Human primary calvarial suture mesenchymal cells
Human primary calvarial suture mesenchymal cells, a heterogeneous population derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull, were grown from human suture samples as described previously  and were obtained from patients undergoing surgical treatment for craniosynostosis at the Australian Craniofacial Unit of the Women’s and Children’s Hospital, Adelaide, South Australia. This work was approved by the Research Ethics Committee of the Women’s and Children’s Hospital (REC1033/10/2014) and was performed in compliance with the Helsinki Declaration for research involving human subjects. Written, informed consent was obtained from the child, parent or guardian according to the guidelines of the Research Ethics Committee of the Women’s and Children’s Hospital. Briefly, for experimental purposes, cells were grown in minimal medium (MM) consisting of high glucose Dulbecco’s modified essential medium (DMEM, Invitrogen Life Technologies, Gaithersburg, MD), supplemented with L-Glutamine (584 mg/l), 10 % foetal bovine serum (Invitrogen, Carlsbad, CA, USA), 1 % antibiotics (penicillin 100 IU/ml), streptomycin (100 μg/ml) and 1 % antibiotic:antimycotic (both purchased from Sigma-Aldrich, MI, USA) by incubating at 37C in an humidified incubator with 5 % CO2 in air. The cultures comprise cells from the suture mesenchyme and adjacent bone growth fronts that exhibit a pre-osteoblastic or osteoprogenitor phenotype.
Transfection of human primary calvarial suture mesenchymal cells using various lipid-based methods
In preparation for transfection using various lipid based methods, cells were grown in 175 cm² flasks to 70-80 % confluency. On the day of transfection, cells were washed once with PBS and trypsinized by adding 2 ml of 1X trypsin (Sigma-Aldrich, St Louis, USA) and incubating the flask at 37C for 2 mins. The cells were stained with trypan blue and counted using a haematocytometer. Transfections using DOTAP (N-[1-(2,3-Dioleoyloxypropyl] N,N,N -trimethyl ammonium methyl sulfate; Roche Applied Biosciences, Indianapolis, USA), Lipofectamine 2000 (Life Technologies, Invitrogen, Australia), Turbofect (Fermentas Inc., Maryland, USA), X-Fect (Clontech Laboratories Inc., CA, USA), Endofectine (GeneCopoeia Inc., MD, USA) and Metafectine (Biontex Laboratories, GMbH, Germany) were carried out as described in manufacturer’s supplied protocols and as previously described [46–48]. Briefly for transfection, human primary suture cells (50,000 cells / well) were seeded in a 24-well tray and grown in 400 μl of minimal medium supplemented with 10 % fetal bovine serum. The cells were attached by incubating at 37C for 24 hrs. For each triplicate transfection, 750 ng pmaxGFP (green fluorescent protein) expression construct (3.49 kb: Amaxa Biosystems, now known as Lonza) was mixed with 30 μl of 20 mM Hepes buffer pH 7.4 in an eppendorf tube and, in another tube, the specific transfection agent was diluted with 30 μl of Hepes buffer to the recommended concentrations. The contents of these tubes were mixed and incubated at room temperature for 20 mins for DNA-lipid complex formation and 20 μl of this complex was aliquotted into three wells of a 24-well tissue culture tray. The next day the medium was removed and the cells washed once with 400 μl of phosphate-buffered saline (PBS) and replaced with 400 μl of medium, then incubated at 37C for another 24 hrs. Microscopy of the GFP-transfected cells was carried out using a Nikon Eclipse TE2000U inverted microscope attached with twin CCD cameras and 20x objective. Cells were then counted as indicated in the appropriate figure legends in an haematocytometer after trypan blue staining to calculate cell survival. Flow cytometric analysis of GFP-positive cells was conducted to determine transfection efficiency.
The expression vector, pmaxGFP, was transfected or nucleofected into human primary calvarial suture mesenchymal cells. At 24 hrs post transfection cells were washed twice with PBS and dislodged from the tissue culture plates with 1X trypsin. Cells were then centrifuged at 1300 rpm for 10 mins and resuspended in 100 μl of PBS. 10 μl of these cells were used for trypan blue staining to calculate cell survival and 90 μl of the cells were further diluted to 200 μl in PBS for analysis of transfection efficiency by flow cytometry. GFP fluorescence was analyzed using a BD Bioscience FACS-AriaII. Dead cells were gated out using 7-Aminoactinomycin D (7-AAD) (5 μg/ml) staining. Data were analyzed using Flowjo Version 7 (Free Star Inc., USA).
Nucleofection of human primary calvarial suture mesenchymal cells using Amaxa transfection kits
Nucleofection of human primary calvarial suture mesenchymal cells was carried out using the Amaxa-II Nucleofector method and available transfection kits. No kit was specifically available for the human primary calvarial suture mesenchymal cells under investigation in this study; therefore the Amaxa Cell Line Optimization Nucleofector Kit was tested for nucleofection of human primary suture cells. Briefly, in preparation for nucleofection, suture cells were dislodged from the tissue culture flask by trypsin and counted in a haematocytometer after trypan blue staining. All centrifugations were carried out with maximum g force of 150 g. For each nucleofection, suture cells were resuspended in 100 μl of V or L Nucleofector solution and 20 μl of the Amaxa Supplement in an eppendorf tube. The appropriate amount of pmaxGFP was also mixed as indicated in the respective tables and figure legends. Immediately, the mixture of suture cells, pmaxGFP and Nucleofector solution was transferred into an Amaxa cuvette and nucleofection conducted using the recommended program. The nucleofected cells were resuspended in 400 μl of minimal media and plated into a 24-well tissue culture tray. The cells were incubated overnight at 37C. The next day the medium was removed and the cells were washed three times with 400 μl of PBS and the medium replaced with serum-free DMEM and the cells incubated at 37C for a further 24 hrs. The following day, microscopy of the GFP-transfected cells was carried out and then cells were trypsinized and resuspended in 100 μl of PBS. Cells (10 μl) were counted in a haematocytometer after trypan blue staining to calculate cell survival. The remaining cells were analysed for GFP expression by flow cytometry analysis and transfection efficiency was determined.
Construction of Gpc3 expression vector
A mouse ORF cDNA (1.74 kb) for glypican 3 (Gpc3) and the gateway cloning vector, pcDNA-DEST40 (7.1 kb) were purchased (GeneCopoeia Inc., MD, USA). Gateway cloning was carried out essentially as described in the manufacturer’s protocol. The construct was verified using restriction digestion analysis and DNA sequence analysis (Applied Biosystems, CA, USA).
Gene expression analysis of nucleofected Gpc3 by RT-PCR
The Gpc3 expression construct (8.84 kb) was nucleofected using optimized transfection conditions (Amaxa transfection kit L and program T030) for human primary calvarial suture mesenchymal cells. Gpc3 nucleofected cells were grown in 24 well tissue culture tray for 24 hrs then washed three times with PBS and grown overnight. RNA was extracted using an RNAeasy mini kit (Qiagen, CA, USA) and cDNA synthesized from 200 ng RNA using a Superscript-III First Strand Synthesis kit (Invitrogen, CA, USA). Primers for Gpc3 (forward 5’-ggttagccagatcattgacaaac-3’ and reverse 5’-cttcatcatcaccgcagtctc-3’) were synthesized (Geneworks, Adelaide, South Australia). The primers were specific for nucleofected mouse Gpc3. PCR reactions were carried out using 2 μl cDNA, 100X SYBR green (Abgene, Epson, UK), 1X Amplitaq PCR buffer, 2 mM MgCl2, 1 unit of Amplitaq (Applied Biosystems, Foster City, CA, USA), 0.4 mM dNTPs (Invitrogen, Carlsbad, CA, USA) and 5.625 μM primers in 25 μl. PCR was carried out using a Gene Amp PCR System 9700 (Applied Biosystems). An expected PCR product (174 bp) was resolved on a 1.5 % agarose gel. The PCR product was sequenced for confirmation (Institute of Medical and Veterinary Sciences, Adelaide, Australia).
Functional analysis of BMP2 and BMP3 using promoter-reporter assays
For transient transfection analysis, human primary calvarial suture mesenchymal cells were grown in 175 cm² flasks to 70-80 % confluency. On the day of transfection cells were washed once with PBS and trypsinized by adding 2 ml of 1X trypsin and incubating the flask in a 37C incubator for 2 mins. The cells were stained with trypan blue and counted using a haematocytometer. Transfection was conducted using the Nucleofector kit L and program T030. In preparation for transfection, 1.5 x 106 cells were resuspended in 100 μl of Nucleofector transfection solution with 3 μg of a BMP responsive promoter luciferase construct, pID183-Luc (5.78 kb)  or a TGF beta responsive promoter luciferase construct, p3TP-Lux (6.55 kb)  together with 500 ng of pRLTK-Luc construct (4.05 kb: Promega Corporation, USA) as a control to normalize transfection efficiency. The transfected cells were transferred to 3 ml of pre-warmed medium and 200 μl of cells were aliquotted to 96-well plates and incubated for 24 hrs in a 37C incubator with 5 % CO2. The following day cells were washed 3 times with PBS and then incubated with 200 μl of serum-free DMEM with antibiotic and BMP2 and BMP3 (R&D Systems, USA) as indicated in figure legends and cells were cultured for up to 48 hrs. Luciferase activity in cell lysates was determined using the Dual Luciferase Assay kit and a Luminometer as described previously [48, 49].
Results are presented as mean value of 2 to 4 biological replicates with standard deviation (SD) where appropriate. Specific assays were statistically analyzed using Analysis of Variance (ANOVA). The differences were considered significant when the P value was less than 0.05.