1 Department of Medical Molecular Biology, Medical University of Lübeck,
D-23538 Lübeck, Germany
2 In Vitro Differentiation Group, IPK Gatersleben, D-06466 Gatersleben,
Germany
* Author for correspondence (e-mail: rohwedel{at}molbio.mu-luebeck.de)
Accepted 11 September 2002
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Summary |
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Key words: Mouse embryonic stem cells, Chondrogenesis, Osteogenesis, Mesenchymal cells, Dedifferentiation, Redifferentiation
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Introduction |
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Differentiation processes of chondrogenic cells can be studied by in vitro
differentiation of mouse embryonic stem (ES) cells
(Kramer et al., 2000). In
vitro differentiation of ES cells closely recapitulates early embryonic
developmental processes, and this system has been used to study
differentiation of many cell types, such as cardiogenic, skeletal muscle,
haematopoietic, neurogenic, epithelial, vascular smooth muscle, adipogenic and
chondrogenic cells (reviewed by Keller,
1995
; Rathjen et al.,
1998
; Guan et al.,
1999
; Wobus et al.,
2001
). This model system offers the possibility of tracing
differentiation from the pluripotent stem cell to terminally differentiated
cell types. ES cells have the potential to develop into cells of all three
primary germ layers owing to their origin from the inner cell mass of
blastocysts (Evans and Kaufman,
1981
; Martin,
1981
). ES cell differentiation has also been discussed as an
experimental avenue to generate cells for transplantation. ES-cell-derived
cardiomyocytes, neuronal cells and insulin-secreting cells have been
transplanted into animals and were able to integrate specifically
(Klug et al., 1996
;
Brüstle et al., 1999
;
McDonald et al., 1999
;
Arnhold et al., 2000
;
Liu et al., 2000
;
Soria et al., 2000
). Because
ES cells differentiate spontaneously into a heterogeneous population of cells,
it is necessary to use selection strategies to obtain pure cultures of a
specific cell type. However, it is questionable how pure these populations
are, because they originate from pluripotent and developing cells and
therefore may contain cells of different developmental states.
Here, we used the ES cell model to study terminal differentiation of chondrocytes and to analyze their differentiation plasticity. We show that chondrocytes differentiated from mouse ES cells via embryoid bodies (EBs) progress further into hypertrophic chondrocytes, which later expressed markers of calcifying cells and present evidence that ES-derived chondrogenic cells continue to display a particular differentiation plasticity.
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Materials and Methods |
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Experiments were done at least in triplicate, and data analysis was performed using the Sigma Plot 5.0 software (Jandel, Corte Madeira, CA). For statistical analysis the Student's t-test was used.
Isolation and differentiation analysis of chondrocytes
For isolation of chondrogenic cells, EBs were cultivated as described
above. Chondrogenic cells developed in dense, matrix-bordered areas called
nodules. These nodules were cut off the EB outgrowths with a microscalpel
under sterile conditions and collected for collagenase (0.1%) treatment for 50
minutes at 37°C to obtain single cell suspensions. Cells were centrifuged
for 5 minutes at 180 g, resuspended in differentiation medium
and plated at high density onto gelatin- or collagen-II- (SIGMA, Taufkirchen,
FRG) coated 6 cm tissue culture plates (at a density of
1-2x105 cells) or two-well (21.3x20 mm/well) Lab Tek
chamber slides (at a density of 2.1x104 cells/well) for total
RNA isolation, indirect immunostaining and Sudan III-staining,
respectively.
To study the effect of the growth factor TGF-ß3 on isolated
chondrogenic cells, either 10 or 50 ng/ml of recombinant human TGF-ß3
(R&D SYSTEMS, Wiesbaden, Germany), respectively, was added to the
differentiation medium after replating of isolated chondrogenic cells derived
from EBs, and differentiation was compared to control cultures without the
growth factor. The concentration of TGF-ß3 was in the range previously
shown to induce chondrogenesis in cultures of human mesenchymal stem cells
(Yoo et al., 1998;
Mackay et al., 1998
;
Pittenger et al., 1999
).
For clonal analysis of differentiation, 20, 200, 400, 1000 and 2000 chondrogenic cells isolated from EBs were plated onto each well of a four-well (20x8.5 mm/well) gelatin-coated Lab Tek chamber slide (Nunc, Wiesbaden, Germany), cultivated for 15 to 24 days and analyzed by immunostaining and Sudan III staining, respectively.
To study their differentiation capacity after replating, cell samples were isolated from chondrocyte cultures by trypsinization using 8x8 mm cloning cylinders (SIGMA, Taufkirchen, Germany). The cells were plated onto two-well (21.3x20 mm/well) gelatin-coated Lab Tek chamber slides (Nunc, Wiesbaden, Germany), cultivated for 18 to 24 days and analyzed by indirect immunostaining and Sudan III staining, respectively.
Sudan III staining
To detect adipocytic cells, the lipid stain Sudan III was used. Cells
plated onto Labtek chamber slides were washed with PBS followed by staining
for 3 minutes with a 0.2% solution of Sudan III (Sigma) in 70% ethanol.
Cryosections of isolated chondrogenic nodules
Chondrogenic nodules were isolated (see above), embedded in Tissue-Tek
O.C.T. (Sakura Finetechnical, Tokyo, Japan) and frozen at -20°C.
Cryosections (10 µm) were prepared using a cryostat (Leica, Bensheim,
Germany) and placed onto Vectabond-coated slides. Sections were air-dryed,
fixed in acetone for 10 minutes at -20°C and washed in PBS. Indirect
immunostaining was performed after fixation as described below.
Detection of gene expression by semi-quantitative RT-PCR
analysis
EBs (n=10) or isolated chondrocytes grown on 6 cm tissue culture
plates were collected at different time points after plating, washed twice
with phosphate-buffered saline (PBS), and total RNA was isolated using the
RNeasy Mini-Kit (Qiagen, Hilden, Germany). The RNA concentrations were
determined by measuring the absorbance at 260 nm. Samples of 500 ng of RNA
were reverse transcribed using oligo-dT primer and Superscript II reverse
transcriptase following the manufacturers recommendations (Life Technologies,
Eggenstein, Germany).
Aliquots of 1 µl from the reverse transcriptase reactions were used for
amplification of transcripts using primers specific for the analyzed genes and
Vent DNA polymerase (New England Biolabs, Schwalbach, Germany) according to
the manufacturer's instructions. Reverse transcriptase reactions were
denatured for 2 minutes at 95°C, followed by 34-45 cycles of 40 seconds
denaturation at 95°C, 40 seconds annealing at the primer-specific
temperature (see below) and 50 seconds elongation at 72°C. Expression of
the following genes was studied (oligonucleotide sequences are given in
brackets in the order antisense-, sense-primer followed by the annealing
temperature used for PCR, length of the amplified fragment and a reference):
genes encoding collagen II [5'-AGGGGTACCAGGTTCTCCATC-3',
5'-CTGCTCATCGCCGCGGTCCTA-3', 60°C, 432 bp (splice variant A)
and 225 bp (splice variant B)
(Metsäranta et al.,
1991)], collagen X [5'-ATGCCTTGTTCTCCTCTTACTGGA-3',
5'-CTTTCTGCTGCTAATGTTCTTGACC-3', 61°C, 164 bp
(Elima et al., 1993
)],
osteocalcin [5'-ATGCTACTGGACGCTGGAGGGT-3',
5'-GCGGTCTTCAAGCCATACTGGTC-3', 64°C, 330 bp
(Desbois et al., 1994
)],
Cbfa-1 [5'-ATCCATCCACTCCACCACGC-3',
5'-AAGGGTCCACTCTGGCTTTGG-3', 63°C, 371bp
(Ducy et al., 1997
)] and the
`house-keeping' gene hypoxanthine guanine phosphoribosyl transferase
[HPRT, 5'-GCCTGTATCCAACACTTCG-3',
5'-AGCGTCGTGATTAGCGATG-3', 63°C, 507 bp
(Konecki et al., 1982
)]. The
latter was used as an internal standard. Electrophoretic separation of PCR
products was carried out on 2% agarose gels. The fragments were analyzed by
computer-assisted densitometry in relation to HPRT gene expression.
RNA from limb buds or limbs of 16 day p.c. old mouse embryos were used as
positive controls. Distilled water was always included as a negative
control.
Indirect immunostaining
EBs or chondrogenic cells cultivated on chamber slides and rinsed twice
with PBS were fixed for 5 minutes with methanol:acetone (7:3) at room
temperature, washed three times with PBS again and incubated at 37°C for
30 minutes with 10% goat serum. Specimens were then incubated for 1 hour with
the first antibody in a humidified chamber at 37°C. The following
monoclonal antibodies (mAbs) diluted in PBS were obtained from the
Developmental Studies Hybridoma Bank, University of Iowa, USA (designation of
the mAb, the dilution used and a reference are given in brackets): Collagen II
[II-II-6B3, 1:20 (Linsenmayer and Hendrix,
1980)], osteopontin [MPIIIB 101,1:10
(Dorheim et al., 1993
)],
collagen X [X-AC9, 1:20 (Schmid and
Linsenmayer, 1985
)] and bone sialoprotein I + II [WVID1(9C5), 1:10
(Dorheim et al., 1993
)]. To
detect cytokeratins, anti-pan cytokeratin, diluted 1:100 (Sigma), for
immunostaining of sarcomeric
-actinin mAb EA-53 (Sigma) diluted 1:20
and for collagen I, a polyclonal antiserum (Chemicon, Temecula, CA) diluted
1:100 were used. After rinsing three times with PBS, slides were incubated for
45 minutes at 37°C with either FITC- (1:200) or Cy3- (1:400) labeled
anti-mouse IgG (Dianova, Hamburg, Germany), respectively. Slides were washed
three times in PBS and briefly washed in distilled water. Specimens were
embedded in Vectashield mounting medium (Vector, Burlinggame, CA) and analyzed
with the fluorescence microscope Axioskop (Zeiss, Oberkochen, Germany).
Fluorescence in situ hybridization for collagen X mRNA
For fluorescence in situ hybridization of collagen X mRNA, a
modified procedure of Yamada et al.
(Yamada et al., 1994) was
used. EBs (n=10) plated on chamber slides were rinsed twice with PBS
and fixed with 4% (w/v) paraformaldehyde, 4% (w/v) sucrose in PBS for 20
minutes at room temperature. Prior to incubation at 70°C in 2xSSC
for 15 minutes, cells were washed twice with PBS for 5 minutes. After rinsing
once again with PBS followed by 2xSSC, the EBs were fixed for 5 minutes
before washing with PBS and 2xSSC were repeated. The cells were
subsequently dehydrated at room temperature for 2 minutes each in 50%, 70%,
95% and twice in 100% ethanol. Prehybridization was performed in a buffer
containing 5xSSC, 5xDenhardt's, 50% formamide, 250 µg/ml
yeast-t-RNA, 250 µg/ml denatured salmon sperm DNA and 4 mM EDTA in a
humidified chamber at 45°C for 3 hours. For hybridization with
digoxigenin-labeled sense and antisense probes against collagen X (1
ng/µl) the same buffer but without salmon sperm DNA was used. Hybridization
was carried out at 45°C in a humidified chamber overnight. After
hybridization, specimens were washed twice with 2xSSC for 15 minutes,
once with 0.2xSSC for 15 minutes and twice with 0.1xSSC for 15
minutes at 45°C and rinsed in PBS. FITC-conjugated sheep F(ab) fragments
against digoxigenin (Boehringer, Mannheim, Germany) diluted 1:800 in PBS were
added. After incubation for 1 hour at 37°C, slides were washed three times
in PBS and once in distilled water, embedded in Vectashield mounting medium
and analyzed with the fluorescence microscope Axioskop (ZEISS, Oberkochen,
Germany).
Hybridization probe
To obtain a hybridization probe for RNA in situ hybridization, we amplified
a fragment of collagen X cDNA from RNA isolated from 16 day p.c.
mouse limb buds following the protocol of RT-PCR as described above. The
fragment was blunt-end cloned into the plasmid vector pCR-BluntII-TOPO using
the TOPO II cloning kit according to the manufacturers protocol (Invitrogen,
Groningen, NL) and the sequence was verified by sequencing.
Digoxigenin-labeled RNA probes of either sense or antisense orientation of
collagen X were synthesized from linearized plasmids of the cloned
collagen X cDNA fragment by in vitro transcription using the T7- or
SP6-RNA polymerase following the protocol supplied by the manufacturer
(Boehringer, Mannheim, Germany).
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Results |
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The disappearance of Alcian blue staining was accompanied by differential expression of marker genes, which are indicative of hypertrophic and/or calcifying chondrocytes as demonstrated by semi-quantitative RT-PCR analysis (Fig. 2). Transcription of the prechondrogenic splice variant of collagen II (collagen IIA) was detected throughout EB cultivation and the level did not change significantly over time (Fig. 2A), whereas mRNA transcription level of the adult or chondrocyte-specific splice variant of collagen II (collagen IIB) increased significantly (t-test=0.0002) from 5+7 d to 5+17 d (Fig. 2A). The gene encoding collagen X, specifically upregulated during chondrocyte hypertrophy in vivo, was expressed throughout EB cultivation, although a significant (t-test=0.02) increase of the mRNA level was detected from day 5+7 to 5+17 (Fig. 2B). The osteoblast-specific osteocalcin mRNA showed a significant (t-test=0.004) shift from 5+10 d to 5+22 d and thereafter was continuously transcribed on a high level up to 5+30 d (Fig. 2C). The mRNA of the osteoblast-related transcription factor Cbfa-1 was present in substantial amounts at the day of EB plating and, except for two phases of downregulation at 5+4 d and 5+22 d, showed an overall increase up to 5+30 d (Fig. 2D).
|
To demonstrate differentiation at the cellular level we performed RNA in situ hybridization and immunostaining for marker molecules of hypertrophic and calcifying chondrocytes as well as osteogenic cells (Fig. 3). Collagen X mRNA as well as the matrix proteins osteopontin and bone sialoprotein were detected in nodules of 5+16 d to 5+30 d EB outgrowths (Fig. 3A,C,E). Besides these positively stained cells organized in nodules we also found clusters of single cells expressing these marker (Fig. 3B,D,F). Apparently, two types of osteogenic cells appeared in EB outgrowths: (i) osteogenic cells derived from hypertrophic and calcifying chondrocytes organized in nodules and (ii) a second population differentiating as single cell cluster.
|
Chondrocytes isolated from EBs dedifferentiated but re-express
chondrogenic markers
To test whether chondrocytes isolated from ES-cell-derived nodules are
phenotypically stable in culture, nodules were cut from 5+16 d EB outgrowths
using a microscalpel, dissociated into single cells using collagenase and
replated. Because the nodules were surrounded by a rigid membraneous structure
of extracellular matrix proteins, intact nodules could be isolated, virtually
avoiding contamination by neighboring cells. As a test for the developmental
stage, cryosections from isolated nodules were immunostained for chondrocyte
marker proteins. The isolated nodules showed expression of collagen II,
osteopontin and bone sialoprotein I and II and collagen X
(Fig. 4A-D), and cells inside
of the nodules showed the typical round-shaped morphology of hypertrophic
chondrocytes (Fig. 4E-H).
Single cell suspensions obtained from such nodules by dissociation with
collagenase were plated onto gelatin- and collagen-II-coated dishes to test
whether different substrates may influence the differentiation characteristics
and marker gene expression profiles of the cells in culture. During
cultivation, the expression of chondrogenic differentiation markers was
analyzed using semi-quantitative RT-PCR and immunostaining
(Fig. 5). mRNA levels of
late-phase marker genes such as the adult splice variant of collagen
II (collagen IIB) and collagen X were significantly
downregulated during the first four days after isolation of chondrogenic cells
from EBs (Fig. 5IA,B). However,
two weeks after isolation, the transcriptional levels of collagen IIB
and collagen X increased again
(Fig. 5IA,B) indicating
redifferentiation of chondrocytes. Freshly isolated cells showed a
fibroblast-like morphology (Fig.
5IIA) and poor expression of collagen II
(Fig. 5IIB), whereas expression
of collagen I, characteristically expressed in dedifferentiated chondrocytes
was prominent (Fig. 5IIC). When
cultivated for four days, the dedifferentiated cells formed monolayers of
fibroblastoid cells including areas of compact and distinct cellular entities
(Fig. 5IID) consisting of cells
positively stained for collagen II (Fig.
5IIE), whereas collagen I expression decreased
(Fig. 5IIF). During further
cultivation the size of these cell formations increased and they developed
into dense aggregates (Fig.
5IIG) expressing collagen II
(Fig. 5IIH) but not collagen I
(Fig. 5III). Together, these
results are in line with the observation that the isolated chondrogenic cells
initially undergo transient dedifferentiation and later redifferentiate into
mature chondrocytes.
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|
Multilineage differentiation of ES cell-derived chondrocytes
The majority of the cells in cultures derived from chondrocyte nodules
redifferentiated into chondrogenic condensations. After prolonged cultivation
times, up to 14-20 days, occasionally other cell types appeared. Cells
carrying large lipid droplets were found. Staining with Sudan III revealed
that these cells were adipogenic cells
(Fig. 6A). Moreover, muscle
cells staining positively for sarcomeric -actinin
(Fig. 6B), and large flattened
cells staining positive for pan cytokeratins characteristic of epithelial
cells (Fig. 6C) were detected.
These additional cell types formed colonies. The number of these colonies and
the amount of cells were low in comparison to the number of chondrogenic
condensations and the number of chondrocytes, respectively. Representative
samples of approximately 2.1x104 cells plated to form a cell
monolayer and analyzed after 14 days of culture contained (per 1000 cells) 9.1
chondrogenic condensations of 50-60 chondrocytes, 0.67 colonies of 20-40
epithelial cells, 0.43 colonies of 3-6 skeletal muscle cells and 0.19 colonies
of 5-30 adipocytes, representing 51.4%, 2.3%, 0.29% and 0.25%, respectively,
of the analysed cells (Table
1). The rest of the cells showed a fibroblastic morphology.
|
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To exclude the possibility that the observed mesenchymal cell types originated from contaminating progenitor cells, two different approaches were used. First, various areas of the redifferentiated cell monolayers were isolated using cloning cylinders and again tested for differentiation, and second, single clones of cells isolated from nodules 16 days after EB plating were analyzed. Cells recovered from the redifferentiated monolayers after cultivation for up to 18 days still showed a fibroblastic morphology and we were not able to detect any differentiated cell by immunostaining for collagen II, sarcomeric actinin or cytokeratins. After 24 days of cultivation, differentiating cell types appeared. In seven samples of 8x103 cells the majority of the cells (94.76%) still appeared to be fibroblastoid but we found per 1000 cells 2.37 chondrogenic condensations of 10-30 chondrocytes, 0.75 colonies of 1-10 epithelial cells, 0.62 colonies of 2-4 skeletal muscle cells and 4.12 colonies of 2-3 adipocytes, representing 3.6%, 0.45%, 0.19% and 1%, respectively, of the analysed cells (Table 2). Four samples showed differentiation into all analyzed cell types. The remaining three samples showed poor differentiation into chondrocytes, skeletal muscle cells and adipocytes or epithelial cells and adipocytes or no differentiation at all. These results show that the capacity of the isolated dedifferentiated chondrocytic cells for redifferentiation was remarkably reduced after replating, but other cell types were still observed in the cultures.
|
We analyzed single clones by plating diluted suspensions of cells isolated from nodules 16 days after EB plating. Cells (n=20, 200, 400, 1000 and 2000) were plated onto chamber slides generating up to 55 clones per slide. After 15 days in culture, most of the cells appeared to be morphologically fibroblastoid. Histo- and immunochemical staining showed that only rarely could some single differentiated cells be found in some of the clones. 24 days after cell plating more differentiated cells appeared. Among 422 clones of approximately 1-3x102 cells, we did not find any clone that was entirely composed of a single differentiated cell type as judged by their morphology. Histochemical analysis and immunostaining showed that almost 90% of the clones analyzed by Sudan III staining contained adipocytes and approximately one third of those immunostained for collagen II carried chondrocytes, whereas skeletal muscle cells and particularly epithelial cells were only rarely found (Table 3). The number of differentiated cells per positive clone was very low with mean values from 32 chondrocytes to 1.6 epithelial cells. In total, a number of 2627 differentiated cells was found, from which 49% were collagen-II-positive chondrocytes, 48% Sudan-III-positive adipocytes, 2.7% sarcomeric actinin-positive skeletal muscle cells and only 0.3% cytokeratin-positive epithelial cells.
|
TGF-ß3 inhibited chondrogenic differentiation in chondrocyte
cultures
TGF-ß3 is able to promote chondrogenic differentiation in cultures of
human mesenchymal stem cells (Mackay et
al., 1998). We therefore analyzed a potentially similar effect in
our mouse system by application of TGF-ß3 (10 ng/ml and 50 ng/ml) to
cultures of chondrogenic cells isolated from EBs. For both experimental
conditions, semi-quantitative RT-PCR analysis showed that at day 8 and even
more prominently at day 14 after replating, chondrogenic cells treated with
TGF-ß3 still showed signs of dedifferentiation in contrast to untreated
control cultures. A significant decrease in the mRNA levels of the gene
encoding the prechondrogenic collagen II splice variant
(Fig. 7A) and the late-phase
marker genes collagen IIB (Fig.
7B), collagen X (Fig.
7C) and osteocalcin
(Fig. 7D) was observed in
TGF-ß3 treated cultures at day 14 after replating in comparison to
untreated control cells. This effect was found in the presence of 10 ng/ml
TGF-ß3 as well as 50 ng/ml, the latter being somewhat stronger.
|
Immunostaining for collagen I as a marker of dedifferentiation and collagen II as a marker of differentiated chondrocytes confirmed that the TGF-ß3-treated cells continued to display a fibroblast-like morphology and did not recover a chondrogenic phenotype 8 days after replating (Fig. 8D,G). The cells lost expression of collagen II (Fig. 8E,H) but showed prominent collagen I expression (Fig. 8F,I). By contrast, untreated cultures formed chondrogenic condensations (Fig. 8A), expressed collagen II (Fig. 8B) and showed only trace amounts of collagen I (Fig. 8C).
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Discussion |
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|
It has been reported that differentiation of chondrocytes into osteoblasts
occurs in vivo and in vitro (Moskalewski
and Malejczyk, 1989; Descalzi
et al., 1992
; Ishizeki et al.,
1997
). This hypothesis is in accordance with our results
demonstrating that hypertrophic chondrocytes organized in nodules start to
express osteogenic markers. In line with our results it was shown recently
that ES cells are able to differentiate into bone nodules in vitro
(Buttery et al., 2001
).
Interestingly, besides those osteogenic cells differentiated in nodules, we
observed clusters of single cells expressing osteogenic markers. It is
conceivable that these cells are derived from an alternative osteogenic
lineage of cells. We tested for the expression of osteocalcin, an
osteoblast-specific marker (Hauschka and
Wians, 1989
; Ducy et al.,
1996
) and found that osteocalcin was already upregulated
10 days after EB plating, which is earlier than collagen X, the
marker of hypertrophic chondrocytes. This indicates that at least some
osteoblasts may have bypassed the chondrocytic state
(Fig. 9). The hypothesis that
ES-cell-derived osteoblasts are differentiated from two different types of
precursors agrees with the in vivo situation where skeletal elements are
formed either by replacing a cartilaginous template as most bones of the
skeleton or directly from mesenchyme such as the flat bones of the skull
(Erlebacher et al., 1995
).
Differentiation plasticity of isolated chondrocytes
Dedifferentiation of primary cultures of chondrocytes isolated from mature
cartilage is a well known process (von der
Mark et al., 1977) that can be repressed by cultivation of
chondrocytes in suspension, at high density or in agarose cultures
(Castagnola et al., 1986
;
Bruckner et al., 1989
). We
prepared chondrocytes from EB nodules to test how these cells behave after
release from their EB environment. In tissue culture, after initial
dedifferentiation they showed rapid redifferentiation, indicating that they
possess a high potential for regeneration. However, additional mesenchymal
cell types such as adipocytes, epithelial cells and muscle cells appeared in
the cultures. This may be explained either by the presence of contaminating
mesenchymal progenitor cells in the chondrocyte cultures or by
transdifferentiation of chondrocytes. The fact that we did not obtain clones
of a single discrete cell type from the isolated chondrocytes excludes the
possibility that the additional cell types were derived from distinct
precursor cells. The additional cell types could, however, still be derived
from mesenchymal progenitor cells, which display a broader differentiation
capacity. It is well known that such progenitor cells are able to
differentiate at least into chondrocytes, osteocytes and adipocytes
(Berry et al., 1992
;
Pittenger et al., 1999
;
Muraglia et al., 2000
;
Dennis et al., 2002
). They
share these characteristics with other cells or cell lines derived from
mesenchymal tissues such as 10T1/2 (Taylor
and Jones, 1979
), RCJ 3.1
(Grigoriadis et al., 1988
) or
human trabecular bone-derived cells
(Sottile et al., 2002
). The
cell lines 10T1/2 and RCJ 3.1 in addition are also capable of differentiating
into myocytes. Recently it has been shown that clones of mesenchymal
progenitor cells differentiate into a mixture of mesenchymal cell types
(Muraglia et al., 2000
). If
such mesenchymal progenitor cells were present as a contamination in the
chondrocyte cultures derived from the EBs we would expect a majority of the
clones to redifferentiate into chondrocytes and rarely into clones with
additional cell types. We found that adipocytes differentiated in most of the
clones, whereas epithelial cells and myocytes were observed to only
differentiate to a minor extent. This indicates that the adipocytes, at least,
were not derived from contaminating mesenchymal stem cells. Furthermore, the
chondrocytic cells isolated from EBs did not exhibit characteristics of
mesenchymal progenitor cells such as expression of the stromal cell marker
Stro-1 (data not shown) or inducibility of chondrocyte differentiation by
TGF-ß3 (Mackay et al.,
1998
). By contrast, we found that the cells expressed collagen I,
a marker for dedifferentiated chondrocytes, and TGF-ß3 kept the cells in
a dedifferentiated state.
Cells reisolated from cultures of chondrocytes initially isolated from EBs
showed a drastically reduced differentiation efficiency into chondrocytes
whereas differentiation into adipocytes increased
(Table 2). Also clonal growth
of chondrocytes isolated from EBs resulted in a reduced differentiation into
chondrocytes compared with high-density cultures and into enhanced
differentiation into adipocytes. Probably, when the cells are plated at a low
density they change differentiation into the adipocytic direction. In fact, it
has been observed in several studies that differentiation of primary cultures
of chondrocytes was affected by the density of cells (e.g.
Castagnola et al., 1986;
Bruckner et al., 1989
). The
observation that mesenchymal cells are able to transdifferentiate into other
mesenchymal cell types together with the finding that mesenchymal
differentiation is regulated by multiple inductive or repressive factors
rather than a set of specific master genes resulted in a stochastic
repression/induction model for mesenchymal cell differentiation
(Dennis and Charbord, 2002
).
Stochastic activation and repression events affecting genes encoding
transcription factors could account for the plasticity of differentiating
mesenchymal cells. The chondrocytic cells released from the EBs are placed
into a new environment with an altered composition of activating and
repressive factors. This new environment could have induced additional
mesenchymal cell types such as the adipocytes in the ES-cell-derived
chondryocyte cultures (Fig. 9).
For the epithelial cells and myocytes, however, we can not completely rule out
the possibility that they were generated by contaminating precursor cells.
Consideration of ES-cell-derived chondrocytes for therapeutic
applications
The regenerative capacity of articular cartilage is limited.
Transplantation methods used to treat cartilage lesions rely mainly on primary
cultures of chondrocytes that do not restore the original hyaline cartilage
(Chen et al., 1999). ES cells
are considered to be an alternative source for generating cells used in cell
therapy for two reasons: first they can almost indefinitely divide in culture
and second they are able to differentiate into cell types of all three germ
layers. Furthermore, because ES cells recapitulate early embryonic
developmental phases during in vitro differentiation it should be possible to
isolate cells at different developmental stages, which could be beneficial for
therapeutic approaches. However, as shown in this paper, chondrocytes derived
from ES cells exhibit a certain differentiation plasticity after release from
EBs probably because the correct combination of determining factors was
missing. In line with these results it has been shown that differentiation,
survival and maintenance of ES-cell-derived dopaminergic neurons was enhanced
by survival-promoting factors present in the culture medium
(Rolletschek et al., 2001
).
Together these results indicate that it crucially depends on the appropriate
culture and selection strategies to obtain defined cell types from ES cells
that show a stable phenotype to be used as cellular grafts.
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Acknowledgments |
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