Human Induced Pluripotent Stem Cells Differentiated into Chondrogenic Lineage Via Generation of Mesenchymal Progenitor Cells.

Abstract

Human induced pluripotent stem cells (hiPSCs) exhibit pluripotency, proliferation capability, and gene expression similar to those of human embryonic stem cells (hESCs). hESCs readily form cartilaginous tissues in teratomas in vivo; despite extensive effort, however, to date no efficient method for inducing mature chondrocytes in vitro has been established. hiPSCs can also differentiate into cartilage in vivo by teratoma formation, but as with hESCs, no reliable system for in vitro chondrogenic differentiation of hiPSCs has yet been reported. Here, we examined the chondrogenic differentiation capability of hiPSCs using a multistep culture method consisting of embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and 3-dimensional pellet culture. In this culture process, the cell density of monolayer culture was critical for cell viability and subsequent differentiation capability. Monolayer-cultured cells exhibited fibroblast-like morphology and expressed markers for mesenchymal stem cells. After 2-3 weeks of pellet culture, cells in pellets exhibited a spherical morphology typical of chondrocytes and were surrounded by extracellular matrix that contained acidic proteoglycans. The expression of type II collagen and aggrecan in pellets progressively increased. Histological analysis revealed that over 70% of hiPSC-derived pellets successfully underwent chondrogenic differentiation. Using the same culture method, hESCs showed similar histological changes and gene expression, but differentiated slightly faster and more efficiently than hiPSCs. Our study demonstrates that hiPSCs can be efficiently differentiated into the chondrogenic lineage in vitro via generation of mesenchymal progenitor cells, using a simplified, multistep culture method.

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ORIGINAL RESEARCH REPORT
Human Induced Pluripotent Stem Cells Differentiated
into Chondrogenic Lineage Via Generation
of Mesenchymal Progenitor Cells
Noriaki Koyama,1,2Masako Miura,1Kazumasa Nakao,2Eri Kondo,1Toshihito Fujii,1Daisuke Taura,1
Naotetsu Kanamoto,1Masakatsu Sone,1Akihiro Yasoda,1Hiroshi Arai,1Kazuhisa Bessho,2and Kazuwa Nakao1
Human induced pluripotent stem cells (hiPSCs) exhibit pluripotency, proliferation capability, and gene ex-
pression similar to those of human embryonic stem cells (hESCs). hESCs readily form cartilaginous tissues in
teratomas in vivo; despite extensive effort, however, to date no efficient method for inducing mature chon-
drocytes in vitro has been established. hiPSCs can also differentiate into cartilage in vivo by teratoma formation,
but as with hESCs, no reliable system for in vitro chondrogenic differentiation of hiPSCs has yet been reported.
Here, we examined the chondrogenic differentiation capability of hiPSCs using a multistep culture method
consisting of embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from
EBs, and 3-dimensional pellet culture. In this culture process, the cell density of monolayer culture was critical
for cell viability and subsequent differentiation capability. Monolayer-cultured cells exhibited fibroblast-like
morphology and expressed markers for mesenchymal stem cells. After 2–3 weeks of pellet culture, cells in pellets
exhibited a spherical morphology typical of chondrocytes and were surrounded by extracellular matrix that
contained acidic proteoglycans. The expression of type II collagen and aggrecan in pellets progressively in-
creased. Histological analysis revealed that over 70% of hiPSC-derived pellets successfully underwent chon-
drogenic differentiation. Using the same culture method, hESCs showed similar histological changes and gene
expression, but differentiated slightly faster and more efficiently than hiPSCs. Our study demonstrates that
hiPSCs can be efficiently differentiated into the chondrogenic lineage in vitro via generation of mesenchymal
progenitor cells, using a simplified, multistep culture method.
Introduction
T
social and healthcare problem. Methods for regenerating
chondrocytes and cartilage tissue are expected to transform
conventional therapies for such diseases. Tissue regeneration
approaches also hold promise for treating craniofacial defor-
mities caused by congenital diseases, trauma, or surgical
resection for malignancy that require reconstruction of carti-
lage/bone tissues for cosmetic and functional improvement.
To date, cartilaginous tissue engineering research has focused
largely on the use of mesenchymal stem cells (MSCs) and
mature chondrocytes as resources. Indeed, bone marrow-
derived MSCs (BMMSCs) are currently being tested in clinical
trials for several orthopedic applications, including articular
cartilage repair [1–3]. MSCs can be isolated from various
postnatal tissues [4–8] and do not trigger an immunological
he increasing prevalence of degenerative cartilage
diseases,particularlyosteoarthritis,presentsanimportant
reaction after transplantation. However, they possess limited
proliferation capability and differentiation directions and
decrease their differentiation potential with increasing donor
age [9,10]. Moreover, the invasive procedure required to
harvest MSCs presents another hurdle to widespread clinical
application.
Human induced pluripotent stem cells (hiPSCs) have been
generated from somatic cells by introducing Oct3/4 and
Sox2 along with either Klf4 and c-Myc or Nanog and Lin28,
using retroviruses or lentiviruses [11–13]. hiPSCs exhibit
pluripotency, proliferation capability, and gene expression
similar to those of human embryonic stem cells (hESCs), but
do not present the same ethical problems. Immune rejection
can be avoided by the establishment of hiPSC banks from
donors with various human leukocyte antigen (HLA) types.
Moreover, to reduce the risk of tumorigenicity, new methods
for generating iPSCs without viral vectors have been devel-
oped [14–16]. Therefore, hiPSCs are viewed as a promising
Departments of1Medicine and Clinical Science and2Oral and Maxillofacial Surgery, Kyoto University Graduate School of Medicine,
Kyoto, Japan.
STEM CELLS AND DEVELOPMENT
Volume 00, Number 00, 2012
? Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2012.0127
1

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new tool for regenerative medicine, disease pathogenesis
studies, and drug screening. hESCs readily form cartilagi-
nous tissues in teratomas in vivo, but the proportion of
chondrocytes arising from spontaneous differentiation via
embryoid body (EB) formation in vitro is very low. Three-
dimensional (3D) pellet culture and micromass culture have
been used successfully for in vitro chondrogenic differenti-
ation of MSCs and mature chondrocytes [17–19]. These cul-
ture systems allow cell–cell interactions analogous to those
that occur in precartilage condensation during embryonic
development, and can induce terminal differentiation of
mesenchymal progenitor cells into hypertrophic chon-
drocytes. Besides pellet culture and micromass culture, many
attempts have been made to induce chondrocytes from ESCs
in vitro, including coculture with irradiated articular chon-
drocytes [20] or limb bud progenitor cells from a developing
embryo [21], culture with a conditioned medium [22], genetic
manipulation (e.g., Sox9 expression) [23], and use of bio-
materials [24]. However, both coculture with other animal
cells and culture with a conditioned medium lack repro-
ducibility and carry the risk of pathogen transmission. Ge-
netic manipulation using virus-based vectors poses risks for
clinical applications. Thus, in spite of the many efforts, an
efficient culture method for inducing mature chondrocytes
from hESCs has not yet been established.
hiPSCs have been reported to generate cartilaginous tissue
in teratoma in vivo [11–13], but limited data exists at present
regarding the in vitro chondrogenic differentiation of
hiPSCs. When dissociated EB cells generated from fetal
neural stem cell-derived hiPSCs were grown with a chon-
drogenic medium on agarose-coated wells, they expressed
chondrogenic differentiation markers detectable by immu-
nofluorescence staining [25]. However, immunofluorescence
did not clearly reveal morphological characteristics of
chondrocytes; a large and spherical shape with clear cyto-
plasm surrounded by an extracellular matrix [25]. Thus, a
reproducible method for in vitro chondrogenic differentia-
tion of hiPSCs must be established. Since hiPSC-derived
chondrocytes can be used to study the pathogenesis of ge-
netic disorders, such as skeletal dysplasia, and to develop
drug screening systems for cartilage diseases, it would be
desirable to develop in vitro differentiation methods that
mimic physiological differentiation processes and do not
require genetic manipulation.
In the current study, we examined the chondrogenic dif-
ferentiation capability of hiPSCs by combining a pellet cul-
ture system, the most reliable method reported to date, with
spontaneous differentiation via EB formation, which mimics
early embryonic development by forming the 3 germ layers
(mesoderm, ectoderm, and endoderm). We also compare and
discuss the chondrogenic cells induced from hiPSCs and
hESCs using our culture protocol.
Materials and Methods
Cell culture
hiPSC line 201B7 (B7) was previously generated by in-
troducing 4 reprogramming factors (Oct3/4, Sox2, Klf4, and
c-Myc) into dermal fibroblasts from the facial dermis of a 36-
year-old Caucasian woman [11]. The hESC line H9 was ob-
tained from the WiCell Research Institute (Madison, WI,
www.wicell.org) [26]. These cells were maintained as pre-
viously described [11,27]. Briefly, murine embryonic fibro-
blasts (MEFs) were grown in the Dulbecco’s modified
Eagle’s medium (DMEM; Invitrogen, Grand Island, NY,
www.invitrogen.com), supplemented with 10% (vol/vol)
fetal bovine serum (FBS; HyClone, Logan, UT, www.hyclo-
ne.com), 100U/mL penicillin, and 100mg/mL streptomycin
(Invitrogen). MEF feeder layers were prepared by chemically
inactivating subconfluent cultures with 10mg/mL mitomycin
C (Kyowa, Tokyo, Japan, www.kyowa.co.jp) for 3h at 37?C
and reseeding on 0.1% (wt/vol) gelatin-coated dishes.
hiPSCs and hESCs were grown on inactivated MEF feeder
layers in the primate ESC medium (ReproCELL, Kanagawa,
Japan, www.reprocell.com) containing 4ng/mL of basic fi-
broblast growth factor (bFGF) (ReproCELL).
Chondrogenic differentiation
For EB formation, hiPSC and hESC colonies were har-
vested by treating with 1mg/mL collagenase type IV, and
then plated onto suspension culture dishes, where they were
allowed to aggregate in a maintenance medium without
bFGF. After 7 days as a suspension culture, EBs were
transferred to gelatin-coated dishes and cultivated for 1
week in DMEM containing 10% FBS, 100U/mL penicillin,
and 100mg/mL streptomycin. EBs and cells sprouted from
EBs were harvested and dissociated with 0.25% trypsin/
EDTA (Life Technologies, Inc., Grand Island, NY, www
.lifetechnologies.com). Dissociated cells were passed through
a 100-mm cell strainer (BD Biosciences, Bedford, MA,
www.bdbiosciences.com) to remove cell aggregates and
cultivated in monolayers for another week in DMEM con-
taining 10% FBS, 100U/mL penicillin, and 100mg/mL
streptomycin. To induce chondrogenic differentiation, we
used a previously described pellet culture system [18,28].
Cell suspensions containing 3·105cells per 15-mL poly-
propylene tube were centrifuged to form a cell pellet. Cell
pellets were cultivated at 37?C with 5% CO2for up to 21
days in the chondrogenic induction medium consisting of
DMEM/F12 supplemented with 10% FBS, ITS+? Premix
(BD Biosciences), 50mg/mL ascorbic acid 2-phosphate (Wako
Pure Chemical Industries, Osaka, Japan, www.wako-chem
.co.jp), 100mg/mL sodium pyruvate, 100nM dexamethasone,
10ng/mL transforming growth factor-b3 (TGF-b3) (Pepro-
Tech Inc., Rocky Hill, NJ, www.peprotech.com), 100U/mL
penicillin, and 100mg/mL streptomycin. The medium was
replaced every 3 days. Pellet cultures were repeated and
evaluated histologically 11 times using hiPSCs (B7) and 13
times using hESCs (H9).
Flow cytometry
Cells were incubated with saturating levels of antibodies
for 1h at 4?C. The following fluorescein isothiocyanate
(FITC)- or phycoerythrin (PE)-conjugated antibodies recog-
nizing human antigens were used: TRA-1-60 (No. 560380),
CD44 (No. 348052), CD90 (No. 555596), CD106 (No. 555647),
CD146 (No. 550315), CD166 (No. 559263), CD34 (No.
348057), and CD45 (No. 347463) (BD Biosciences); CD73 (No.
12-0739) and CD105 (No. 12-1057) (eBioscience, Inc., San
Diego, CA, www.ebioscience.com); and CD140a (No. 323505)
(BioLegend, San Diego, CA, www.biolegend.com). The anti-
Stro-1 monoclonal antibody (MAB1038) was purchased from
2 KOYAMA ET AL.

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R&DSystems,Inc.(Minneapolis,MN).Eachanalysisincluded
corresponding FITC- or PE-conjugated isotype controls. Sam-
pleswererunonaBectonDickinsonFACScanflowcytometric
system (BD Biosciences), and analysis was completed using
FlowJo Software (FlowJo, Ashland, OR, www.flowjo.com).
Real-time reverse transcription–polymerase
chain reaction
Total RNA was extracted from cell pellets using the
RNeasy?Mini Kit (Qiagen, Chatsworth, CA, www.qiagen
.com). To ensure the complete removal of DNA, we included
a 15-min DNase I (Qiagen) treatment before the washing
step. First-strand cDNA synthesis was performed using
TaqMan?Reverse Transcription Reagents (Applied BioSys-
tems, Carlsbad, CA, www.appliedbiosystems.com) with ran-
dom hexamers. Real-time reverse transcription–polymerase
chain reaction (RT-PCR) was performed in a StepOne? real-
time PCR System (Applied BioSystems). Complementary
DNA was mixed with TaqMan Universal PCR Master Mix
(Applied BioSystems) and TaqMan Gene Expression Assay
(Applied BioSystems) primers: Sox9 (SOX9, Assay ID;
Hs00165814_m1), type II collagen (COL2A1, Hs01060345_m1),
aggrecan (ACAN, Hs00153936_m1), type X collagen (COL10A1,
Hs00166657_m1), and GAPDH (GAPDH, Hs99999905_m1).
All RNA samples were titrated to yield equal amplification of
GAPDH as an internal normalization control. Reactions for
each sample were performed in triplicate. After an initial de-
naturation step (95?C for 10min), amplification was performed
for 45 cycles (15-s denaturation at 95?C and 60-s extension
at 60?C).
Histological analysis
Cell pellets were fixed in 2% paraformaldehyde, dehy-
drated, and embedded in paraffin. The sections were cut at a
thickness of 4mm and stained with Alcian blue and toluidine
blue, as previously described [28]. For immunohistochemical
analysis, a labeled streptavidin–biotin staining kit (LSAB2
System-HRP) (Dako, San Antonio, TX, www.dako.com) was
used. The sections were incubated overnight at 4?C with the
following primary antibodies: anti-type II collagen (F-57;
Daiichi Fine Chemical, Takaoka, Toyama, www.daiichi-
fcj.co.jp), anti-aggrecan (sc-70333; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA, www.scbt.com), and anti-mitochondria
(MAB1273; Millipore, Billerica, MA, www.millipore.com).
Normal human articular chondrocytes from knee cells
(NHAC-kn) (Lonza Walkersville, Inc., Walkersville, MD,
www.lonza.com) were grown using the same pellet culture
protocol as that used for hiPSCs and hESCs, and were used
as a positive control. Cell pellets derived from human
BMMSCs (Lonza Walkersville, Inc.) were cultivated in
DMEM/F12 supplemented with 10% FBS, 100U/mL peni-
cillin, and 100mg/mL streptomycin, and were used as a
negative control.
Statistical analysis
Real-time RT-PCR experiments were performed indepen-
dently at least 3 times and gave highly similar results. Data
are presented as the mean–SD. Statistical analysis was
performed using one-factor analysis of variance. Differences
were considered statistically significant when P<0.05.
Results
Mesenchymal differentiation of hiPSCs
via EB formation
We used a multistep culture method combining sponta-
neous differentiation via EB formation, cell outgrowth from
EBs on gelatin-coated dishes, monolayer culture after cell
dissociation into single cells, and 3D pellet culture (Fig. 1).
Chondrogenic progenitor cells are derived from MSCs,
which originate from the mesoderm and neural crest.
Therefore, we first tried to promote differentiation of undif-
ferentiated hiPSCs into the mesenchymal lineage. hiPSCs
contained in EBs retained pluripotency in vitro; we used
immunofluorescence analysis to confirm the expression of
markers for mesoderm (a-smooth muscle actin), ectoderm
(b3-tubulin), and endoderm (forkhead box A2; FOXA2) on
cultured EBs (Supplementary Fig. S1; Supplementary Data
FIG. 1.
(hiPSCs) and human embryonic stem cells (hESCs). The differentiation culture protocol consists of 4 steps: (1) Embryoid body
(EB) formation in suspension culture dishes; (2) Cell outgrowth from EBs on gelatin-coated dishes; (3) Monolayer culture
under mesenchymal stem cell (MSC) growth conditions after dissociation into single cells by trypsin/EDTA; and (4) Three-
dimensional pellet culture in the chondrogenic induction medium. During pellet culture, histological and gene expression
analyses were performed at days 3, 7, 14, and 21.
Schematic diagram of the culture protocol for chondrogenic differentiation of human induced pluripotent stem cells
CHONDROGENIC DIFFERENTIATION OF HUMAN IPS CELLS3

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are available online at www.liebertpub.com/scd). Before
proceeding to pellet culture, we included a monolayer cul-
ture step to exclude residual undifferentiated cells present in
the EBs as well as to expand cells committed to the mesen-
chymal lineage. These cells were able to attach to regular
culture dishes and proliferate in the MSC growth medium
consisting of DMEM supplemented with 10% FBS. In our
experience, the initial cell density was critical for monolayer
culture. To determine the ideal cell density, we used trypsin/
EDTA to dissociate cells that had sprouted from EBs and
seeded them at serial dilutions ranging from 0.05 to 20·104
cells/cm2. We found that a cell density of 1–2·104cells/cm2
was ideal for cell proliferation in monolayer culture (Fig. 2).
Seeding at a low cell density induced mitotic arrest and a
large, flattened, and irregular cell morphology. Inversely,
when seeded at a high cell density, cells formed clusters and
proliferated in 3 dimensions. The cell proliferation rate in
monolayers decreased, but did not drop to zero, after 7 days
of culture (Supplementary Fig. S2). The culture period in the
monolayer had no apparent effect on the differentiation ca-
pability (Supplementary Fig. S3). Therefore, we seeded cells
at 1–2·104cells/cm2and cultivated the resulting monolay-
ers for 7 days in the remaining experiments.
Next, before proceeding to pellet culture, we analyzed the
expression of cell surface markers on hiPSCs in monolayer
culture. Flow cytometric analysis demonstrated that the
majority of hiPSCs expressed CD90 (Thy-1), CD44, and
CD166 (activated leukocyte cell adhesion molecule) (Fig. 3).
A small fraction of hiPSCs was positive for CD146 (MUC18),
CD73, CD105 (endoglin), and CD140a (platelet-derived
growth factor receptor a [PDGFRa]) (Fig. 3). CD73, CD90,
CD105, CD44, CD166, and CD146 are known to be mark-
ers for MSCs [29,30]. PDGFRa is a marker for paraxial
mesodermal cells, from which cartilage tissues originate,
especially in mouse development [31]. TRA-1-60, an undif-
ferentiated cell marker both for hiPSCs and hESCs, was not
detected in monolayer-cultured hiPSCs (Fig. 3), suggesting
that the multistep culture process promoted differentiation of
hiPSCs. CD34 and CD45, hematopoietic markers that are not
expressed in MSCs [29], were also not detected in mono-
layer-cultured hiPSCs (Fig. 3). However, we did not detect
the expression of the MSC markers Stro-1 and CD106. Stro-1
has been established as a monoclonal IgM derived from mice
immunized with human CD34-positive bone marrow cells
[32]. Recently, it was reported that Stro-1 is a 75-kDa endo-
thelial antigen, and that its expression on MSCs might result
from induction of MSCs to endothelial lineage [33]. Mono-
layer-cultured hiPSCs in our method may commit to differ-
entiating into mesenchymal lineage cells, which do not
contain endothelial antigens. Furthermore, few reports have
demonstrated the expression of Stro-1 on MSCs derived from
hiPSCs and hESCs, although many attempts have been made
to induce MSCs from hiPSCs and hESCs [34]. In a recent
report describing one-step derivation of MSC-like cells from
hiPSCs, MSC-like cells did not express Stro-1, although the
cells clearly expressed CD73, CD90, CD105, CD144, and
CD166 [35]. A Nestin(+)/CD271(-)/Stro-1(-) cell popula-
tion from hESCs was recently reported to be mesenchymal-
like precursors [36]. MSCs induced from hiPSCs or hESCs in
vitro may exhibit different characteristics, including surface
marker expression, from those of MSCs isolated primarily
from bone marrow and other organs. CD106 is also known
as vascular cell adhesion molecule-1 [37]. Several groups
have reported changes in the expression of cell surface
markers, including CD106, following prolonged cultivation
[37,38]. Treatment with hyaluronan, a major glycosamino-
glycan ligand of CD44, has also been reported to upregulate
the expression of CD106 in MSCs [39]. Thus, it is possible
that our culture method, which consists of 3 weeks of culture
under 3 different conditions, attenuates the expression of
FIG. 2.
hiPSCs for monolayer culture.
Phase-contrast micrograph of
monolayer cultured hiPSCs at
40· magnification 7 days af-
ter plating onto adhesive cul-
ture dishes. To determine the
ideal cell density, sprouted
cells from EBs were dissoci-
ated by trypsin/EDTA and
serially diluted to yield cell
densitiesof
cells/cm2. A cell density at 1–
2·104cells/cm2was deter-
mined to be ideal for cell
proliferation without cell ag-
gregation.
Ideal cell density of
0.05–20·104
4 KOYAMA ET AL.

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CD106. Nonetheless, expression of CD90, CD44, CD166,
CD146, CD73, CD105, and CD140a and the lack of CD34 and
CD45 suggest that at least a fraction of hiPSCs in monolayer
culture differentiated along the mesenchymal lineage. In-
deed, osteogenic differentiation
hiPSCs was confirmed using a previously described induc-
tion medium [7] (data not shown).
of monolayer-cultured
Chondrogenic differentiation of hiPSCs
by pellet culture
The original in vitro chondrogenic differentiation method
was initially established using rabbit and human bone
marrow-derived mesenchymal progenitor cells [17,18]. The
high-density 3D microenvironment is thought to facilitate
cell–cell and cell–matrix interactions and to mimic in vivo
limb development, in which mesenchymal condensation
occurs before chondrogenic induction. In a pellet or micro-
mass culture system, cells change their morphology, express
chondrogenic differentiation markers, and produce an ex-
tracellular matrix that contains acidic proteoglycans, which
stain positive for Alcian blue and toluidine blue. In a pre-
vious report, micromass culture using hESCs dissociated
from EBs exhibited chondrogenesis superior to that of a two-
dimensional (2D) EB direct-plating outgrowth system [40].
In the present study, we dissociated EBs and selected only
the cells that proliferated in monolayer culture in the MSC
growth medium. After 3 days of pellet culture, little Alcian
blue staining was observed, and hiPSCs inside the pellet
exhibited fibroblast-like morphology (Fig. 4A). At day 7 of
induction, pellets showed partial Alcian blue staining and
contained some large spherical cells with typical chon-
drocyte-like morphology (Fig. 4A). At day 14 of induction,
spherical cells and interstitium, which was clearly stained
FIG. 3.
growth conditions for 7 days, and then dissociated by trypsin/EDTA for pellet culture. Flow cytometric analysis of cell
surface marker expression on dissociated hiPSCs revealed that the major fraction of hiPSCs expressed CD90, CD44, and
CD166, and that a fraction of hiPSCs expressed CD146, CD73, CD105, and CD140a. hiPSCs were negative for TRA-1-60, a
marker of undifferentiated cells, and for hematopoietic markers (CD34 and CD45). Gray lines show staining with the relevant
isotype-matched control antibodies.
Expression of MSC markers by monolayer cultured hiPSCs. Cells were cultivated in monolayer culture under MSC
CHONDROGENIC DIFFERENTIATION OF HUMAN IPS CELLS5

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with Alcian blue, were evident throughout the pellet (Fig.
4A). At day 21 of induction, the entire pellet was intensely
stained with Alcian blue, suggesting maturation of cartilag-
inous extracellular matrix (Fig. 4A).
Before chondrogenic induction by pellet culture, hiPSCs in
monolayer culture were small and fibroblast-like (Fig. 4B).
However, at day 21 of induction, hiPSCs located inside the
pellet exhibited large spherical morphology (Fig. 4C) and
were surrounded by Alcian blue–positive (Fig. 4D) and to-
luidine blue–positive (Fig. 4E) acidic proteoglycans. At day
28 of induction, many pellets exhibited internal necrosis
(data not shown). Thus, in our method, the differentiation of
hiPSCs appeared to reach a plateau at day 21 of induction.
The cell morphology and positive staining for Alcian blue
and toluidine blue indicated that hiPSCs differentiated into
the chondrogenic lineage. To confirm the chondrogenic dif-
ferentiation capability of hiPSCs, we examined 2 more hiPSC
lines. One line (iPS-TIG107) was generated from TIG-107
FIG. 4.
cell pellets stained with Alcian blue/hematoxylin/eosin (HE) revealed progressive chondrogenic differentiation of hiPSCs.
The positive area and intensity of Alcian blue staining, which indicated the existence of acidic proteoglycans, increased
progressively (A). hiPSCs in monolayer culture before pellet culture exhibited small size and fibroblast-like morphology (B).
After pellet culture for 21 days, staining with HE revealed spherical cell morphology and interstitium (C). Micrographs at
high magnification revealed positive staining with Alcian blue (D) and metachromasy of toluidine blue (E) in interstitium of
pellets cultured in the chondrogenic induction medium for 21 days. (A), 100· magnification; (B), 40· magnification; (C–E),
200· magnification.
Histological analysis of chondrogenic differentiation from hiPSCs (B7). Histological analysis of paraffin-embedded
FIG. 5.
stained with Alcian blue/HE indicated progressive chondrogenic differentiation similar to that observed with hiPSCs. The
positive area and intensity of Alcian blue staining increased progressively (A). hESCs in monolayer culture before pellet
culture exhibited morphology similar to that of hiPSCs in monolayer culture (B). After pellet culture for 21 days, staining with
HE revealed spherical morphology typical of chondrocytes and interstitium (C). hESCs also presented positive staining with
Alcian blue (D) and metachromasy of toluidine blue (E) in interstitium of pellets cultured with chondrogenic induction
medium for 21 days. (A), 100· magnification; (B), 40· magnification; (C–E), 200· magnification.
Histological analysis of chondrogenic differentiation from hESCs (H9). Histological analysis of hESC-derived pellets
6KOYAMA ET AL.

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fibroblasts derived from the skin of an 81-year-old Japanese
woman by introducing 4 reprogramming factors using ret-
roviruses. Another line, 253G1 (G1), was derived from the
same donor as B7, but generated by introducing 3 factors
(Oct 3/4, Sox2, and Klf4, but not c-Myc) using retroviruses
[41]. These 2 iPSC lines (iPS-TIG107 and G1) exhibited
chondrogenic differentiation capability and time course
similar to those of B7 (Supplementary Fig. S4 and S5), al-
though the level of matrix synthesis appeared to be smaller
in G1-derived pellets.
Comparative analysis of chondrogenic
differentiation capability of hiPSCs and hESCs
We performed the procedure described above with hESCs
(H9) and compared their chondrogenic differentiation capa-
bility to that of hiPSCs. During 21 days of induction, hESCs
demonstrated a sequence of histological changes similar to
that observed with hiPSCs (Fig. 5A). At day 3 of induction,
some areas of hESC-derived pellets were faintly stained with
Alcian blue, and some hESCs already exhibited chondrocyte-
like spherical morphology. At day 7 of induction, the whole
hESC-derived pellet was stained with Alcian blue. In com-
parison, hiPSC-derived pellets showed only partial staining
at this time. At day 14 of induction, the whole pellet was
more strongly stained with Alcian blue. At day 21 of in-
duction, the appearance of cells with chondrocyte-like mor-
phology and Alcian blue staining of the interstitium were
more apparent, although the difference between days 14 and
21 was not significant. Thus, compared to hiPSCs, hESCs
exhibited slightly faster progression of chondrogenic induc-
tion. Before chondrogenic induction by pellet culture, the
morphology of monolayer-cultured hESCs was also fibro-
blast-like, similar to that of hiPSCs (Fig. 5B).
hESCs began showing morphological changes at day 3,
and matured progressively thereafter (Fig. 5C). At day 21,
the hESC-derived pellets showed intense Alcian blue
staining (Fig. 5D) and toluidine blue metachromasy (Fig.
5E), as was the case for hiPSCs. At day 28 of induction,
many hESC-derived pellets also exhibited internal necrosis
(data not shown). About 85% of hESC-derived pellets (11
successful pellets/13 experiments), compared to 73% of
hiPSC-derived pellets (8 successful pellets/11 experiments),
exhibited successful chondrogenic differentiation. We also
applied this culture protocol to another hESC line, KhES-1
[42], and found that it exhibited similar chondrogenic dif-
ferentiation (Supplementary Fig. S6). These data demon-
strate that in our multistep culture method, both hiPSCs
and hESCs differentiate into the chondrogenic lineage, al-
though hESCs exhibited slightly faster progression and in-
creased efficiency of chondrogenic differentiation compared
to hiPSCs.
Time course of gene expression of chondrogenic
differentiation markers
We analyzed the time course of chondrogenic differenti-
ation marker expression by real-time RT-PCR. The chon-
drogenic transcription factor Sox9 is required for precartilage
condensation and directly regulates the transcription of type
II collagen and aggrecan [43–45]. Type II collagen is an early
chondrogenic differentiation marker, and aggrecan is a major
sulfated proteoglycan of the cartilage matrix and a highly
specific marker of differentiated chondrocytes [45]. SOX9
expression was already present in hiPSCs at day 0 of
monolayer culture (Fig. 6A), temporarily decreased at day 3
before the elevation of expression of COL2A1 and ACAN,
and gradually increased thereafter. COL2A1 exhibited low
expression at day 0, but was gradually upregulated at day 3
and 7, and was over 60-fold upregulated by day 14 (Fig. 6B).
The expression of ACAN was also quite low initially (at days
0 and 3), but was dramatically upregulated at day 7 and
increased further thereafter (Fig. 6C). The expression of
COL10A1, a specific marker for hypertrophic chondrocytes,
FIG. 6.
differentiation markers was analyzed by real-time reverse transcription–polymerase chain reaction. Both hiPSCs and hESCs in
monolayer culture exhibited high expression of SOX9 (A and D). After the expression of SOX9 was downregulated, the expression
of type II collagen (COL2A1) (B and E) and aggrecan (ACAN) (C and F) increased progressively, especially from day 7 onward. The
expression of SOX9 was increased again at later stages (day 14 and 21). No type X collagen (COL10A1) expression was detected in
either hiPSCs or hESCs (data not shown). *Significant change relative to control (day 0) (n=3). Data are shown as the mean–SD.
Marker gene expression during chondrogenic differentiation of hiPSCs and hESCs. The expression of chondrogenic
CHONDROGENIC DIFFERENTIATION OF HUMAN IPS CELLS7

Page 8

was not detected at any time during the culture period (data
not shown). hESCs showed similar trends in SOX9, COL2A1,
and ACAN expression, but the increase in COL2A1 levels
was more modest, and the increase of ACAN was larger in
hESCs than in hiPSCs (Fig. 6D–F). COL10A1 expression was
not detected in hESCs at any time during the culture period
(data not shown).
Immunohistochemical analysis of chondrogenic
differentiation markers
After confirming the expression of chondrogenic differ-
entiation markers in both hiPSC- and hESC-derived culture
pellets at the mRNA level, we next examined the expression
of these markers by immunohistochemistry. Sections of
hiPSC-derived pellets stained with hematoxylin/eosin (HE)
exhibited spherical cell morphology characteristic of chon-
drocytes (Fig. 7A). Although we tried several different
antibodies, we detected no type II collagen signal in hiPSC-
derived pellets at day 21 of induction (Fig. 7B). Weak ag-
grecan expression was detected in hiPSC-derived pellets at
day 21 of induction (Fig. 7C). Staining of NHAC-kn-derived
pellets with HE revealed large spherical cell morphology and
rich interstitium, suggesting greater maturation in NHAC-
kn-derived pellets than in hiPSC-derived pellets (Fig. 7E).
Unlike hiPSC-derived pellets, NHAC-kn-derived pellets ex-
hibited strong positive staining both for type II collagen and
aggrecan (Fig. 7F, G). Human BMMSC (hBMMSC)-derived
pellets were cultivated in DMEM/F12, which contained only
10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin,
for 21 days as negative control. hBMMSC-derived pellets
did not exhibit cell morphological change (Supplementary
Fig. S7A) or the expression of chondrogenic markers;
Type II collagen (Supplementary Fig. S7B) and aggrecan
(Supplementary Fig. S7C). Positive signals for human-specific
mitochondria in the hiPSC-, NHAC-kn-, and hBMMSC-derived
specimens (Fig. 7D, H, Supplementary Fig. S7D) suggested that
lack of staining for chondrogenic markers was not due to a
general loss of antigenicity during specimen processing. In the
specimens from hESC-derived pellets, type II collagen was not
detected, and aggrecan was weakly detected, as with the
hiPSC-derived pellets (data not shown). Taken together, these
data suggested that hiPSCs and hESCs differentiated into
chondrogenic lineage cells with similar characteristics, but
neither hiPSCs nor hESCs yielded fully mature chondrocytes.
Discussion
In this study, we developed a simplified, reproducible
method for differentiating hiPSCs into the chondrogenic
lineage in vitro. Our method does not involve genetic ma-
nipulation, making it suitable for studying the mechanism of
idiopathic genetic diseases, such as skeletal dysplasia, and
for evaluating drug effects on chondrocytes. Our protocol
consists of 4 steps: (1) EB formation, which involves spon-
taneous differentiation and mimics the formation of the 3
germ layers in vitro; (2) Cell outgrowth from EBs to increase
cell number; (3) Monolayer culture to remove undifferenti-
ated cells and select cells that can adapt to MSC growth
conditions; and (4) 3D pellet culture, which is important for
cell–cell and cell–matrix interactions. Our pellet culture me-
dium was supplemented with TGF-b3 and dexamethasone,
since these factors were previously demonstrated to produce
the highest chondrogenic potential in 3D culture reported to
date [46]. In our protocol, differentiation into the mesen-
chymal lineage during EB formation and monolayer culture
was critical. In addition, cell density was a crucial factor for
monolayer culture. The ideal cell density for monolayer
FIG. 7.
from paraffin-embedded hiPSC pellets cultivated for 21 days in the chondrogenic induction medium were stained with HE
(A). Normal human articular chondrocytes from knee cells (NHAC-kn) cultivated for 21 days according to the same pellet
culture protocol, used as a positive control, were also stained with HE (E). Sections in both (A) and (E) contained large
spherical cells typical of chondrocytes and rich interstitium that was positive for Alcian blue (data not shown). Im-
munohistochemical staining of hiPSC-derived pellets failed to detect expression of type II collagen (B). Staining for aggrecan
was faintly positive (C). The sections from NHAC-kn stained strongly for both type II collagen (F) and aggrecan (G). Human-
specific mitochondrial staining was detected in both the hiPSC-derived pellet (D) and NHAC-kn–derived pellet (H). (A–H),
100· magnification.
Immunohistochemical analysis of chondrogenic differentiation markers in pellets derived from hiPSCs. Sections
8 KOYAMA ET AL.

Page 9

culture in our method was the same as that previously re-
ported for induction of chondrocytes from hESCs in an
arginine-glycine-aspartate–modified hydrogel combined with
EB formation and monolayer culture [47], suggesting that
the optimal culture condition for chondrogenic differentia-
tion of hiPSCs is very close to that of hESCs.
Other attempts to induce MSCs from ESCs and iPSCs have
also been reported. For instance, MSCs or MSC-like cells
have been derived from hESCs by either transfection of a
human telomerase reverse transcriptase (hTERT) gene [48] or
by coculture with mouse OP9 cells [34]. However, the use of
exogenous genetic material or mouse cells in those protocols
introduces, respectively, the risk of tumorigenicity or con-
tamination by animal pathogens. To overcome these prob-
lems, hESCs have been plated directly on gelatin-coated
dishes without EB formation and cultured with bFGF and
PDGF-AB [49]. In that study, after 1 week, CD105-positive
and CD24-negative cells were sorted as MSCs. Those cells
expressed MSC markers, such as CD44, CD49a, CD105, and
CD166. However, expression of type II collagen mRNA de-
creased progressively after chondrogenic induction, even
though the immunostaining for type II collagen was positive.
Recently, mesenchymal progenitor cells were induced from
murine iPSCs using a method similar to ours [50]. In that
method, murine iPSC-derived EBs were transferred to cul-
ture dishes and cultivated in DMEM supplemented with 10%
FBS and 10-6M all-trans retinoic acid. Sprouted cells from
EBs were dissociated with trypsin/EDTA, seeded onto
gelatin-coated dishes, and cultivated in the MesenPRO RS?
Medium (Invitrogen) supplemented with bFGF. The result-
ing induced mesenchymal progenitor cells expressed mark-
ers for MSCs. Thus, a culture protocol consisting of EB
formation, cell outgrowth from EBs, and monolayer culture
after dissociation into single cells appears to be suitable for
mesenchymal differentiation from iPSCs and ESCs. This
method is simple, reproducible, and results in good cell vi-
ability, although some details still need to be refined. In the
present study, to reduce the risk of cell damage, we did not
use cell sorting. However, purification of mesenchymal
progenitor cells by cell sorting may improve the efficiency of
chondrogenic differentiation, and should be combined with
our approach in future studies.
The expression of sequential markers of chondrogenic
differentiation in vitro is inconsistent among studies. Sox9
belongs to the Sry-related high-mobility-group box (Sox)
transcription factors and directly regulates the expression of
several chondrogenic markers, including type II collagen and
aggrecan [43–45]. Sox9 was identified as a causative gene of
campomelic dysplasia, with heterozygous patients exhibiting
disproportionately short stature, bowing of the limbs, low
ears, depressed nasal bridge, talipes equinovarus, long
philtrum, and micrognathis [51,52]. Heterozygous Sox9
mouse mutants exhibited the same skeletal abnormalities as
campomelic dysplasia patients, and histological analysis re-
vealed smaller and delayed chondrogenic mesenchymal
condensation and enlargement of the hypertrophic zone in
association with premature mineralization [53]. During
mouse embryogenesis, Sox9 is expressed in all chon-
droprogenitors and chondrocytes except for hypertrophic
chondrocytes. Thus, Sox9 is required for the commitment of
undifferentiated MSC to the chondrogenic lineage, for mes-
enchymal condensation, and for inhibiting hypertrophic
conversion of proliferating chondrocytes. Gong et al. re-
ported that micromass culture using hESCs (H9) resulted in
chondrogenic differentiation, with Sox9 expression elevated
at the early stage and strikingly downregulated at the in-
termediate stage of inductive culture [54]. In their report, no
type X collagen expression was detected even at late stages
of induction, which the authors deemed favorable, because
chondrocytes in articular cartilage do not normally undergo
hypertrophic maturation. Meanwhile, a report by Toh et al.,
in which hESCs (line H1 and H9) were differentiated into
hypertrophic chondrocytes following micromass culture of
EB-derived cells, demonstrated the expression of type X
collagen, which was detected by both PCR and immuno-
histochemistry [40]. However, in another report Toh et al.
induced chondrogenic differentiation of hESCs (H9) by a
combination of micromass culture of EB-derived cells and
pellet culture after cell dissociation; they demonstrated that
Sox9 expression increased continuously for 21 days in mi-
cromass culture, whereas no type X collagen expression was
detected even after pellet culture for another 4 weeks [55]. In
a recent study, chondrocytes could be induced from hESCs
in 2D culture by a multistep process that applied different
cell density and different combinations of growth factors to
each stage [56]. In that study, differentiated cells expressed
Sox9, CD44, and type II collagen, but did not express type X
collagen. In our experiments, the expression of SOX9 was
already elevated at the start of pellet culture, decreased
promptly, and increased again at a later stage. Because Sox9
inhibits hypertrophic conversion of proliferating chon-
drocytes [53], increased expression of SOX9 at a late stage
might explain why the expression of type X collagen was not
detected at any time during the culture period. Moreover,
negative expression of type X collagen is not necessarily
unfavorable, because hyaline cartilage, which constitutes
articular cartilage, does not express type X collagen [54].
The cause of the discrepancy between gene expression and
protein expression in cell pellets needs to be elucidated. Al-
though RT-PCR revealed that expression of type II collagen
and aggrecan mRNAs was obviously increased, immuno-
histochemistry detected only weak staining for aggrecan and
no staining for type II collagen. The reason for negative
immunostaining of type II collagen is not known. However,
a discrepancy between gene expression and protein expres-
sion of type II collagen has been reported previously, sug-
gesting that mRNAs containing AU-rich elements are
destabilized when translated [49]. There is a possibility that
translation might be destabilized in our culture conditions.
Another possibility is that cells in pellets are still immature
chondrocytes, and do not produce enough collagen to be
detected by immunohistochemistry. To achieve in vitro
chondrogenic differentiation, 3D pellet culture in the pres-
ence of TGF-b superfamily members seems to be essential.
While pellet culture is definitely an effective tool, it is not
without its limitations. Another critical factor is oxygen
tension. The physiological environments of both articular
cartilage and bone marrow exist within a range of 1%–7%
oxygen. Previous studies have shown that hypoxia promotes
chondrogenic differentiation of MSCs [57,58] and that hyp-
oxia increases the expansion potential of MSCs [59,60]. Ad-
ditionally, Sox9 is upregulated by hypoxia, resulting in
increased expression of type II collagen and aggrecan in
human articular chondrocytes. Thus, it is possible that
CHONDROGENIC DIFFERENTIATION OF HUMAN IPS CELLS9

Page 10

hypoxic culture conditions would promote chondrogenic
differentiation of hiPSCs in combination with our protocol.
In addition, the combination of TGF-b in the induction me-
dium with various growth factors, such as growth and dif-
ferentiation factor 5 [61], cell sorting by MSC markers, and
subjecting cultures to cyclic hydrostatic pressure [62] might
also improve maturation of chondrocytes in our method,
resulting in enhanced expression of type II collagen and
aggrecan protein.
In our method, the ability of hESCs to differentiate into the
chondrogenic lineage was similar to that of hiPSCs, although
hESCs exhibited slightly faster progression and greater effi-
ciency of chondrogenic differentiation compared to hiPSCs.
However, a slight difference in the progression and efficiency
of chondrogenic differentiation might arise from clonal var-
iation, since different ESC and iPSC lines can vary in their
characteristics [63,64]. In our study, G1-derived pellets
seemed to produce less matrix than B7 and iPS-TIG107 lines.
It is difficult for our method to control evenly the differen-
tiation time course and the characteristics of induced chon-
drocytes from different cell lines.
In conclusion, we have developed a simplified and re-
producible culture method that allows hiPSCs and hESCs to
be differentiated into the chondrogenic lineage via genera-
tion of mesenchymal progenitor cells. Although the induc-
tive culture method still needs improvement, our study
provides an important foundation for cartilaginous tissue
engineering, which will benefit regenerative medicine,
pathogenesis studies of idiopathic skeletal dysplasia, and the
development of drug screening systems for cartilaginous
diseases.
Acknowledgments
We thank J. Toguchida and A. Nasu, Center for iPS Cell
Research and Application, Kyoto University, and H. Yao,
Department of Transfusion Medicine and Cell Therapy,
Kyoto University, for technical advice. This work was sup-
ported by the project for realization of regenerative medicine
and Grant-in-Aid for Scientific Research (22591009) from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan, Grant-in-Aid for Scientific Research from the
Ministry of Health, Labor, and Welfare of Japan, and Novo
Nordisk Study Award for Growth and Development 2011.
Author Disclosure Statement
The authors indicate no potential conflict of interests.
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Address correspondence to:
Dr. Masako Miura
Department of Medicine and Clinical Science
Kyoto University Graduate School of Medicine
54 Shogoin-Kawaharacho, Sakyo-ku
Kyoto 606-8507
Japan
E-mail: mmiura@kuhp.kyoto-u.ac.jp
Received for publication March 14, 2012
Accepted after revision July 18, 2012
Prepublished on Liebert Instant Online XXXX XX, XXXX
12 KOYAMA ET AL.