Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation
- Yoshihisa Matsumoto†1, 2, 3,
- Yohei Hayashi†4,
- Christopher R Schlieve†4, 5, 6,
- Makoto Ikeya2Email author,
- 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, 7Email author and
- Edward C Hsiao4, 6Email author
© Matsumoto et al.; licensee BioMed Central Ltd. 2013
Received: 14 May 2013
Accepted: 2 December 2013
Published: 9 December 2013
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.
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.
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.
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.
KeywordsInduced pluripotent stem cells Fibrodysplasia ossificans progressiva FOP Disease model in a dish Endochondral ossification
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 activation of endochondral bone formation in soft tissues from congenital disorders, such as fibrodysplasia ossificans progressiva (FOP; OMIM #135100) or from trauma or central nervous system injury  cause severe disability and pain. Although bone morphogenetic proteins (BMPs) are key regulators of osteogenesis  and are implicated in pathologic calcification , their specific functions in humans are not well understood. Human induced pluripotent stem cells (iPS cells) derived from patients with genetic mutations affecting key signaling pathways provide a unique opportunity to dissect human development and disease pathogenesis in models amenable to experimental manipulation .
FOP is a rare but debilitating disease of heterotopic bone formation associated with mutations in the Activin A Type I receptor (ACVR1) , which is activated by BMPs. A single amino acid change (R206H) commonly resulting from a single base mutation (617G > A) is found in the majority of FOP patients and can increase sensitivity of the receptor to BMP ligands . Patients are born without heterotopic ossification yet characteristically form large amounts of bone either spontaneously or after trauma . The heterotopic ossification follows an endochondral bone formation 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.
Materials and methods
Cell lines used in this study
Human dermal fibroblasts
Foreskin (Stemgent 08–0027)
Fibroblast (Cell Applications, 1323)
Fibroblast (Cell Applications, 1388)
Collection of Research Bioresources, Japan
Skin Fibroblast, Corielle GM00513
Skin Fibroblast, Corielle GM00783
Control retroviral iPS cell lines (wildtype ACVR1)*
FOP retroviral iPS cell lines (ACVR1 R206H)*
Control episomal iPS cell lines (Wildtype ACVR1)**
FOP episomal iPS cell lines (ACVR1 R206H)**
Retroviral  and episomal integration-free  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)-coated plates (150–300 μg/ml, 30 min coating) or in primate ES cell medium (ReproCELL, Tokyo, Japan) on mitomycin C-treated or irradiated SNL feeder cells . 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 was done 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 and Embryonic Stem Cell Research Committee, or by the Ethics Committee of the Department of Medicine and Graduate 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 amino acids, and 0.1 mM 2-mercaptoethanol) and mTeSR1, and supplemented with 10 μM Y-27632. Cells were plated onto ultra-low attachment plates without medium changes for seven 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 four days. EBs were then transferred to gelatinized plates and cultured until day 15 for RNA collection in Trizol (Invitrogen).
iPS cells grown in six-well Matrigel-coated plates to 100% 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 . 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 the Division of Technical Support of the Institute for Frontier Medical Sciences in Kyoto University. All mouse studies 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.
Primary human MSCs were cultured in OB mineralization medium (DMEM with 20% FBS, glycerol-2-phosphate, 4 μM dexamethasone, 0.1 mM 2-mercaptoethanol, and 50 μg/ml L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate ). We could detect mineralization activity after 12 days as increased black staining by von Kossa (Additional file 2: Figure S1B), which was the preferred staining method used because the black mineralization nodules could be easily distinguished from the expected 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-well plates, 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 . DMH1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline; from Dainippon-Sumitomo Pharma or Nacalai Tesque, Tokyo, Japan) was used 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.
Pellet chondrogenic cultures were performed as described . Briefly, human iPS cells collected with a scraper were suspended 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-coated tissue-culture dishes, grown to confluence, released with 0.25% trypsin/EDTA, filtered through a 70-μm cell strainer, and seeded onto new gelatin-coated dishes. Once confluent, cells were collected and 2.5 × 105 cells were placed 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 and maintained 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 analysis of endogenous pluripotency genes, persistent expression of the retroviral iPS cell induction factors, episomal 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 the primers or probesets listed in Additional file 3: Table S2. Each reaction was run in technical triplicates on an Applied Biosystems 7900HT or Viia7 thermocycler (Life Technologies) and normalized to GAPDH as an endogenous control using protocols recommended by ABI.
Cells or EBs were fixed with 4% PFA/PBS for 10 min at room 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 the following 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), α-fetoprotein (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).
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.
ACVR1 R206H mutation is not sufficient to induce mineralization of fibroblasts
FOP patients show dramatic heterotopic bone formation in their soft tissues, yet do not ossify their skin. We asked if the ACVR1 R206H mutation  could induce mineralization in primary cells that do not normally form 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 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 non-mineralizing HDF maintenance medium, even with an additional 6 days of culture (Additional file 2: Figure S1C) and despite detectable increases in BMP-related phospho-SMAD levels in FOP HDFs (data not shown). Thus, the ACVR1 R206H mutation was not sufficient to induce spontaneous mineralization in HDFs in vitro.
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 , 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.
Retroviral FOP iPS cells show a trend to increased mineralization
FOP iPS cells show increased chondrogenesis
Integration-free FOP iPS cells also show increased mineralization
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 the mineralization that occurred without altered osteogenic markers, further experiments are needed to delineate the distinct effects of ACVR1 R206H on mineralization and osteogenesis 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 , the decreasing levels 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 is down-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 bone formation and identify a new therapeutic target that would complement strategies to modulate the signaling properties of the ACVR1 receptor.
Although we could identify differences in mineralization by 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  and in some mesenchymal stem cell culture conditions . 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 is consistent with reports indicating that BMP signaling increases mineralization activity in other cell types . Bone formation and mineralization can also occur in the absence 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, and in 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 of osteogenesis, including those that initiate heterotopic bone formation and lead to the formation of mature 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 the R206H ACVR1 mutation promotes skeletogenesis by affecting cell fate. Several observations support this notion. Studies in connective tissue progenitor cells from discarded primary teeth (SHED cells) of FOP patients with the ACVR1 R206H mutation show enhanced osteogenic differentiation . In addition, introducing the R206H ACVR1 mutation into endothelial cells can increase endothelial-mesencymal transition and promote entry into an osteogenic lineage , possibly contributing to the bone formation in FOP. Finally, BMP activation induces differentiation in human ES cells . 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 can be 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 . This could be partially mitigated 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 that different 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, the steps needed to remove replicating virus from cultures and the sensitivity of the viral RNA replicase to transgene sequences may contribute to different overall efficiencies of iPS cell generation . Furthermore, the conditions under which iPS cells are derived, including the specific feeder cells used, can affect important aspects such as gene expression, reprogramming efficiency, and X-inactivation . These observations indicate that the 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, our study 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 useful for screening drug modulators that target different stages of human osteogenesis, particularly once the chondrogenic potential of the episomal iPS cells has been determined. Further studies to identify if cell fate stability is affected 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.
Our iPS cell strategy provides a key first step towards dissecting the cellular and molecular mechanisms of human skeletal 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, and epigenetic influences. Creation of new isogenic controls by gene correction , combined with translational genetic studies and correlations with murine models, provides exciting venues for new strategies to understand the stages of 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 proof-of-concept for further development of human iPS cells as disease models for studying human skeletal diseases.
Bone morphogenetic protein
- ES cells:
Embryonic stem cells
Fibrodysplasia ossificans progressiva
Human dermal fibroblast
- iPS cells:
Induced pluripotent stem cells
Mesenchymal stem cells.
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 Basil O’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 the scientific advisory boards of iPierian, iPS Academia Japan, Megakaryon Corporation, and HEALIOS K.K., Japan. BRC is on the Scientific Advisory Board of iPierian, Inc. The funders had no role in study design, data collection/analysis, decision to publish, or preparation of the manuscript.
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