Matsumoto et al. Orphanet Journal of Rare Diseases 2013, 8:190
http://www.ojrd.com/content/8/1/190

RESEARCH

Open AccessInduced pluripotent stem cells from patients withhuman fibrodysplasia ossificans progressiva show
increased mineralization and cartilage formation
Yoshihisa Matsumoto1,2,3†, Yohei Hayashi4†, Christopher R Schlieve4,5,6†, Makoto Ikeya2*, Hannah Kim6,
Trieu D Nguyen4, Salma Sami4, Shiro Baba4, Emilie Barruet6, Akira Nasu1,2,7, Isao Asaka8, Takanobu Otsuka3,
Shinya Yamanaka4,9, Bruce R Conklin4,10, Junya Toguchida1,2,7* and Edward C Hsiao4,6*

Abstract
Background: Abnormal activation of endochondral bone formation in soft tissues causes significant medical diseases
associated with disability and pain. Hyperactive mutations in the bone morphogenetic protein (BMP) type 1 receptor
ACVR1 lead to fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterized by progressive ossification
in soft tissues. However, the specific cellular mechanisms are unclear. In addition, the difficulty obtaining tissue samples
from FOP patients and the limitations in mouse models of FOP hamper our ability to dissect the pathogenesis of FOP.
Methods: To address these challenges and develop a “disease model in a dish”, we created human induced pluripotent
stem cells (iPS cells) derived from normal and FOP dermal fibroblasts by two separate methods, retroviral integration or
integration-free episomal vectors. We tested if the ability to contribute to different steps of endochondral bone formation
was different in FOP vs. control iPS cells.
Results: Remarkably, FOP iPS cells showed increased mineralization and enhanced chondrogenesis in vitro. The
mineralization phenotypes could be suppressed with a small-molecule inhibitor of BMP signaling, DMH1. Our results
indicate that the FOP ACVR1 R206H mutation favors chondrogenesis and increases mineral deposition in vitro.
Conclusions: Our findings establish a FOP disease cell model for in vitro experimentation and provide a proof-of-concept
for using human iPS cell models to understand human skeletal disorders.
Keywords: Induced pluripotent stem cells, Fibrodysplasia ossificans progressiva, FOP, Disease model in a dish,Endochondral ossification

Background
Tissue mineralization is one of the most dramatic transitions in normal development and disease. Hard tissues,
such as bone and teeth, maintain body structure, protect
vital organs, and facilitate eating. Abnormal activationof endochondral bone formation in soft tissues fromcongenital disorders, such as fibrodysplasia ossificans* Correspondence: mikeya@cira.kyoto-u.ac.jp; togjun@frontier.kyoto-u.ac.jp;
Edward.hsiao@ucsf.edu†
Equal contributors
2
Department of Cell Growth and Differentiation, Center for iPS Cell Researchand Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku
606-8507, Kyoto, Japan1
Department of Tissue Regeneration, Institute for Frontier Medical Sciences,
Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku 606-8507, Kyoto, Japan
4
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
Full list of author information is available at the end of the article

progressiva (FOP; OMIM #135100) or from trauma or
central nervous system injury [1] cause severe disability
and pain. Although bone morphogenetic proteins(BMPs) are key regulators of osteogenesis [2] and are
implicated in pathologic calcification [3], their specificfunctions in humans are not well understood. Human
induced pluripotent stem cells (iPS cells) derived frompatients with genetic mutations affecting key signaling
pathways provide a unique opportunity to dissect humandevelopment and disease pathogenesis in models amenable to experimental manipulation [4].
FOP is a rare but debilitating disease of heterotopic
bone formation associated with mutations in the Activin
A Type I receptor (ACVR1) [5], which is activated by
BMPs. A single amino acid change (R206H) commonly© 2013 Matsumoto et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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resulting from a single base mutation (617G > A) is foundin the majority of FOP patients and can increase sensitivityof the receptor to BMP ligands [6]. Patients are born without heterotopic ossification yet characteristically form large
amounts of bone either spontaneously or after trauma [7].
The heterotopic ossification follows an endochondral boneformation pathway that starts with a pre-cartilaginous
fibro-proliferative anlage and eventually mineralizes. The
ferocity of these episodes makes it nearly impossible to obtain tissue samples prospectively and significantly complicates surgical management.
Here, we created a series of iPS cells from dermal fibroblasts of donors with FOP by either DNA-integrating
retroviral or integration-free episomal plasmid methods[4,8]. The FOP iPS cells showed increased mineral deposition and chondrogenesis, potentially reflecting a predisposition towards endochondral bone formation in
FOP. Our findings provide a valuable foundation for human stem cell-based systems to delineate the mechanisms of normal and pathologic skeletal formation.

Table 1 Cell lines used in this studyvWT-TIG120-4f1

HDF

Materials and methods

vWT-201B2

HDF

Cell culturevWT-201B7

HDF

Primary human dermal fibroblasts (HDFs) from commercial sources or from 3-mm skin biopsies carefully obtained
from donors (without causing deep tissue injury) or fromsurgical excess were cultured [9] and are described inTable 1 and Additional file 1: Table S1. HDFs less than fivepassages old were used for iPS cell reprogramming. Presence or absence of the ACVR1 mutation was sequenced
and verified as described [5]. Primary human mesenchymal stem cells (hMSCs) were prepared from iliac bone as
described previously [10] and expanded as a monolayer.
Retroviral [4] and episomal integration-free [8] iPS cells
were derived as described. H9 human embryonic stem(ES) cells were from WiCell Research Institute (Madison,
WI). All pluripotent cell lines were maintained in mTeSR1
medium (StemCell Technologies, Vancouver, Canada) on
growth-factor-reduced Matrigel (BD Biosciences)-coatedplates (150–300 μg/ml, 30 min coating) or in primate ES
cell medium (ReproCELL, Tokyo, Japan) on mitomycin Ctreated or irradiated SNL feeder cells [11]. SNLs were
carefully removed by at least one passage in feeder-free
conditions before use in differentiation assays. The ROCK
inhibitor Y-27632 (10 μM, Tocris Bioscience, Minneapolis,
MN) dissolved in DMSO was added to mTeSR1 at passaging and removed the following day. Karyotyping wasdone by Cell Line Genetics (Madison, WI) or Nihon Gene
Research Laboratories (Sendai, Japan). Cells exposed to recombinant BMP4 protein (R&D Systems, Minneapolis,
MN) were treated for 45 minutes.
All human tissue collection, human stem cell studies,
procedures, and written consents were approved by the
UCSF Committee on Human Research, the UCSF Gamete

FOP retroviral iPS cell lines (ACVR1 R206H)*

Name

OriginHuman dermal fibroblasts
HDF-WTaSkin

HDF-WTb

Skin

FF-WT-BJ

Foreskin (Stemgent 08–0027)

HDF-WT-1323

Fibroblast (Cell Applications, 1323)

HDF-WT-1388

Fibroblast (Cell Applications, 1388)

HDF-WT-TIG120

Collection of Research Bioresources, Japan

HDF-FOP1

Skin Fibroblast, Corielle GM00513

HDF-FOP2Skin Fibroblast, Corielle GM00783

HDF-FOP3Skin

HDF-FOP4Skin

HDF-FOP5

Skin

Control retroviral iPS cell lines (wildtype ACVR1)*
vWTa

HDF

vWTb

HDF

vFOP1-1

HDF-FOP1

vFOP1-4

HDF-FOP1

vFOP2-1

HDF-FOP2

vFOP2-2

HDF-FOP2

vFOP4-1

HDF-FOP4

vFOP4-3

HDF-FOP4

vFOP5-22

HDF-FOP5

Control episomal iPS cell lines (Wildtype ACVR1)**
eWT-1323-2

HDF-1323

eWT-1323-4

HDF-1323

eWT-BJ2

FF-BJ

eWT-BJ4

FF-BJ

FOP episomal iPS cell lines (ACVR1 R206H)**
eFOP1-1

HDF-FOP1

eFOP1-10

HDF-FOP1

eFOP2-3

HDF-FOP2

eFOP2-8

HDF-FOP2

eFOP3-2

HDF-FOP3

eFOP3-4

HDF-FOP3

Summary of the FOP and control human dermal fibroblasts and iPS cell lines
used in this study, including naming, classification (control vs. FOP), andoriginal tissue source. HDF, human dermal fibroblast; FF, foreskin fibroblast.
Please see Additional file 1: Table S1 for a detailed summary of the iPS cellcharacterization. Method of making iPS cells: *, Retroviral OSKM ; **, Epi(Y4).

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and Embryonic Stem Cell Research Committee, or by the
Ethics Committee of the Department of Medicine andGraduate School of Medicine, Kyoto University.
Embryoid body formation

Embryoid bodies (EBs) were formed from iPS cells or human ES cells once their cultures reached 80% confluence.
After washing with PBS, Accutase (StemCell Technologies, Vancouver, Canada) was applied for two minutes
to remove cells from the plate. Cells were centrifuged at
175 × g for two minutes and then resuspended in a
4:1 mix of EB differentiation medium (80% Knockout
DMEM, 20% FBS, 1% Glutamax, 1% non-essential aminoacids, and 0.1 mM 2-mercaptoethanol) and mTeSR1, andsupplemented with 10 μM Y-27632. Cells were plated onto
ultra-low attachment plates without medium changes forseven days. On day eight, EBs were collected and allowed
to settle in a conical tube for 30 minutes. The mixed
medium was removed and replaced with 100% EB differentiation medium (Knockout DMEM supplemented with 20%
FBS, 1% Glutamax, 1% non-essential amino acids, and
0.1 mM 2-mercaptoethanol) changed every three to fourdays. EBs were then transferred to gelatinized plates and cultured until day 15 for RNA collection in Trizol (Invitrogen).

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light golden-yellow staining of the cell layer. Human iPS
cells maintained in feeder-free conditions were plated in
20% mTeSR1 mixed with 80% OB mineralization medium
and Y-27632 (10 μM) at 2 million, 400,000, or 37,500
cells per well of gelatin-coated 6-, 24-, or 96-wellplates, respectively. Medium was replaced on day two
with 100% OB mineralization medium and changed
every other day. Samples for von Kossa staining were
fixed in 4% paraformaldehyde (PFA), stained in 5% silver nitrate for 15 minutes, and developed in 5% sodium
carbonate/9.25% formaldehyde for two minutes. Regions of increased mineralization (dark black staining)can be seen at the edges of the well, where the culture
surface meets the well wall. This was observed for all
cell types and was excluded from staining intensity analysis. ImageJ (ver. 1.44o) was used for staining intensity
analysis [13]. DMH1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline; from DainipponSumitomo Pharma or Nacalai Tesque, Tokyo, Japan) wasused at indicated concentrations; levels above 10 μM
caused cell death. Scanning electron microscopy was performed on an Olympus TM3000 scanning electron microscope by the Gladstone Microscopy Core.
Chondrogenic differentiation

Teratoma formation

iPS cells grown in six-well Matrigel-coated plates to100% confluence were released with Accutase for 30
secs, rinsed twice with PBS, and resuspended in mTeSR1
supplemented with 10 μM Y-27632. Cells (1 × 106 in
20 μl) were injected into 8–14 week-old male CB17
SCID mice (Charles River) under the testis capsule as
described [4]. A minimum of six testes were injected per
iPS cell line. Tumors were collected 8–12 weeks after injection, fixed with 10% neutral buffered formalin for
24 hours, and processed for paraffin-embedded sections
the Gladstone Histology and Microscopy Core or at theDivision of Technical Support of the Institute for Frontier Medical Sciences in Kyoto University. All mousestudies were approved by the UCSF Institutional Animal
Care and Use Committee, or performed in strict accordance with the Regulations on Animal Experimentation at
Kyoto University.
Mineralization assay

Primary human MSCs were cultured in OB mineralization
medium (DMEM with 20% FBS, glycerol-2-phosphate,
4 μM dexamethasone, 0.1 mM 2-mercaptoethanol, and50 μg/ml L-ascorbic acid 2-phosphate sesquimagnesium
salt hydrate [12]). We could detect mineralization activity
after 12 days as increased black staining by von Kossa(Additional file 2: Figure S1B), which was the preferredstaining method used because the black mineralization
nodules could be easily distinguished from the expected

Pellet chondrogenic cultures were performed as described[14]. Briefly, human iPS cells collected with a scraper weresuspended as clumps in EB formation medium (DMEM,
10% Knockout Serum Replacement (KSR, Invitrogen),
10% FBS) and cultured for seven days on non-adherent
bacterial petri dishes. The medium was changed every
three days. EBs were landed onto 10-cm gelatin-coatedtissue-culture dishes, grown to confluence, released with
0.25% trypsin/EDTA, filtered through a 70-μm cellstrainer, and seeded onto new gelatin-coated dishes. Once
confluent, cells were collected and 2.5 × 105 cells wereplaced into a 15-ml polypropylene tube, centrifuged at
1200 rpm for three min, and re-suspended in chondrogenic medium (high-glucose DMEM, with human 10 ng/
ml TGF-β3, 100 nM dexamethasone, 6 μg/ml insulin,
100 μM ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 6 μg/ml transferrin, 0.35 mM proline, and 1.25 mg/
ml bovine serum albumin). Cells were re-centrifuged andmaintained as a small pellet in the conical tubes for 28 days
with medium changes every three days. Glycosaminoglycan content was quantified with BLYSCAN Dye and Dissociation reagents (BIOCOLOR, Belfast, UK). DNA
content was quantified using a PicoGreen dsDNA quantitation kit (Invitrogen).
RT-PCR and quantitative PCR expression analysis

Semi-quantitative reverse transcription expression analysisof endogenous pluripotency genes, persistent expressionof the retroviral iPS cell induction factors, episomal

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factors, or episomal gene expression, were performed on
DNase-treated RNAs with the primers and PCR conditions as described [4,8]. Quantitative PCR reactions were
performed using 10 ng of reverse-transcribed cDNA in a
final volume of 5 μl with ABI’s TaqMan Universal PCR
Master Mix (Life Technologies) or ABI’s Syber Green PCR
Master Mix at the recommended reagent ratios with theprimers or probesets listed in Additional file 3: Table S2.
Each reaction was run in technical triplicates on an Applied Biosystems 7900HT or Viia7 thermocycler (LifeTechnologies) and normalized to GAPDH as an endogenous control using protocols recommended by ABI.

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did not contain any exogenous BMPs that might mask
the effects of the ACVR1 R206H mutation. This medium
still promoted mineralization in primary human MSCs
(Additional file 2: Figure S1B) when compared with standard non-mineralizing MSC maintenance medium. However, FOP HDFs showed no differences in mineralization
activity when compared to HDFs cultured in nonmineralizing HDF maintenance medium, even with anadditional 6 days of culture (Additional file 2: Figure S1C)
and despite detectable increases in BMP-related phosphoSMAD levels in FOP HDFs (data not shown). Thus, theACVR1 R206H mutation was not sufficient to induce
spontaneous mineralization in HDFs in vitro.

Immunocytochemistry

Cells or EBs were fixed with 4% PFA/PBS for 10 min atroom temperature, permeabilized with PBS/0.1% Triton
X-100 for 10 min at room temperature, then blocked with
1% BSA. Representative lineages were detected with thefollowing primary antibodies: SSEA3 (0.5 μg/ml, eBiosciences), TRA-1-60 (0.5 μg/ml, eBiosciences), TRA-1-81(0.5 μg/ml, eBiosciences), NANOG (2 μg/ml, AF1997,
R&D Systems), βIII-TUBULIN (1:100, MAB1637, Millipore), glial fibrillary acidic protein (1:500, Z0334, DAKO),
α-smooth muscle actin (pre-diluted, N1584, DAKO), αfetoprotein (2 μg/ml, MAB1368, R&D Systems), and tyrosine hydroxylase (1:100, AB152, Chemicon). Secondary
antibodies were obtained from Invitrogen: Alexa488 or
555-conjugated goat anti-mouse IgG (1:200), Alexa488 or
555-conjugated goat anti-rabbit IgG (1:200), and Alexa488
or 555-conjugated donkey anti-goat IgG (1:200). Nuclei
were stained with DAPI in the Vectashield set (Vector
Laboratories).
Statistical analysis

Because each iPS cell line was derived clonally, and thus
may display different behaviors, we treated each cell line
as an individual biological replicate and pooled our results into controls or FOP categories. p values were calculated using the Student’s t-test or Dunnett multiple
comparisons t-test. p values ≤ 0.05 were considered statistically significant.

Results
ACVR1 R206H mutation is not sufficient to induce
mineralization of fibroblasts

FOP patients show dramatic heterotopic bone formationin their soft tissues, yet do not ossify their skin. We
asked if the ACVR1 R206H mutation [6] could induce
mineralization in primary cells that do not normallyform bone. Human dermal fibroblasts (HDFs) from control donors without known skeletal disease and FOP donors were cultured, genotyped (Additional file 2: Figure
S1A), and propagated in HDF maintenance medium. For
our mineralization assays, we used a culture medium that

Human iPS cells with the ACVR1 mutation are pluripotent

Isolating primary tissues from FOP patients is extremely
difficult since injuries, including surgical procedures, induce bone formation flares. iPS cells derived from a small
skin biopsy or excess surgical material provide a potential
way to create a continuous supply of diseased tissues
in vitro for experimentation. Since BMPs induce human
ES cell differentiation [15], activated BMP signaling by the
R206H ACVR1 mutation could adversely affect our ability
to create FOP iPS cells. Fortunately our standard iPS cell
culture media (mTeSR1) is free of BMPs, thus minimizing
the potential activation of the hypersensitive R206H
ACVR1 receptor by low levels of BMP.
We first created iPS cell lines from banked FOP skin fibroblasts (FOP1 and FOP2). Retroviruses with OCT4, SOX2,
KLF4, and C-MYC [4] were transfected into two control andtwo FOP fibroblast lines. We found a significant number ofalkaline phosphatase-positive ES cell-like colonies from the
FOP fibroblasts. Two control iPS cell lines (vWTa andvWTb) and four FOP iPS lines (vFOP1-1, vFOP1-4,
vFOP2-1, and vFOP2-2) were characterized in detail andshowed the expected genotypes (Figure 1A-D). All of theretroviral iPS cell lines suppressed the C-MYC and OCT3/4
transgenes (Figure 1E). Although low levels of exogenous
KLF4 and SOX2 were detected in some lines, the iPS
cell lines still expressed genetic markers of pluripotency(Figure 1F) and could form all three germ layers in teratomas (Figure 1G, Additional file 4: Figure S2A) and in EBs(Additional file 4: Figure S2B). While large amounts of cartilage were evident in several of the teratomas derived from
FOP iPS cells, comparing the skeletal elements was not possible because of the heterogeneity of the internal structures,
overall sizes, and maturity of each individual tumor, evenfrom those derived from the same iPS cell line. All lines
retained normal karyotypes (data not shown).
Retroviral FOP iPS cells show a trend to increased
mineralization

Since FOP patients develop heterotopic endochondralbone formation, we tested if FOP and control iPS cells

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Figure 1 Characterization of retroviral iPS cell lines. (A) Representative photos of FOP fibroblasts. (B) Subsequent retroviral iPS cell colonies
on mouse feeder cells (colony indicated by dashed circle). (C) Retroviral iPS cell colonies after transition to feeder-free conditions. Black scalebar = 50 μm. (D) Retroviral iPS cell lines are heterozygous for the ACVR1 R206H (617 G > A) mutation. Control lines vWTa and vWTb1 showed awildtype ACVR1 gene. (E) The OCT3/4 and C-MYC retroviral transgenes are silenced in the human iPS cells, but KLF4 and SOX2 show detectablelevels of expression in some lines. (F) Pluripotent iPS cells show higher expression of pluripotency markers TERT and NANOG as compared todifferentiated cultures (15-day-old EBs). Gene expression is normalized to GAPDH and relative to H9 differentiated EB levels. Error bars are average
expression +/− 1 SD of technical triplicates. Gene expression studies were repeated in triplicate. (G) Teratoma images showing representative
tissues from all three germ layers. Yellow scale bar = 200 μm.

might have different predisposition to tissue mineralizationin vitro. Retroviral FOP iPS cells cultured in mineralizing
conditions showed a modest trend (p = 0.15 at 15 days) towards increased mineralization as assessed by von Kossa
staining (Figure 2A-B). Using electron microscopy, we
confirmed the presence of collagen-like fibers embeddedin the mineral (Figure 2C-D), suggesting that the increased
mineralization detected by von Kossa was not due tosimple calcium/phosphate precipitation that can occur
in cultured cell models [16]. qPCR of the mineralizing
iPS cells showed minimal changes in the expressionof RUNX2, OSX, and OC in FOP and control iPS cells(Figure 2E), consistent with the relatively mild increase inmineralization. Thus, the vFOP iPS cells showed a trend
towards increased mineral deposition by histology staining
but surprisingly mild changes in gene expression patterns.

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Figure 2 Increased mineralization in retroviral FOP iPS cells. (A) von Kossa staining of retroviral control and vFOP1 and vFOP2 iPS cells after
culture in mineralizing conditions. Mineral is detected as black staining by von Kossa. (B) Quantitation of mineralization intensity. Error bars
represent the average of the two control or four FOP iPS lines, with at least three replicates at each time, +/− 1 SEM. (C, D) Electron microscopyof day 24 mineralization cultures from vWTb or vFOP1-4 iPS cells showing collagen-like fibers (c) embedded within the mineralizing matrix (m).
Scale bar = 500 nm. (E) Quantitative PCR expression analysis of the mineralizing cultures. Two control and three FOP lines were pooled together
for the expression analysis from triplicates for each time point. Error bars represent average expression +/− 1 SEM.

Persistent expression of exogenous transgenes in iPS
cells has been reported and may be associated with partial
reprogramming [17]. To test if the ACVR1 R206H mutation favored iPS cells that retained expression of the inducing transgenes, and thus created partially-reprogrammed
cells that were unable to fully show an osteogenic phenotype, we derived a second cohort of retroviral FOP iPS celllines from fresh HDFs carefully isolated from two additional FOP patients (FOP4 and FOP5; Figure 3A-C). We
used the vWT-TIG120-4f1, vWT-201B2, and vWT-201B7
iPS cell lines [4,18] (here, preceded by vWT- for clarity) as
controls. In this additional cohort, OCT3/4, SOX2, andKLF4 transgene expression was suppressed (Figure 3D),
although a very faint band of C-MYC could be detectedin vFOP4-1 by RT-PCR. However, quantitative PCR
for transgene expression showed significant silencing of alltransgenes relative to fibroblasts transduced with retrovirus (Additional file 5: Figure S3A). The vFOP4-1,
vFOP4-3, and vFOP5-22 lines also formed teratomas containing all three germ layers (Additional file 5: Figure
S3B), had normal karyotypes (Additional file 5: Figure
S3C), expressed markers of pluripotency (Additional file 5:
Figure S3D), and retained the ACVR1 R206H (617G > A)
mutation (Figure 3C). Furthermore, differentiation of
the iPS cells in the same mineralizing conditions usedpreviously showed increased mineralization by histology(Figure 3E, F) and a similar gene expression pattern(Additional file 5: Figure S3E) as seen in the first cohort ofretroviral iPS cells. Treating vFOP4-1 and vFOP5-22
iPS cells with the BMP signaling inhibitor DMH1 [19,20]
during culture could block the mineralization phenotype(Figure 3G, H).
FOP iPS cells show increased chondrogenesis

Since the second cohort of retroviral iPS cells showed better retroviral silencing characteristics, we used this set totest if the FOP mutation might affect chondrogenesis in a
directed in vitro chondrogenesis assay. vFOP iPS cells cultured in 3-dimensional pellet cultures supplemented with
TGF-β and dexamethasone [14] produced larger pellets
with more ECM-containing chondrocyte-like cells thancontrol iPS cells (Figure 4A, B). Glucose-amino-glycan(GAG) levels were 2–3-fold higher in vFOP iPS cell chondrocyte pellets than in controls (not shown). The vFOP4
and vFOP5 pellets also showed higher numbers of larger
chondrocyte-like cells by morphology (Figure 4C-D). Quantitative PCR showed elevated mRNA levels of SOX9 and
COL2a1 (markers of immature chondrocytes) and COMP
(marker of mature chondrocytes) in the pellets (Figure 4E).
Integration-free FOP iPS cells also show increased
mineralization

Since the degree of increased mineralization variedamong the different batches of retroviral iPS cells with

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Figure 3 Increased mineralization in a second cohort of retroviral FOP iPS cells. (A, B) Representative photos of control and vFOP4 iPS cellcolonies on feeders after retroviral iPS cell generation. White scale bar = 200 μm. (C) Retroviral iPS cell lines vFOP4-1, vFOP4-3, and vFOP5-22
show the ACVR1 R206H (617G > A) mutation. (D) Retroviral transgene expression in vFOP4 and vFOP5 iPS cell lines by RT-PCR. (E) vFOP4, vFOP5,
and control iPS cells grown in mineralizing culture conditions. Mineral is detected as black staining with von Kossa. Representative images from
triplicate assays with similar results. (F) Quantitation of mineralization intensity. Error bars represent the average of the three control cell lines (H9,vWT-TIG120-4f1, and vWT-201B7) or three vFOP iPS lines (vFOP4-1, vFOP4-3, and vFOP5-22), +/− 1 SD. n ≥ 6 measurements from per condition ateach timepoint. **, p < 0.01; ***, p < 0.001 by Student’s t-test. (G) Increased mineralization by vFOP4 and vFOP5 iPS cells after 15 days of culture
can be blocked by treatment with the BMP inhibitor DMH1. Representative experiment from triplicate assays with similar results. (H) Quantitationof mineralization intensity after DMH1 treatment of FOP iPS cell lines. n = 3 measurements for each DMH1 concentration and timepoint. *, p < 0.05;**, p < 0.01 by Dunnett’s multiple comparisons t-test compared to the no DMH1 treatment group.

different levels of transgene silencing (Figures 2A and3E), we created integration-free iPS cells to test if retroviral transgenes confound our ability to detect the effectsof the ACVR1 R206H mutation. We used integrationfree episomal vectors to introduce the iPS cell transformation factors SOX2, KLF4, OCT4, L-MYC, LIN28,
and p53 shRNA into control and FOP dermal fibroblasts[8]. We included an expanded collection of control
dermal fibroblasts and FOP fibroblasts from skinremoved during a medically-necessary surgical procedure (FOP3). We obtained a large number of FOP iPS cellcolonies with morphology consistent with human ES cells(Figure 5A). Four lines from two control donors (eWT1323-2, eWT-1323-4, eWT-BJ2, and eWT-BJ4) and sixlines from three FOP donors (eFOP1-1, eFOP1-10,
eFOP2-3, eFOP2-8, eFOP3-2, and eFOP3-4) were characterized in detail. All had normal karyotypes (Additional
file 6: Figure S4A), retained the R206H ACVR1 mutation

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Figure 4 Increased chondrogenesis in vFOP iPS cells in pellet cultures. (A) Histology of chondrogenic pellets from vFOP4 and vFOP5 iPS
cells show larger chondrocytes and increased proteoglycan. Blue, alcian blue; red, nuclear fast red. Black scale bar = 200 μm; red scale bar = 50 μm.
(B) Chondrocyte pellet size (calculated volume). Error bars are averages of triplicate experiments, +/− 1 SD. (C) Cell size in the chondrogenic pellets.
Error bars are averages of triplicate experiments, +/− 1 SD. (D) Percentage of chondrocyte-like cells within a culture. Error bars are averages of triplicate
experiments, +/− 1 SD. (E) Chondrogenic (SOX9, COL2a1, and COMP) gene expression in the pellets. Error bars are averages of triplicate measurements
on individual pellets, +/− 1 SD. **, p < 0.01; ***, p < 0.001 by Student’s t-test.

(Figure 5B), and expressed pluripotency markers by immunostaining (Additional file 7: Figure S5). Five of the six
FOP lines had normal teratoma formation (Figure 5C,
Additional file 6: Figure S4B). Line eFOP2-8 lacked clear
mature endodermal elements and was excluded from further analyses. Expression analysis suggested eFOP3-4 had
low levels of OCT4 plasmid expression; however, noretained episomal vectors were found by genomic DNA
qPCR for the EBNA gene which is located in the episome
backbone (Additional file 6: Figure S4C, D). The FOP lines
had higher levels of phospho-SMAD 1/5/8 before andafter stimulation with BMP4 (Figure 5D), showing responsiveness to exogenous BMP.
eFOP iPS cells cultured in mineralization medium hadsignificantly increased mineralization activity at day 6 by
von Kossa staining (Figure 6A, B), but this difference
narrowed by day 12. We found no statistical differencein wildtype ACVR1 expression even though the R206H
ACVR1 allele expression decreased significantly over thecourse of the experiment (Figure 6C). ID1 and DLX5,
both activated by BMP signaling, were increased at day
6, indicating that the ACVR1 R206H mutation increased

activation of BMP signaling (Figure 6D). Expression analysis showed a trend towards transient increases of chondrogenic markers COLLAGEN 2 (COL2a1) as well as
increased expression of the osteogenic marker OSTEOCALCIN (OC) at day 6 (Figure 6E, F). The FOP iPS cells
showed no significant increase in expression of earlymarkers of chondrogenesis (SOX9) or osteogenesis(RUNX2) at the time points we assayed; in fact, levels ofthese early markers were higher in control iPS cells ascompared to the FOP iPS cells (Figure 6E, F). FOP iPS
cells also showed increased expression of genes associated with mineralization activity (Figure 6F), such as alkaline phosphatase. In addition, we found increased
expression of TFIP11, a ubiquitously expressed gene thatsuppresses chondrocytic development, may function
during the transition from a cartilage template towards
the formation of ossification centers [21] and may regulate tooth enamel deposition [22]. These in vitro results
suggest that the ACVR1 R206H mutation confers increased propensity towards mineral deposition, possibly
via a mineralized cartilage mechanism with transient increases in osteogenic markers.

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Figure 5 Integration-free FOP iPS cells. (A) Representative images of FOP3 dermal fibroblasts subsequently converted to human iPS cellcolonies in feeder-free conditions. Black scale bar = 50 μm. (B) Human integration-free iPS cells from donors with FOP are heterozygous for the
ACVR1 R206H (617G > A) mutation but control lines are not. (C) Representative teratomas from one eFOP iPS cell line showing tissues representing
all three germ layers (n, neuronal/primitive neural tube like structure; c, cartilage; g, primitive gut). Additional cell lines are shown in Additional file 6:
Figure S4. Black scale bar = 50 μm. (D) Western blot showing total SMAD1/5/8 and phospho-SMAD1/5/8 (pSMAD) levels in FOP iPS celllines relative to control lines with (+) and without (−) 45 minutes of BMP4 exposure. GAPDH, loading control. Representative blot of
triplicate experiments.

Discussion
Here, we use human iPS cells derived from FOP donors
to establish a cellular foundation to understand themechanisms underlying this dramatic disease (Figure 7).
Our FOP iPS cells are a valuable in vitro human model
system for understanding how human bone develops
and identifying what control points may be amenable formanipulating normal and pathologic bone formation.
The iPS cells will also facilitate identifying the triggers ofheterotopic bone formation and strategies to block the
different steps of mineralization or ossification, particularly by providing the ability to create key human cell
types not directly available from the FOP patients orfrom patient progenitor cells. These iPS cells also provide a unique human-specific perspective that complements the in vivo studies of the FOP mouse model [23].
In this study, we created and analyzed three sets of
independently-derived iPS cell lines. Our results suggestthat generating iPS cells with integration-free methods
rather than retroviral methods may decrease the likelihood of confounded results from persistent transgene
expression, as has been described for C-MYC [24].
Our findings indicate that the ACVR1 R206H mutation can allow human iPS cells to form chondrocytes
and mineralize in vitro without a clear fibro-proliferative
stage as in FOP patients. Since the fibro-proliferative

cells in an early FOP lesion are likely a collection of diverse cell types, we speculate that more primitive cell
types or early skeletal precursors, such as those potentially in our iPS cell model, could be major contributors
to the early FOP lesion. Although this might explain themineralization that occurred without altered osteogenic
markers, further experiments are needed to delineate thedistinct effects of ACVR1 R206H on mineralization andosteogenesis in our iPS cell model.
Our results also suggest that the ACVR1 R206H mutation may have greater influence earlier in endochondral bone formation (i.e., at the chondrogenesis stage)
based on gene expression patterns. Although ACVR1
can be regulated by miR-148a [25], the decreasinglevels of ACVR1 R206H mRNA during mineralizing
culture suggest that the 617 G > A mutation confers
allele-specific regulation of RNA transcripts. However,
the heterogeneity of our iPS cell mineralization cultures
precluded us from identifying if a specific cell type isdown-regulating ACVR1 R206H expression during culture. Delineating this potential regulatory function
using FACS-purified cells or iPS cell-derived cell types
could identify more specific roles for ACVR1 in boneformation and identify a new therapeutic target thatwould complement strategies to modulate the signaling
properties of the ACVR1 receptor.

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Figure 6 Increased mineralization capacity in integration-free FOP iPS cells. (A) eFOP iPS cells show greater mineralization than controls by
von Kossa staining. (B) Quantitation of mineralization. n = 46–51 wells for each condition, combining at least three replicates of each of the four
control iPS lines or five FOP iPS lines. Error bars, +/− 1 SD. *, p < 0.05. †, p < 0.1. (C) Quantitative PCR of ACVR1 (wildtype and R206H mutant)during osteogenic culture. (D–F) Quantitative PCR during osteogenic culture of genes downstream of BMP signaling (ID1 and DLX5), chondrogenic(SOX9, ACAN, COL2a1, and COMP), osteogenic (RUNX2, SP7, and OC), and mineralization (AP and TFIP11) markers. n = 3–6 samples per iPS cell line percondition, each measured in triplicate; Error bars, +/− 1 SEM; *, p < 0.05. †, p < 0.1.

Although we could identify differences in mineralizationby the FOP iPS cells, our simple culture conditions may
not fully recapitulate the requirements for forming mature
osteogenic cells from iPS cells. Similar challenges for creating mature cell types have been identified using current
culture methods in other types of ES or iPS cell-derived
progeny [26] and in some mesenchymal stem cell culture
conditions [27]. This could account for the low expression
levels of osteogenic genes. Instead, our culture conditions
may better reflect the early steps of matrix mineralization
in injured cartilage. The increased expression of genes
associated with mineralization activity, such as TFIP11
and alkaline phosphatase, suggest the ACVR1 R206H

mutation can direct mineral deposition in tissues. This isconsistent with reports indicating that BMP signaling increases mineralization activity in other cell types [28].
Bone formation and mineralization can also occur in theabsence of osteoblasts in Osx-deficient mice [29,30].
Future studies will benefit from newer 3D scaffolds,
the creation of multi-cellular bone models with iPS cell–
derived cell types, better defined culture conditions, andin vivo translational assays which may favor the formation of mature skeletal cells. These directions will help
identify the factors that may act at different stages ofosteogenesis, including those that initiate heterotopic
bone formation and lead to the formation of mature

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Figure 7 Human iPS cells for disease modeling. Our results suggest an increased disposition towards mineralization and chondrogenesis,
consistent with modeling the early developmental and heterotopic bone formation processes. Our in vitro model will allow detailed interrogationof the mechanisms regulating bone formation and triggers of FOP flares, potentially identifying novel treatments for treating pathologic bonediseases.

skeletal elements in humans. In addition, our results
suggest that blocking mineralization induced by ACVR1
R206H may be a useful treatment strategy for preventing
full mineralization of heterotopic bone lesions in FOP
patients, thus preserving some limited joint mobility
even if cartilage formation still occurs.
The increased chondrogenesis and mineralization, as
well as the trend towards increased expression of endothelial cell gene markers, raise the possibility that theR206H ACVR1 mutation promotes skeletogenesis by affecting cell fate. Several observations support this notion. Studies in connective tissue progenitor cells fromdiscarded primary teeth (SHED cells) of FOP patientswith the ACVR1 R206H mutation show enhanced osteogenic differentiation [31]. In addition, introducing theR206H ACVR1 mutation into endothelial cells can increase endothelial-mesencymal transition and promoteentry into an osteogenic lineage [32], possibly contributing to the bone formation in FOP. Finally, BMP activation induces differentiation in human ES cells [15].
These results suggest that activating the BMP signaling
pathway may affect a cell’s ability to retain cell-fate commitment under specific circumstances.

Our studies showed that pluripotent FOP iPS cells canbe generated without a BMP inhibitor using either the
retrovirus or episomal methods. Prior attempts to make
iPS cells from FOP fibroblasts by the Sendai virus method
showed cellular instability [33]. This could be partiallymitigated by culturing the cells with a BMP inhibitor,
making the iPS cell formation process a useful assay for
identifying small molecules that affect ACVR1 signaling.
Although our study was not designed to directly compare
the different iPS cell-generating methods, our results identify two major implications. First, our findings suggest thatdifferent pluripotency induction techniques, culture conditions, or embryonic fibroblast feeder cells could influence the success of iPS cell generation. Although Sendi
virus can efficiently express reprogramming factors, thesteps needed to remove replicating virus from cultures
and the sensitivity of the viral RNA replicase to transgenesequences may contribute to different overall efficienciesof iPS cell generation [34]. Furthermore, the conditions
under which iPS cells are derived, including the specificfeeder cells used, can affect important aspects such as
gene expression, reprogramming efficiency, and Xinactivation [35]. These observations indicate that theMatsumoto et al. Orphanet Journal of Rare Diseases 2013, 8:190
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specific iPS cell derivation conditions may significantly
affect the ability to generate iPS cell lines particularly if a
disease mutation affects cell fate stability. Second, ourstudy applies the FOP iPS cells as a disease model to demonstrate a potential function for ACVR1 in chondrogenesis and mineralization. These two endpoints may be usefulfor screening drug modulators that target different stagesof human osteogenesis, particularly once the chondrogenic potential of the episomal iPS cells has been determined. Further studies to identify if cell fate stability isaffected in non-pluripotent cells such as chondrocytes or
osteoblasts may also reveal if cell fate plays a key role in
the pathogenesis of heterotopic ossification in FOP.

Conclusions
Our iPS cell strategy provides a key first step towards dissecting the cellular and molecular mechanisms of humanskeletal disease pathogenesis in FOP. Human iPS cells will
also be a valuable tool that reflects the diversity of patients
we see in the clinic since patients, and thus, their iPS cells
show clinical variability, genetic background effects, andepigenetic influences. Creation of new isogenic controls bygene correction [36], combined with translational genetic
studies and correlations with murine models, provides exciting venues for new strategies to understand the stagesof normal and pathologic human skeletal development.
Our studies demonstrate the creation of iPS cells from patients with FOP, identify that ACVR1 may have a new role
in regulating mineralization activity, and provide a proofof-concept for further development of human iPS cells as
disease models for studying human skeletal diseases.
Additional files
Additional file 1: Table S1. Characteristics of the iPS cells used in thisstudy. This table summarizes the characterization results for the FOP andcontrol iPS cell lines used in this study and the original fibroblast source
numbers for the iPS cell lines.
Additional file 2: Figure S1. FOP dermal fibroblasts do notspontaneously mineralize. (A) Human dermal fibroblasts (HDFs) frompatients with FOP are heterozygous for the ACVR1 R206H (617G > A)
mutation. (B) Primary human mesenchymal stem cells (MSCs) cultured inmineralizing media for 12 days show an increase in von Kossa staining.
Mineralized deposits appear as the darker brown or black staining. The
light golden staining is background. Standard MSC maintenance medium
is used as a comparison for non-mineralizing conditions. (C) HDFs from
FOP donors do not mineralize (black staining) when cultured in mineralizing
medium for 18 days. Standard HDF medium without osteogenic supplements
is used as a comparison for non-mineralizing conditions.
Additional file 3: Table S2. Quantitative and RT-PCR primers used inthis study.
Additional file 4: Figure S2. Characterization of retroviral FOP iPS cells.
(A) Teratoma formation showing representatives of the three germ layers.
Scale bars = 200 μm. (B) Quantitative PCR gene expression analysis
showing expression of pluripotency markers by the iPS cells. Error bars
are average expression +/− 1 SD of technical triplicates. Gene expression
studies were repeated in triplicate.

Page 12 of 14

Additional file 5: Figure S3. Characterization of additional retroviral
FOP iPS cells. (A) Relative expression of retroviral transgenes in vFOP4-1,
vFOP4-3, and vFOP5-22 iPS cells analyzed by quantitative RT-PCR. The
value of each transgene 6 days after infection of wild-type dermal fibroblast(DF 4 F D6) was set to 1, demonstrating suppression of transgene
expression. (B) Teratoma formation showing representatives of the threegerm layers. Scale bars = 200 μm. (C) Karyotypes of the vFOP4 and vFOP5
iPSC lines are normal. (D) RT-PCR gene expression analysis showing
expression of pluripotency markers by the iPS cells. (E) Quantitative PCRgene expression analysis showing expression of RUNX2, SP7/OSX, andOC genes during iPS cell mineralization culture. Error bars are average
expression +/− 1 SD of measurements pooled from vFOP4-1, vFOP4-3,
and vFOP5-22 iPS cell lines. n = 3 per time point. *, p < 0.05.
Additional file 6: Figure S4. Characterization of episomal iPS cell lines.
(A) FOP human dermal fibroblasts and the derived eFOP iPS cells havenormal karyotypes. (B) Teratoma formation showing representatives of
the three germ layers. b, bone; c, cartilage; g, primitive gut; m,melanocytes; n, neuronal tube like structures. Line eFOP2-8 showed no
identifiable endodermal structures, and so was excluded from further
analysis. Black scale bar = 50 μm. (C) Quantitative RT-PCR analysis of
episomal transgene expression in the integration-free iPS cell lines. HDFs
electroporated with the episomal vectors are used to determine thecontrol levels of total mRNA present. Log10 scale. (D) Quantitative PCR
analysis of the EBNA gene in genomic DNA from the integration-free iPS
cell lines. Note that although episomal transgene expression of OCT4
was detectable in the eFOP3-4 cell line, the level is low and no EBNA
integration into the genome was detected.
Additional file 7: Figure S5. Immunohistochemistry of episomal iPS
cell colonies show expression of pluripotency markers NANOG, OCT3/4,
SSEA3, TRA1-60, and TRA1-81.
Abbreviations
BMP: Bone morphogenetic protein; EB: Embryoid body; ES cells: Embryonicstem cells; FOP: Fibrodysplasia ossificans progressiva; HDF: Human dermal
fibroblast; iPS cells: Induced pluripotent stem cells; MSC: Mesenchymal stem
cells.
Competing interests
The authors have no competing interests to declare.
Authors’ contributions
ECH, JT, TO, SY, and BC conceived and designed the experiments. ECH, YM,
YH, CRS, HK, TDN, SS, SB, EB, AN, IA, and MI derived the iPS cell lines andperformed the experiments. ECH and MI prepared the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
We thank Gary Howard, Matt Spindler, Kenta Nakamura, Wint Lwin, Will
Collins, Kathy Ivey, Ashley Urrutia, Nathalie Gaborit, Sanae Nagata, Deepak
Srivastava, Eileen Shore, the Gladstone Stem Cell Core, and the UCSF Stem
Cell Core for valuable technical assistance and discussions. We gratefully
acknowledge the contributions of the participants in our study. This work
was supported by NIH R01 HL60664 to BRC; NIH K08 AR056299, California
Institute of Regenerative Medicine/Gladstone Institutes CIRM Fellowship(#T2-00003), UCSF Department of Medicine, and March of Dimes BasilO’Connor Starter Grant (#5-FY12-167) to ECH; the Duke-NUS Graduate
Medical School Singapore 3rd-year Research Program to CRS; a California
Institute of Regenerative Medicine/UCSF CIRM Fellowship (#TG2-01153) to
EB; Grants-in-aid for Scientific Research from JSPS (#23791636, #25293320),
the Leading Project for Realization of Regenerative Medicine from MEXT, and
The Program for Intractable Diseases Research utilizing Disease-specific iPS
cells from JST in part to MI and JT; the Uehara Memorial Foundation and
USCF Program for Breakthrough Biomedical Research to YH; and the Leading
Project of MEXT, Funding Program for World-Leading Innovative R&D on
Science and Technology (FIRST Program) of the JSPS, Grants-in-Aid for
Scientific Research of the JSPS and MEXT, Program for Promotion of
Fundamental Studies in Health Sciences of NIBIO, L.K. Whittier Foundation,
and Rodenberry Foundation to SY. SY is a member without salary of thescientific advisory boards of iPierian, iPS Academia Japan, Megakaryon
Corporation, and HEALIOS K.K., Japan. BRC is on the Scientific Advisory Board

Matsumoto et al. Orphanet Journal of Rare Diseases 2013, 8:190
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Page 13 of 14

of iPierian, Inc. The funders had no role in study design, data collection/
analysis, decision to publish, or preparation of the manuscript.
15.
Author details
1
Department of Tissue Regeneration, Institute for Frontier Medical Sciences,
Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku 606-8507, Kyoto,
Japan. 2Department of Cell Growth and Differentiation, Center for iPS Cell
Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin,
Sakyo-ku 606-8507, Kyoto, Japan. 3Department of Orthopaedic Surgery,
Graduate School of Medical Sciences, Nagoya City University, Nagoya
467-8601, Japan. 4Gladstone Institute of Cardiovascular Disease, San
Francisco, CA 94158, USA. 5Department of Medicine, Duke-NUS Graduate
Medical School, Singapore 169857, Singapore. 6Department of Medicine,
Division of Endocrinology and Metabolism and the Institute for Human
Genetics, University of California-San Francisco, 513 Parnassus Ave., HSE901G,
San Francisco, CA 94143-0794, USA. 7Department of Orthopaedic Surgery,
Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
8Department of Fundamental Cell Technology, Center for iPS Cell Researchand Application, Kyoto University, Kyoto 606-8507, Japan. 9Department of
Reprogramming Science, Center for iPS Cell Research and Application, Kyoto
University, Kyoto 606-8507, Japan. 10Department of Medicine and Cellular
and Molecular Pharmacology, University of California-San Francisco,
San Francisco, CA 94143, USA.

16.

17.

18.

19.

20.

21.
Received: 14 May 2013 Accepted: 2 December 2013
Published: 9 December 2013
22.
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Cite this article as: Matsumoto et al.: Induced pluripotent stem cells
from patients with human fibrodysplasia ossificans progressiva show
increased mineralization and cartilage formation. Orphanet Journal ofRare Diseases 2013 8:190.

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