGFß3 was replaced with 50 ng/ml BMP4 after the cells were treated with TGFß3 for 10 to 12 days (Fig. 7C). This resulted in a large `full' cartilage particle filled with a cartilage matrix and COL2 at a frequency of 75-100% (Table 3) in the following 7 to 8 days of culture (Fig. 7B).
Thus, the exogenously added BMP4 synergistically enhanced the TGFß-induced cartilage formation from the ES-cell-derived mesodermal cells. However, TGFß treatment seemed to be pre-requisite for BMP4 to stimulate chondrogenesis.
Potential inhibitory effects of TGFß on chondrocyte
maturation
The cartilage nodules formed in the presence of TGFß3 stained fainter
with Toluidine blue than those formed in the absence of it
(Fig. 2A,B). This raised the
possibility that TGFß suppresses chondrocyte maturation at a later stage
of cartilage formation, as is the case for the embryonic limb mesenchymes.
Therefore, TGFß3 was removed on days 10 to 12 of culture, and the
resulting particles were examined on days 17 to 20
(Fig. 7C). Under these
conditions, the flk-1PDGFR+ cells gave
rise to particles containing a larger cartilaginous area that consisted of
larger chondrocytes and displayed stronger metachromatic staining with
Toluidine blue. The `full' cartilage was also obtained at a frequency of 26%
(Table 3). The
TGFß+PDGF-induced chondrogenic culture gave rise to a `full' cartilage
particle in 17 to 20 days (Fig.
5). When both TGFß3 and PDGF-BB were removed on days 10 to
12, no increase in either the COL2 level or the strength of Toluidine blue
staining was noted on days 17 to 20. However, an increase in the size of the
chondrocytes and a reduction in the COL1 level in the cartilaginous area were
apparent (Fig. 8A).
These observations suggest that, in a later phase of the chondrogenic culture of the ES-cell-derived mesodermal cells, the continuous presence of TGFß3 prevented or retarded the chondrocyte maturation and, even in the presence of PDGF-BB, maintained the COL1 expression within the cartilage matrix, thereby preventing the hyaline cartilage formation.
Hyaline cartilage formation and induction of mineralization
When TGFß3 and PDGF-BB were removed, a distinct, thin layer of cells
that strongly stained with the anti-COL1 antibody, but did not stain with
either Toluidine blue or the anti-COL2 antibody, became apparent on the
surface of a particle (Fig.
7Cc, Fig. 8Ad-f).
It was composed of spindle-shape cells resembling undifferentiated mesenchymal
cells. Upon the addition of 50 ng/ml BMP4, the thin cell layer became a
thicker layer containing round chondrocytic cells and a matrix that was rich
in COL2 and stained lightly with Toluidine blue
(Fig. 7Cd,
Fig. 8Ag,i). Moreover, BMP
treatment resulted in a reduced level of COL1 in the surface cell layer as
well as in the interior cartilaginous area
(Fig. 8Ah), resulting in a
hyaline cartilage particle. A similar thick layer that stained lightly with
Toluidine blue was evident when the cells were cultured from the beginning
with TGFß3 and 20-50 ng/ml BMP4 (Fig.
7Bc,d,f).
The hyaline cartilage particles formed by culturing the
flk-1PDGFR+ cells or the
flk-1+PDGFR
cells with TGFß3 and
PDGF-BB for 10 days, and with BMP4 alone for the following 6 days, were
further tested for mineralization. In the presence of T3 and
b-glycerophosphate an area in which the intercellular space was stained with
von Kossa became apparent within the particle after 5 days of culture,
indicating that mineral deposits in the cartilage matrix were induced
(Fig. 8B). The anti-COL10
antibody detected COL10 deposition as a larger area, in which the mineralizing
matrix was included and a lower level of COL2 was accumulated.
These observations indicate that the ES-cell-derived mesodermal cells are able to form a hyaline cartilage particle and that the hyaline cartilage generated with the ES-cell-derived mesodermal cells mature and mineralize the matrix. They also suggest that the COL1+ mesenchymal cells in the surface layer could be BMP-sensitive chondroprogenitors that serve as the source of chondrocytes during the growth of the cartilage particle, which is equivalent to the perichondrium.
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Discussion |
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In vitro chondrogenesis of the ES-cell-derived mesodermal cells as a
faithful culture model for chondrogenesis
First, to address whether chondrogenesis was achieved through a
developmental pathway similar to that in vivo, we examined the roles of three
factors that are known to be involved in embryonic chondrogenesis: TGFß,
PDGF and BMP.
TGFß and BMP are implicated in several steps during limb cartilage
development, and their effects are both positive and negative, depending on
the developmental stage. For example, TGFß has an early positive function
and a late negative function. Our pellet culture of either
flk-1+PDGFR or
flk-1PDGFR
+ EB cells produced similar
TGFß effects: (1) supporting aggregate growth and formation of
cartilaginous areas within the particle generated (Figs
2,
5) and (2) inhibiting of the
generation of hypertrophic chondrocytes and the formation of hyaline
cartilage, both of which were induced by removing TGFß later
(Fig. 7C, Fig. 8A). However, the
detection of the COL10 transcript by PCR suggested that some degree of
hypertrophic differentiation might occur in the presence of TGFß
(Fig. 3). BMP is also involved
in multiple steps of chondrogenesis. We provided evidence that (1) BMP is
involved in TGFß-induced as well as TGFß+PDGF-induced chondrogenesis
(Fig. 6, Fig. 7C), (2) exogenous BMP
enhanced the cartilage matrix deposition in the TGFß-treated particles,
leading to a dramatic increase in the volume of the cartilage particles
(Fig. 7), and (3) BMP
suppressed the COL1 level and elevated COL2 expression, resulting in a hyaline
cartilage particle (Fig. 8A), thus supporting the positive roles of BMP. It is worth mentioning that no
significant changes in mineral deposition within such hyaline cartilage
particles were observed after 5 days of hypertrophic differentiation induced
with or without 100 ng/ml BMP6, although COL10 accumulation was enhanced by
BMP6 (data not shown).
Despite the positive effects of BMP4 at a late stage of chondrogenesis
culture, BMP4 added alone from the beginning was rather detrimental
(Fig. 7A), which implies that
chondrogenesis of the ES-cell-derived mesodermal cells occurs through two
steps requiring different proteins: TGFß followed by BMP. This two-step
model agrees with the previous notion that, during the micromass culture of
chicken limb mesenchymal cells, a stimulatory effect of BMP4 is observed 24-48
hours after culture, whereas that of TGFß3 was readily detectable from
the beginning of the culture (Roark and
Greer, 1994).
PDGF-BB also has striking synergy with TGFß and forms a large `full'
cartilage particle (Fig. 5)
through BMP action (Fig. 6B).
Therefore, the role of PDGF may also be placed upstream of that of BMP. The
direct effect of PDGF on chondrogenesis has been demonstrated with primary
mouse somitic cells (Tallquist et al.,
2000). PDGF-BB, and to a lesser degree PDGF-AA, increased the
accumulation of Alcian-blue-positive cartilage matrix in the absence of
TGFß without stimulating cell proliferation. Enhancement of COL2
accumulation by PDGF-BB but not by PDGF-AA
(Fig. 4C) is consistent with
this observation, although our experiment was done in the presence of
TGFß3.
Thus, as judged by the effects of TGFß, BMP and PDGF, the serum-free chondrogenesis culture using isolated ES-cell-derived mesodermal cells seems to reproduce some, if not all, aspects of the in vivo process.
The ES-cell-derived PDGFR+ mesodermal cells are
chondrogenic but not hemogenic
The TGFß-independent chondrogenic activity was highly enriched in the
flk-1PDGFR+ EB cell fraction
(Fig. 4), although no
lymphohematopoietic cell potential was detected
(Fig. 1), suggesting that the
flk-1PDGFR
+ cells are committed to
non-hematopoietic cell lineages, including the chondrocyte lineage. This is
consistent with the notion that the PDGFR
signaling is essential for
normal axial skeletogenesis (Soriano,
1997
) and that PDGFR
is highly expressed in the somites and
later in the sclerotomal mesenchymes, the condensing limb mesenchymes and the
perichondrium, which are considered to be non-hematopoietic, chondrogenic
cells (Orr-Urtreger et al.,
1992
; Schatteman et al.,
1992
).
The biological significance of the two types of chondrogenic activities,
the TGFß-independent activity and the TGF-dependent activity, is not
clear. The flk-1PDGFR+ EB cell fraction
may contain TGFß-producing cells or mature chondroprogenitors that no
longer require TGFß to form cartilage nodules. Candidates for the latter
are the PDGFR
+ sclerotomal mesenchymes and the limb
mesenchymes. Nevertheless, we have concluded that the PDGFR
+
EB cell fraction contains chondrogenic mesoderm.
The ES-cell-derived flk-1+ mesodermal cells containing the
hemangioblasts are also chondrogenic
The ES-cell-derived flk-1+ hemangioblast is characterized by its
ability to form a blast cell colony in the presence of VEGF and to generate
hematopoietic CFCs (Faloon et al.,
2000; Kabrun et al.,
1997
; Nishikawa et al.,
1998a
). In this respect,
flk-1+PDGFR
is likely to be the
hemangioblast fraction, because it was enriched for CFC-generating activity
(Fig. 1B), whereas no
spontaneous chondrogenic activity was detected in it (Figs
2,
4,
5). The TGFß-dependent
chondrogenic activity of the flk-1+PDGFR
cells thus implies that the hemangioblast may be multi-potential at this stage
of development. The observations that the ES-cell-derived flk-1+
cells produce vascular smooth muscle-like cells in the presence of PDGF-BB
(Yamashita et al., 2000
), and
that a purer preparation of hemangioblasts
(flk-1+VE-cadherin+PDGFR
cells)
was also chondrogenic in the presence of TGFß3 and BMP4 (data not shown),
supported this possibility.
Another possibility is that the
flk-1+PDGFR cell fraction is a
heterogeneous mixture of cell types that are differentially committed to
either the hematopoietic/endothelial cell lineages or the chondrogenic cell
lineage. However, the result that
flk-1PDGFR
+ cells, DP cells and
flk-1+PDGFR
cells accumulated similar
levels of COL2 during 8 days of micromass culture in the presence of
TGFß3 and PDGF-BB (Fig.
4C) would argue against the possibility that cross contamination
by neighboring cells, such as DP cells, accounts for the TGFß-dependent
cartilage forming activity of the
flk-1+PDGFR
cells.
An appropriate expansion culture method may have to be established to answer these questions definitively and is currently under way.
In vitro chondrogenesis in the whole EB and with the ES-cell-derived
mesodermal cells
Kramer et al. demonstrated that chondrogenesis can be performed directly
within an EB (Kramer et al.,
2000). The culture medium contained 15% FCS, unlike ours, and a
high concentration of BMP (100 ng/ml) stimulated chondrogenesis when added
within the first 5 days of culture. In contrast, TGFß displayed either no
effect or an inhibitory effect. These results are inconsistent with our
observation that chondrogenesis with the FACS-purified mesodermal cells from
day 3.6-4.6 EBs either required TGFß or was augmented by TGFß
(Fig. 2) and that the effect of
BMP4 (50 ng/ml) was observed even after 10 days of the pellet culture
(Fig. 7C;
Table 3). The discrepancy can
be explained by the serum effect as well as by the difference in the cell
populations used. For example, the requirement of TGFß in our culture
condition may be caused by the removal of TGFß-producing cells and/or a
TGFß-inducing factor, FCS (Mummery
and van den Eijnden-van Raaij, 1999
). Nevertheless, as judged by
the temporal order of the factor requirements, the serum-free chondrogenesis
culture using isolated ES-cell-derived mesodermal cells seems to represent the
in vivo process better. Furthermore, considering the heterogeneity in the DN
cell population (Fig. 2G),
macroscopic cartilage containing a homogeneous population of chondrocytes
would be formed easier with isolated mesodermal cells.
By taking advantage of the ease of obtaining large numbers of purified mesodermal cells, the ES cell differentiation culture described here would provide an important research platform for molecular and cellular dissections of the chondrogenesis pathway in the context of macroscopic cartilage. This method may lead to a better strategy for cartilage repair and cartilage engineering. Applications to the human ES cells may provide an important tissue source for cartilage replacement/repair therapy.
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Acknowledgments |
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