Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem...
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O R I G I N A L R E S E A R C H P A P E R
Analysis of collagen expression during chondrogenic
induction of human bone marrow mesenchymal stem cellsEmeline Perrier Marie-Claire Ronziere
Reine Bareille Astrid Pinzano
Frederic Mallein-Gerin Anne-Marie Freyria
Received: 26 February 2011 / Accepted: 23 May 2011 / Published online: 10 June 2011
Springer Science+Business Media B.V. 2011
Abstract Adult mesenchymal stem cells (MSCs)
are currently being investigated as an alternative to
chondrocytes for repairing cartilage defects. As
several collagen types participate in the formation
of cartilage-specific extracellular matrix, we have
investigated their gene expression levels during MSC
chondrogenic induction. Bone marrow MSCs were
cultured in pellet in the presence of BMP-2 and TGF-
b3 for 24 days. After addition of FGF-2, at the fourth
passage during MSC expansion, there was an
enhancing effect on specific cartilage gene expression
when compared to that without FGF-2 at day 12 in
pellet culture. A switch in expression from the pre-
chondrogenic type IIA form to the cartilage-specific
type IIB form of the collagen type II gene was
observed at day 24. A short-term addition of FGF-2followed by a treatment with BMP-2/TGF-b3
appears sufficient to accelerate chondrogenesis with
a particular effect on the main cartilage collagens.
Keywords Bone morphogenetic protein-2,
chondrogenesis Collagen Fibroblast growth-factor-
2 Mesenchymal stem cells Transforming growth
factor-beta3
Introduction
Cartilage tissue engineering is currently exploring the
potential of adult mesenchymal stem cells (MSCs),
present in different tissues, as an alternative to the use
of autologous chondrocyte transplantation for carti-
lage repair (Khan et al. 2010; Freyria et al. 2008).
Prior to their use for tissue repair, there have been
extensive studies to develop methods to enhance
MSC proliferation and subsequent chondrogenic
Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-011-0653-1) containssupplementary material, which is available to authorized users.
E. Perrier
M.-C. Ronziere
F. Mallein-GerinA.-M. Freyria (&)
Institut de Biologie et Chimie des Proteines, Universite
Lyon 1, Univ Lyon, CNRS FRE 3310, IFR128
BioSciences Gerland-Lyon Sud, 7 Passage du Vercors,
69367 Lyon Cedex 7, France
e-mail: [email protected]
E. Perrier
e-mail: [email protected]
M.-C. Ronziere
e-mail: [email protected]
F. Mallein-Gerin
e-mail: [email protected]
R. Bareille
Inserm Unite 577, Universite Victor Segalen ,
Bordeaux 2, 33076 Bordeaux, France
e-mail: [email protected]
A. Pinzano
Laboratoire de Physiopathologie, Pharmacologie et
Ingenierie Articulaires, UMR 7561 CNRS-Nancy
Universite, 9, avenue de la Foret de Haye,
54505 Vanduvre-Les-Nancy, France
e-mail: [email protected]
123
Biotechnol Lett (2011) 33:20912101
DOI 10.1007/s10529-011-0653-1
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differentiation, using both animal and human models.
Some procedures have now been standardized but
before obtaining a sufficient cell reservoir for labo-
ratory and clinical purposes it is necessary to amplify
MSCs in vitro. These cells represent a minor fraction
of the total nucleated cell population in the bone
marrow (BM), the most common and best-character-ized MSC source, with an approx. frequency of 1
MSC per 5 9 103 mononuclear cells (Kastrinaki
et al. 2008). Fibroblast growth factor-2 (FGF-2) has
been most frequently used as it was known to
maintain the differentiation potential of MSCs after
several mitotic divisions (Karlsson et al. 2007;
Murdoch et al. 2007; Solchaga et al. 2005, 2010;
Varas et al. 2007). Interestingly, FGF-2 treatment of
MSCs during expansion has the potential to delay the
loss of chondrogenic potential (Solchaga et al. 2010).
In addition, to promote chondrogenic differentiation,the expanded MSCs need to be subsequently cultured
in a three-dimensional environment as micromasses
or in scaffold materials and this differentiation also
requires the presence of various compounds such as
vitamins, dexamethasone and members of the TGF-b
superfamily (Karlsson et al. 2007; Marsano et al.
2007; Mehlhorn et al. 2006; Merceron et al. 2009;
Miyanishi et al. 2006; Murdoch et al. 2007; Pelttari
et al. 2006; Solchaga et al. 2005; Varas et al. 2007).
Bone morphogenetic proteins (BMPs) have also been
added to the culture medium of BM-MSCs alone,such as BMP-2 or in various combinations with TGF-
b (Schmitt et al. 2003; Sekiya et al. 2005; Shen et al.
2009). Interestingly, all BMPs enhance the chondro-
genic effect of TGF-b.
These studies have mainly investigated the essen-
tial components of hyaline cartilage to assess and
characterize the chondrogenic induction of MSCs
such as type II collagen and sGAG (Tew et al. 2008).
Regarding the collagens, those characteristic of car-
tilaginous tissues include type II collagen [a1(II)3]
representing about 9095%, as well as types IX[a1(IX) a2(IX) a3(IX)] and XI [a1(XI) a2(XI)
a3(XI)], representing less than 10% of the total
collagen content of the ECM in adult articular
cartilage (Petit et al. 1992). Type II collagen is
synthesized as a larger precursor procollagen mole-
cule under two forms, IIA and IIB, resulting from
alternative splicing of exon 2, which codes for a
cysteine-rich (CR) domain located in the N-propep-
tide region (Sandell et al. 1991). Type IIA procollagen
contains the CR domain and is produced by chondro-
progenitor cells, whereas type IIB procollagen does
not contain this domain and is synthesized only when
chondrocytes are fully differentiated (Aigner et al.
1999; Zhu et al. 1999). Thus, the switch from type IIA
to type IIB procollagen mRNA expression is a marker
of the chondrocyte phenotype. Besides, the fibril-associated collagens present in cartilage and forming
the primary core fibrillar network (types II, IX and XI
collagens), other collagen molecules, such as types VI
[a1(VI) a2(VI) a3(VI) a4(VI) a5(VI) a6(VI)], XII
[a1(XII)3] and XXVII [a1(XXVII)3], have a structural
role in cartilage organization or are temporally or
qualitatively associated with cartilage development
and thus their encoding genes represent potential
reference markers to monitor chondrogenesis in MSC
cultures (Alexopoulos et al. 2009; Gregory et al. 2001;
Hjorten et al. 2007).The aim of the present study was to seek a concise
system for analyzing the effects of the growth factors
on the chondrogenic ability of human BM-MSCs,
with special attention given to collagen expression.
We sought conditions that would produce expression
of the characteristic markers with the minimal and
effective content of growth factors. In order to treat
the cells similarly given by each donor, indepen-
dently of the time of harvest, we chose to expand
MSCs during three passages to obtain a sufficient cell
reservoir for our experimental purposes and to addFGF-2 at the fourth passage. The subsequent level of
cell differentiation in pellet cultures was investigated,
and especially the mRNA expression of spliced forms
of type II procollagen, as well as other collagens. The
occurrence of chondrogenesis was characterized by
the expression of marker genes for chondrocytes,
hypertrophic chondrocytes and by the synthesis of
extracellular matrix proteins.
Materials and methods
Isolation and cell culture of MSCs and human
chondrocytes
Adult bone marrows were obtained from iliac aspi-
rations of three donors (age range: 3766 years)
undergoing total hip replacement, after informed
consent and according to local ethical guidelines. The
BM-MSCs were separated from BM mononuclear
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cells by adherence on plastic (Cournil-Henrionnet
et al. 2008; Vilamitjana-Amedee et al. 1993). Cells
(0.5 9 106/cm2) were then expanded in monolayer
culture in Dubelccos modified Eagles Medium
(DMEM) supplemented with 10% (v/v) fetal calf
serum (FCS), 100 U penicillin/ml and 100 lg strep-
tomycin/ml in humidified incubators at 37C with 5%CO2. As surface markers on MSCs showed little
variation during the culture, the cell surface receptor
profile of the BM-MSCs was not reported in this
paper (Cournil-Henrionnet et al. 2008). Cells were
cultured until 80% confluence and harvested with
0.25% trypsin/1 mM EDTA for 34 min at 37C and
seeded in new flasks for expansion. After the third
subculture, cells were harvested and frozen in a
cryopreservation medium containing 50% FCS, 40%
DMEM and 10% dimethyl sulfoxide (DMSO).
Before the formation of pellets, a fourth passagewas carried out in the presence or absence of 5 ng/ml
of human recombinant FGF-2 (R&D Systems). After
each trypsination, cells were counted with a Cello-
meter Auto T4 (Nexcelom Bioscience) and the
proliferation rate was calculated between the differ-
ent culture conditions.
Articular chondrocytes were isolated from the
macroscopically healthy zone of cartilages obtained,
according to the local ethical guidelines and after
informed consent, from three donors undergoing total
knee replacement. The rationale to compare theinduced MSCs with cultured articular chondrocytes is
that both cells are in a nonmatrix environment, while
with fresh tissue the gene expression represents in
vivo levels. Cells were cultured in monolayer for
36 h (Ach), as previously described (Hautier et al.
2008). Total cell RNA obtained at this stage gave us a
chondrocytic gene expression reference to compare
with the induced MSCs.
Pellet cultures
Cells (0.25 9 106) were seeded in V-bottomed
96-well plates and pelleted for 5 min at 250 g. The
pellets were cultured in 250 ll high glucose DMEM
supplemented with 1% (w/v) insulin/transferring/
selenium/bovine serum albumin/linoleic acid
(ITS ? Premix; BD), 100 U penicillin/ml, 100 lg
streptomycin/ml, 40 lg L-proline/ml, 100 lg sodium
pyruvate/ml, 100 nM dexamethasone, 50 lg ascor-
bate-2-phosphate/ml (Asc-P) and BMP-2 (R&D Sys-
tems) and TGF-b3 (R&D Systems) in combination
[10 ng TGF-b3/ml ? 50 ng BMP-2/ml (BT)]. In a
preliminary experiment, we verified that the combi-
nation TGF-b3/BMP-2 was a better inducer of
chondrogenesis than each growth factor alone. Mediain both groups was changed every 3 days. Pellet sizes
were regularly measured on micrographs taken with a
bright-field Nikon TE300 microscope equipped with
a QICAM Fast 1394 camera (Qimaging).
Gene expression analysis
Four to six pellets for each culture condition and for
each donor were homogenized with TissueLyser
(Qiagen). Total RNA was isolated after 1, 12 and24 days of culture, using the RNeasy kit (Qiagen) and
reverse transcription of 50500 ng total RNA was
performed as previously described (Cortial et al.
2006). Quantitative RT-PCR was performed with an
iCycler iQ (BioRad). Each analysis was carried out in
duplicate. PCR primers (Table 1) were obtained from
Invitrogen. We focused our interest on genes coding
for collagens (types I, II, VI, IX, X, XI, XII and
XXVII), proteoglycan (aggrecan: ACG1), transcrip-
tion factor (SOX9) and one MEC-degrading enzyme
(MMP13). For each cDNA sample the Ct value of thereference gene ribosomal protein L30 (RPL30) was
subtracted from the Ct value of the target gene to
obtain the DCt. The level of expression was then
calculated as 2-DCt and was expressed in relative
quantity.
In addition, the total amounts of type II collagen
transcripts and of procollagen IIA and IIB isoform
transcripts were assessed by conventional PCR after
12 and 24 days of culture, using previously
described parameters (Hautier et al. 2008). Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH)was used as the housekeeping gene. Photographs
of gels obtained using a Baby Imager (Appligene
Oncor, Illkirch, France) were scanned using an
Epson 1640 scanner (Epson France). The relative
ratios of the bands corresponding to type IIA
(475 bp) and type IIB (268 bp) procollagen tran-
scripts were quantified using Image Quant software
(GE Healthcare).
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Immunohistological analysis
Pellets collected after 12 and 24 days of culture were
fixed for 24 h in 4% neutral buffered formalin,
processed in paraffin wax, and then sectioned. Perox-
idase staining was performed, according to the
horseradish peroxidase conjugated Envision methodas previously described (Freyria et al. 2004). Before
their application, polyclonal antibodies to type I, type
II, or type VI collagen and aggrecan (Novotec) were
diluted 1:1000, 1:500, 1:1000 and 1:1000, respec-
tively, in PBS/3% BSA. Sections were lightly coun-
terstained using Harriss haematoxylin stain, washing
in PBS between each step of the procedure. Control
sections without the addition of primary antibodies
were processed in parallel to rule out nonspecific
labeling. Sections were observed with a Leica DMLB
microscope directly coupled to a JVC color camera
(Leica Microsystemes SAS).
For immunofluorescence staining of type II colla-
gen, pellets were rapidly frozen at -20C in Tissue
Tek OCT (Sakura Finetek Microm France). Frozen
sections (5 lm thick) were fixed with acetone,permeabilized with PBS/3% BSA/1% Tween20 and
incubated (45 min at 20C) with a polyclonal anti-
serum recognizing the CR domain in the type IIA
propeptide (Oganesian et al. 1997) and then diluted
1:300 in PBS/3% BSA. Following washes in PBS,
sections were incubated (45 min at 20C) with
secondary antibodies [Alexa-Fluor 488 goat anti-
rabbit IgG (H ? L): AF488, Invitrogen] diluted
1:500. Fluorescent nuclear staining was obtained
Table 1 Nucleotide sequences of primers used for real-time PCR
Genes Primers References
Extracellular matrix proteins
a1 chain of collagen I (COL1A1) Forward CAGCCGCTTCACCTACAGC Hautier et al. (2008)
Reverse TTTTGTATTCAATCACTGTCTTGCC
a1 chain of collagen II (COL2A1) Forward GCCTGGTGTCATGGGTTT NM 001844.4
Reverse GTCCCTTCTCACCAGCTTTG
a1 chain of collagen VI (COL6A1) Forward GAAGAGAAGGCCCCGTTG NM 001848.2
Reverse CGGTAGCCTTTAGGTCCGATA
a1 chain of collagen IX (COL9A1) Forward ACGGTTTGCCTGGAGCTAT NM 001851.4
Reverse ACCGTCTCGGCCATTTCT
a1 chain of collagen X (COL10A1) Forward CAAGGCACCATCTCCAGGAA NM 000493
Reverse AAAGGGTATTTGTGGCAGCATATT
a1 chain of collagen XI (COL11A1) Forward TCCTCTTCCAAGCTAGAGAGGTC NM 080629.2
Reverse GGAGAATTGTGAAAATCTAGTGCT
a2 chain of collagen XI (COL11A2) Forward CCTGAGCCACTGAGTATGTTCATT Khan et al. (2008)
Reverse TTGCAGGATCAGGGAAAGTGA
a1 chain of collagen XII (COL12A1) Forward TGGTCATCCAGCAGTCAGG NM 004370.5
Reverse TGGCAAGCTCATTGTAGTCG
a1 chain of collagen XVII (COL27A1) Forward GGTCTCCTGCAACTTCACTCAT Hjorten et al. (2007)
Reverse GCTTAGCAGGTGCAGGAAATTC
Aggrecan (AGC1) Forward TCGAGGACAGCGAGGCC Hautier et al. (2008)
Reverse TCGAGGGTGTAGCGTGTAGAGA
Transcription factor
SOX9 Forward ACGCCGAGCTCAGCAAGA Hautier et al. (2008)
Reverse CACGAACGGCCGCTTCT
Housekeeping geneRibosomal protein L30 (RPL30) Forward CCTAAGGCAGGAAGATGGTG NM 000989.2
Reverse AGTCTGCTTGTACCCCAGGA
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after incubation with propidium iodide diluted 1:100
in PBS, for 30 min at 20C. In some experiments, a
sequential double immunostaining was carried out
with, first, immunolabeling of type II collagen
(Novotec; antibodies diluted 1:300) detected with
AF488, then with immunolabeling of the type IIA
propeptide (Oganesian; antibodies diluted 1:300)detected by Cy3 conjugated sheep anti-rabbit IgG
(Sigma), diluted 1:100. Sections were observed with
a Zeiss Axioplan 2 Imaging or a Nikon E600 micro-
scope, equipped for epifluorescence.
Statistical analysis
Changes in gene expression were given as fold
change of FGF-2 treated compared with untreated
MSCs. To determine the statistical significance of
induced gene expression (BT) compared with con-trols (CTL), the t-test was used. A value of P\ 0.05
was considered significant.
Results
Increases in cell proliferation and changes in BM-
MSC pellet morphology
BM-MSCs expanded during the fourth passage in the
presence of FGF-2 exhibited a shorter populationdoubling time than those expanded in the absence of
FGF-2 (2.1 vs. 11 days). Moreover in the presence of
the inducers, pellets from FGF-2-treated cells exhib-
ited a larger diameter at day 24 (1.6 mm) in compar-
ison to the pellets from untreated cells (1 mm).
Expansion with FGF-2 accelerates chondrogenic
induction and favors expression of the cartilage-
specific collagens
Since FGF-2 increases not only the growth rate butalso maintains the multidifferentiation potential of
human MSCs, we investigated whether the short-term
addition of FGF-2 during the expansion step, could
influence the effects of BMP-2/TGF-b3 observed in
our preliminary experiment, when cells were subse-
quently cultured in pellets. BM-MSCs were first
expanded in the presence or absence of 5 ng FGF-2/
ml during the fourth passage and then cultured in
pellets in the presence of the BT combination.
In order to evaluate the effect of this culture
condition on gene expression levels, the data are
presented as a ratio (FGF-2?/FGF-2-) for each gene
in Fig. 1. The data were pooled and presented as
means SEM as the gene expression profiles under
all the culture conditions were similar for the three
donors examined. First, we measured the expressionlevel variations for several collagen genes. With
FGF-2?/FGF-2- ratios close to 1, the same expres-
sion levels for COL6A1 and for COL27A1 mRNA
were recorded throughout the study under all culture
conditions (Fig. 1a). A similar pattern of expression
was observed for COL1A1 and COL12A1 mRNA
with an increase in the presence of FGF-2 only on
day 12 under both culture conditions (CTL and BT).
For the other collagen genes, their expression levels
were maximal on day 12 in those (?FGF-2) pellets
that received the inducers and levels decreasedslightly over time in culture. Statistically significant
increases were recorded only on day 12 for COL9A1
(885-fold; P = 0.07), COL11A2 (540-fold; P =
0.009). For COL2A1 (311-fold; P = 0.01 and
23-fold; P = 0.06), COL10A1 (575-fold; P\ 0.001
and 75-fold; P\0.001) and for COL11A1 (33-fold;
P = 0.02 and 12-fold; P = 0.006) increases were
significant both on day 12 and day 24. Second, we
investigated the variations in expression levels for
other components playing a role in ECM production
during chondrogenesis (Fig. 1b). For a gene such asMMP13 the presence of FGF-2 during expansion was
followed by a slight increase in the expression levels
under all the culture conditions. A similar expression
pattern was recorded for SOX9 and AGC1 mRNA
levels, with the highest levels on day 12 in (?FGF-2)
pellets receiving the inducers. When compared to the
control conditions, the largest increases (56-fold;
P\ 0.001) were recorded for the AGC1 mRNA
level. Finally, higher expression levels were observed
for all the genes in the pellets on day 24 compared to
human articular chondrocytes, whatever the cultureconditions (Electronic Supplementary Figs. 1 and 2).
The cartilage-characteristic gene expression levels
measured on day 24 in inducer-treated-pellets were
higher than those in articular chondrocytes (Ach),
strongly suggesting a chondrogenic conversion of
MSCs. Furthermore, the greatest difference was
noted for COL10A1 mRNA, certainly due to the
non-hypertrophic and non-osteoarthritic characteris-
tics of articular chondrocytes.
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Expansion with FGF-2 favors the expression
of the cartilage-specific collagen isoform of type
II procollagen
To examine the extent of this chondrogenic conver-
sion more closely, we analyzed the expression of the
pre-cartilaginous isoform (IIA) and cartilaginous
isoform (IIB) of type II procollagen. First, total type
II procollagen expression was detected, for the three
donors, in the presence of BT at high levels in
(?FGF-2) pellets, on days 12 and 24 (Fig. 2a, Elec-
tronic Supplementary Fig. 3), whereas in (-FGF-2)
pellets it could only be detected on day 24. Second,
both IIA and IIB transcriptswere expressed and a sharp
decrease in IIA expression occurred between days 12
and 24 (Fig. 2a). Indeed, quantitative analysis of the
A
B
0
1
10
100
1000
10000
CTL BT CTL BT CTL BT CTL BT CTL BT CTL BT CTL BT CTL BT CTL BT
COL1A1 COL2A1 COL6A1 COL9A1 COL10A1 COL11A1 COL11A2 COL12A1 COL27A1
0
1
10
100
CTL BT CTL BT CTL BT
AGC1 SOX9 MMP13
Fig. 1 a Effect of FGF-2 during MSC expansion on geneexpression during subsequent culture in pellet. The levels of
collagen genes associated with the chondrogenic phenotype
(COL1A1, COL2A1, COL6A1, COL9A1, COL10A1, COL11A1,
COL11A2, COL12A1, COL27A1) were analyzed by real-time
PCR on total RNA isolated on days 1, 12 and 24 from the
MSCs of three donors, amplified in the absence or presence of
FGF-2 (?) during the fourth passage and further induced
towards chondrogenesis in pellets in the absence (CTL) or
presence of BMP-2 and TGF-b3 (BT). The results are
presented as the ratio of the relative quantities corresponding
to expansion (?FGF-2/-FGF-2), obtained under each condi-
tion for each donor. Data are presented as means SE and
P values are given. b Effect of FGF-2 during expansion ofMSCs on gene expression during subsequent pellet culture.
The levels of genes associated with the formation of
chondrogenic extracellular matrix components (AGC1, SOX9
and MMP13) were analyzed by real-time PCR on total RNA
isolated on days 1, 12 and 24 from MSCs of three donors,
amplified in the absence or presence of FGF-2 (?) during the
fourth passage and further induced towards chondrogenesis in
pellet culture in the absence (CTL) or presence of BMP-2 and
TGF-b3 (BT). The results are presented as the ratio of the
relative quantities corresponding to expansion (?FGF-2/-
FGF-2), obtained under each condition for each donor. Data are
presented as means SE and P values are given
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IIA and IIB amplicons showed that their relative values
switched from 36:64 on day 12 to 5:95 on day 24,
supporting the view that BM-MSCs had entered a
chondrogenic differentiation pathway. Furthermore,
these latter data differed from that measured (25:75)
for human Ach which rapidly and partially dediffer-
entiate in monolayer culture.Finally, immunohistological analysis was carried
out to visualize accumulation and localization of
cartilage matrix components synthesized by the cells
during the course of chondrogenic induction. As
shown on Fig. 2b, type II collagen, including the type
IIA isoform, was progressively synthesized and
deposited in the extracellular matrix of the pellets,
in a time and culture condition-dependent manner.
There was more intense immunofluorescent staining
at the periphery of the (?FGF-2) pellets induced with
BT (Fig. 2b). Immunostaining of type IIA procolla-gen was observed at the periphery of the pellets on
day 12, showing the early presence of chondropro-
genitor-like cells in the pellets, and this staining was
still observed on day 24 (Fig. 2b). When peroxidase
staining was used, both extracellular and intracellular
labeling of type II collagen could be clearly seen on
day 24 although with a non-homogenous deposition
(Electronic Supplementary Fig. 4), suggesting that
the cells were still metabolically active towards
chondrogenic differentiation. It is also interesting to
report that labeling for type VI collagen was presentin the (-FGF-2) pellet more at the periphery on day
24. In the BT-treated pellet this collagen was
deposited at similar locations than type II collagen
and aggrecan (Electronic Supplementary Fig. 4).
Altogether these immunohistological findings also
upheld the results from real-time PCR.
Discussion
BM-MSCs are capable of chondrogenic differentiationin vitro in the presence of various growth factors
provided during the expansion and induction steps. In
this study, we found that expanding the MSCs in the
presence of FGF-2 for a short-term addition during the
fourth passage stimulated the cell chondrogenic con-
version induced by the combination of BMP-2 and
TGF-b3. The switch in expression from the pre-
cartilaginous IIA isoform to the cartilaginous IIB
isoform of the type II procollagen gene and an increase
in gene expression of the main collagen types forming
the primary core fibrillar network of cartilage sup-
ported this conversion.
When we performed cell expansion with growth
medium supplemented with FGF-2, we obtained data
consistent with the findings of other groups concern-
ing the stimulatory effects of this growth factor onBM-MSC proliferation and differentiation (Solchaga
et al. 2005, 2010; Varas et al. 2007). Our data are of
interest as they report the effect of adding FGF-2 for
a short time, at the fourth passage, in comparison to
its presence during all passages in the other studies.
The cell proliferation that we observed, assessed by
the shortening of the population doubling time, is the
mark that the cells after 3 passages in culture were
still responsive to the mitogenic factor FGF-2. As in
many studies the chondrogenic differentiation was
induced in cells expanded with or without a cocktailof growth factors and at different passages (15) our
data are not surprising and they correspond to another
cell amplification condition (Ronziere et al. 2010). In
the presence of the inducers (BMP-2/TGF-b3) the
cells in pellets exhibited a higher chondrogenic
potential with larger pellet derived from FGF-2-
treated cells than those made from control cells
corresponding to a higher matrix synthesis as previ-
ously reported (Solchaga et al. 2005). There was an
enhancing effect of FGF-2-treatment on the gene
expressed in pellet cultures between days 1 and 12mainly for the genes coding for cartilage components.
Changes were about one hundred-fold for the major
cartilage collagens (COL2A1, COL9A1, COL11A2
and COL10A1 mRNA) and about ten-fold for the
genes coding for other cartilage specific components
(AGC1 and COL11A1 mRNA). Only the levels of
expression of 2 chondrogenic-specific genes COL2A1
and COL11A2 increased between days 12 and 24
attesting that it was sufficient to add FGF-2 at the
passage preceding the induction to observe its chon-
drogenic capacity.It is still not clear how FGF-2 plays a role in
chondrogenesis as several signal transduction path-
ways might be activated in addition to the MEK/ERK
(mitogen-activated protein kinase kinase/extracellu-
lar-signal regulated kinase) cascade and the Wnt
signaling by FGFs stimulation described in these cells
(Bobick et al. 2007; Solchaga et al. 2010). As FGFs
and their receptors play fundamental roles regulating
growth morphogenesis and cartilage formation in
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embryonic limbs further studies have to be conducted
to describe and understand the precise role of FGFs
on in vitro MSC chondrogenesis. The chondrogene-
sis, in our culture conditions, was not totally efficient
in the pellets as the ECM was not homogeneously
deposited around the cells in FGF-2 treated samples
contrary to previously reported data with FGF-2
present at all passages (Solchaga et al. 2005). Beside
the differences in cell harvesting, donor variability
and culture conditions in different laboratories these
data likely correspond to the heterogeneity of the cell
population after the expansion steps (Kastrinaki et al.
2008).
Chondrogenic conversion was also clearly shown
on day 12 by the expression of both the IIA and IIB
forms of procollagen type II and on day 24 by the
Fig. 2 a Expression of the type II procollagen gene, COL2A1
(total) and isoforms IIA and IIB in pellet culture. MSCs were
first expanded in the absence or presence of FGF-2 (?) and
further induced towards chondrogenesis in pellet culture in the
absence or presence of BMP-2 and TGF-b3 (BT). Total RNA
were extracted from the pellets after 12 and 24 days of culture
and measured by conventional PCR, using specific primers.
GAPDHwas used as the housekeeping gene. Data are obtained
from one donor representative of all three. b Collagen type II
deposition by MSCs in pellet culture. The pellets were obtained
as described in Fig. 2a. The pellets were fixed and immuno-
stained for type II collagen using a polyclonal antibody against
the triple helical domain (total type II collagen) and a
polyclonal antibody against the CR domain (propeptide of
type IIA collagen) (green collagen, red nucleus). Bar 250 lm.
Inset double-stained immunofluorescence for type II collagen
(triple helix staining, Alexa Fluor 488) and type IIA collagen
(propeptide IIA staining, Cy3 labeled antibodies) under 24-day
culture (green collagen). Bar 500 lm. Results are obtained
from one representative of three independent experiments
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switch in expression from type IIA to type IIB. In
addition, type II collagen synthesis (including the
type IIA form) was only detected in (?FGF-2) pellets
treated with BT. Importantly, the switch was
observed under these culture conditions with cells
obtained from three donors. Indeed, this combination
overtook donor-to-donor phenotypic differences thatwere observed in the other culture conditions (Elec-
tronic Fig. 3). Thus, our data show that measuring the
procollagen IIA: procollagen IIB ratio gives a useful
index of chondrogenic induction during the culture of
MSCs. Moreover, Murdoch demonstrated that only
4 days were necessary for a complete switch of
phenotype in Transwell culture, while more than
12 days were required in pellet culture, implying that
the microenvironment of the MSCs played a role
together with the addition of growth factors (Mur-
doch et al. 2007). Our data also showed that theincrease in gene expression was correlated with an
increase in ECM synthesis, as attested by immuno-
staining of type II collagen and aggrecan in the
pellets. The fact that type IIA procollagen, the non-
chondrogenic form of type II collagen, was still
present at day 24 in the pellet is likely an indicator of
an incomplete or slow chondrogenesis in our culture
model in comparison with, for example, the Trans-
well culture. However, our model may be used to
study the mechanisms involved in the synthesis of
type II collagen and it organization in the ECMduring MSC chondrogenesis.
We also paid particular attention to the expression
of collagens rarely studied in the context of MSCs
and not investigated in an in vitro model of
chondrogenesis. We found that BM-MSC cultures
expressed COL6A1, COL12A1 and COL27A1, three
genes coding for proteins previously described as
playing a structural role in the perichondrocytic
matrix and at the bone cartilage interface (Alexopo-
ulos et al. 2009; Gregory et al. 2001; Hjorten et al.
2007). Interestingly, these three genes appeared not toresponse, contrary to the other collagen genes, to the
addition of FGF-2 during cell expansion. Further-
more, COL12A1 and COL27A1 genes appeared to be
up-regulated by the combination of inducers but not
COL6A1 gene, which was unresponsive to the culture
conditions of the study. Along this study we were
able to visualize type VI collagen which, in normal
articular cartilage, has an exclusive location in the
pericellular matrix together with proteoglycan,
fibronectin and type II and IX collagens (Poole
et al. 1997). Type VI was recently reported as an
integrating molecule in a Col6a1-knockout mice as
its deficiency led to an alteration in the biologic and
mechanical environment of the chondrocyte (Alexo-
poulos et al. 2009). Thus our finding of the presence
of type VI collagen together with type II collagen andaggrecan in the pellets indicates that the cells
accumulate a cartilage-like matrix with an organiza-
tion of the molecules not yet characteristic of native
articular chondrocytes. Chondrogenesis is a dynamic
process where ECM production is constantly chang-
ing. Thus the presence of different type of collagens
described to play a role in the alignment of the fibrils
(type XII collagen) or in the later stages of the
cartilage to bone formation to likely offer a transient
scaffold for cartilage mineralization (type XXVII
collagen) may be required to produce different cellmatrix interactions corresponding to a specific stage
of the chondrogenic program (Gregory et al. 2001;
Hjorten et al. 2007; Plumb et al. 2007). Additional
studies will be required to more completely describe
and understand the role of these collagens in
chondrogenesis.
Conclusion
The competency of MSCs for chondrogenesis, under
treatment with BMP-2 and TGF-b3, is accelerated
and enhanced by the presence of FGF-2, during cell
amplification at the passage preceding the induction.
This was particularly demonstrated by the induction
of a subset of genes coding for collagens forming the
primary core of the fibrillar cartilage network.
However, the persistent expression of COL1A1, a
marker gene for mesenchymal cells, and the induc-
tion of COL10A1 expression observed in our cell
model and in other studies indicate that turning offthese expressions, and thus the deposition of non
cartilage-characteristic proteins, should be one of the
main future aims in cartilage therapy strategies that
use MSCs as a cell source. For instance, the use of
decoy or siRNA strategies targeting transactivators of
the genes could be considered in further studies.
Acknowledgments This work was supported by Cluster
Handicap Vieillissement NeuroSciences (HVN) (Decryp-
tage des interactions des cellules souches avec la matrice
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extracellulaire necessaires a leur conversion en chondrocytes)
from the Region Rhone-Alpes. The authors would like to thank
Dr L J Sandell (Washington University School of Medicine,
St Louis, MO, USA) for rabbit antiserum against recombinant
human type IIA and Sylviane Guerret (Novotec, Lyon, France) for
her expertise in histological analysis. The Analyse Genetique and
Platim platforms of IFR 128 are gratefully acknowledged for the
use of the iCycler iQ (BioRad) and the Nikon E600 microscope.
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