Human umbilical vein endothelial cells (HUVEC) and human cutaneous microvascular endothelial cells (HCMEC) isolated as previously described35
. Cells were tested for purity and found to express no markers for lymphatic endothelial cells or stromal cells (pericytes, smooth muscle cells, etc.)36
. This was reconfirmed by flow cytometry analysis (Supplementary Fig. 11a).
Clonal populations were isolated with PYREX 8×8 mm cloning cylinders (Corning) and shown to respond in a manner identical to that of the cultures used for our experiments (Supplementary Fig. 11b-d).
Cells were grown in culture using EGM-2 medium (Cambrex), containing 10% FBS and 1% Penicillin/Streptomycin, followed by human endothelial serum free medium (Gibco) 24 h prior to all experimental conditions. Bone marrow derived mesenchymal stem cells (ScienCell Research Laboratories) were grown in mesenchymal stem cell medium (ScienCell Research Laboratories). Bone marrow derived hematopoietic stem cells (Lonza) were grown in HPGM medium (Lonza). Human corneal fibroblasts, from a stock provided by Dr. Elizabeth Hay (Harvard Medical School), were grown in RPMI medium, containing 10% FBS and 1% Penicillin/Streptomycin. Primary human osteoblasts, chondrocytes, and adipocytes and their respective growth media were obtained from Cell Applications, Inc. Recombinant TGF-β2, BMP4, and BMP7 proteins (R&D Systems) were added to the serum free culture medium for all relevant experiments at a concentration of 10 ng ml−1
. Recombinant VEGF (R&D Systems) was added to cultures at a concentration of 25 ng ml−1
. Dorsomorphin (Sigma-Aldrich) was added to cultures at a concentration of 5 μM. All experiments for this study were performed at minimum in triplicate.
Plasmids and adenoviral constructs
Human ALK2 expression constructs were generated by insertion of full-length human ALK2 cDNA (GenBank NW_001105) into the pcDNA 3. 1 D V5-His-TOPO vector (Invitrogen). The ALK2 R206H mutant construct was generated by site-directed mutagenesis of the normal ALK2 sequence using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen). The oligonucleotides used to generate the mutant construct were: forward 5′-GTACAAAGAACAGTGGCTCACCA GATTACACTG-3′; reverse 5′-GTGAGCCACTGTTCTTTGTACCAGAAAAGGAAG-3′. SpeI and SphI sites were used to generate the adenoviral vectors through the Clontech Adeno-X System (University of Pennsylvania Vector Core). Expression from these constructs was confirmed by sequence analysis and immunoblot analysis. Viral constructs were added to cultures at a concentration of 20 pfu ml−1.
All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Floxed caALK2 transgenic mice37
were a gift from Dr. Yuji Mishina (University of Michigan). To induce expression of caALK2, AV-Cre (University of Pennsylvania Vector Core; 1 × 1011
particles per mouse) was injected into the left hindlimbs of mice at one month of age. The contralateral limb was injected with empty vector as a control. Heterotopic bone and cartilage were detected by X-ray (Senographe DS technology, General Electric Medical Systems) at 14-21 d following A V-Cre injection. Tie2-Cre and IRG reporter mice were obtained from the Jackson Laboratory. Heterotopic ossification was induced in the offspring of crosses between the Tie2-Cre and IRG reporter mice with BMP4 (provided by Genetics Institute; currently Pfizer) injected at a concentration of 0.05 μg μl−1
, intramuscularly in growth factor-reduced Matrigel (BD Biosciences) Tissues were recovered at 7 and 14 d after implantation. Tissues were fixed in isopentane (2-methylbutane) as previously described23
and cryosections were cut at 10 μm. Counterstaining of sections from Tie2-Cre IRG reporter mice was performed using blue (shown in red) fluorescent AlexaFluor secondary antibodies (Invitrogen). No leakage or aberrant expression of the reporter was found in any tissues (Supplementary Fig. 12).
All FOP patient samples were obtained with informed consent and protocols approved by the Investigational Review Board of the University of Pennsylvania. All biopsies were obtained prior to a diagnosis of FOP since tissue trauma in FOP frequently induces episodes of heterotopic ossification. Normal human bone and cartilage tissue from the hip joint was provided by the Department of Pathology at the Beth Israel Deaconess Medical Center and samples were obtained with informed consent and protocols approved by the Investigational Review Board.
Immunoblotting, immunoprecipitation, and immunofluorescence
Immunoassays were performed using the following antibodies at concentrations (and using protocols) recommended by the respective manufacturers: FSP-1 (H00006275-M01; Stressgen), phospho-Smad2 (3101), Smad2 (3122), phospho-Smad5 (9516), Smad5 (9517; Cell Signaling Technology), ALK1 (sc-19547), ALK2 (sc-25449), ALK3 (sc-20736), ALK4 (sc-31297), ALK5 (sc-398), ALK6 (sc-25455), ALK7 (sc-135001), phospho-tyrosine (sc-7020), VE-cadherin (sc-6458), TIE1 (sc-342), TIE2 (sc-324, sc-9026), STRO-1 (sc-47733), CD10 (58939), CD44 (sc-71220), CD71 (sc-32272), CD90 (sc-9163), CD117 (sc-17806), osteocalcin (sc-74495, sc-23790), SOX9 (sc-20095), PPARγ2 (sc-22022; Santa Cruz Biotechnology), osterix (ab22552), adiponectin (ab22554), N-cadherin (ab76057), NG2 (ab83508), vWF (ab68545; Abcam); CD31 (IR610; Dako), His (A00174; GenScript); α-SMA (A5228), β-actin (A1978; Sigma-Aldrich). Samples were run with Criterion precast SDS-PAGE Gels (Bio-Rad). HRP-conjugated IgG TrueBlot reagents (18-8814, 18-8816, 18-8817; eBioscience) were used at a dilution of 1:1000. TrueBlot IgG beads (eBioscience) were used for immunoprecipitation experiments. AlexaFluor secondary antibodies (Invitrogen) were used at a dilution of 1:200. Images were acquired using a Nikon 80i fluorescence microscope.
Endothelial cells were stained in suspension using antibodies specific for TIE2, FSP-1, STRO-1, α-SMA, NG2, and His (described above) and the protocols provided by their respective manufacturer. Flow cytometry was performed at the Harvard Medical School, Department of Pathology flow cytometry core facility using a FACSDCalibur (BD Biosciences) cell sorter isolating 30, 000 cells per sample.
LEF-1 (Cell Signaling Technology), VE-cadherin (sc-6458), CD44 (sc-71220), CD90 (sc-9163), Snail (sc-10433), ZEB-1 (sc-134159; Santa Cruz Biotechnology), FSP-1 (H00006275-M01; Stressgen), SIP-1 (AV33694; Sigma-Aldrich), Slug (ab27568), and Twist (ab49254; Abcam) antibodies were conjugated to Bio-Plex carboxylated beads with unique optical codes using the Bio-Plex Amine Coupling Kit (BioRad). β-actin antibody (A1978; Sigma-Aldrich) was also conjugated to Bio-Plex carboxylated beads to be used as an internal control. Samples were run on a Luminex 200 multiplex testing system (Luminex) using the Universal Cell Signaling Assay Kit and protoco! (Millipore). Experimental values were divided by the β-actin control values to provide normalized data.
Cells were grown in StemPro osteogenic, chondrogenic, or adipogenic culture medium (Invitrogen). Alkaline phosphatase staining was performed using the alkaline phosphatase kit and protocol (Sigma-Aldrich) on cultures grown in osteogenic medium for 7 d to detect osteoblasts. Alizarin Red (Sigma-Aldrich) staining was performed for 30 min on cultures grown in osteogenic medium for 21 d to detect matrix calcification. Alcian Blue (Sigma) staining was used to stain chondrocyte proteoglycans for 5 minutes in cultures grown in chondrogenic medium for 14 d. Oil Red O (Sigma-Aldrich) staining was performed for 15 min on cultures grown in adipogenic medium for 7 d. For in vivo analysis, endothelial cells were labeled with fluorescent quantum dots using the Qtracker 525 CeΠ Labeling Kit (Invitrogen). Cells were treated in culture to induce EndMT then absorbed into OPLA polylactic acid scaffolds (BD Biosciences). Scaffolds were surgically implanted subcutaneously into immunodeficient nude mice (Nu/Nu strain; Charles River Laboratories). Local injection of StemPro differentiation medium (described above) was performed in the area of the implants every 72 h for 6 weeks. Scaffolds were cryosectioned and stained with Alizarin Red, Alcian Blue, or Oil Red O as described above. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Harvard Medical School.
siRNA gene expression knockdown studies were performed using the TriFECTa RNAi kit (Integrated DNA Technologies) and corresponding protocoL Each 27mer siRNA duplex was transfected into cells using X-tremeGene siRNA transfection reagent (Roche) following the manufacturer′s guidelines. siRNA was synthesized (Integrated DNA Technologies) with the following sequences: ALK1: 5′-CUGGGCUAUUGAAUCACUUUAGGCUUC-3′; ALK2: 5′-GCAACACUGUCCAUUCUUCUUAACCAG-3′; ALK3: 5′-CAUCUCAUGAAUUCCAAGACAGUAUUA-3′; ALK4: 5′-AUGAGGGAUCUUCCAUGUCCAGUCUCU-3′; ALK5: 5′-CUCAGAAUGUUCUUUAGCUACCACCUC-3′; ALK6: 5′-AUCUGAAUCUGCUUAGCUAUAGUCCUU-3′; ALK7: 5′-ACUUAAAUACUGUACUGUCUUAUCUUU-3′; negative control: 5′-UCACAAGGGAGAGAAAGAGAGGAAGGA-3′.
One-way analysis of variance (ANOVA) was performed and confirmed with two-tailed paired student’s t test using GraphPad Prism 4 software. P values less than 0. 05 were considered significant.