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Supplementary material
Fig. S1 Passage 3 hMSCs were characterized using flow cytometric method.
Approximately 98.5% cells were negative for CD34 (A), 89.9% cells were positive
for CD105 (B), and 91.0 cells were positive for CD166 (C).
Fig. S2 Continuous change of GFP expression in Ad-GDF5-infected hMSCs (A) and
the images of GFP expression (B). The cells were transfected with Ad-GDF5 (MOI
50, 100, 200, and 300), and incubated in SM for 1, 3, 5, 7, 9, 11, and 13 days
respectively, and then analyzed by fluorescence-activated cell sorter at each time
point (A). Error bars represent mean±SD (n=3). A small part of cells was dead and
detached from the culture plate (arrows) at MOI 300 (a) 5 days after infected with
Ad-GDF5, while there were nearly no dead cells at MOI 200 (b). Bar is 200µm
Supplementary method
Construction of recombinant adenovirus Ad-GDF5 and Ad-GFP
Ad-GDF5 was constructed using the AdEasy adenoviral vector system
(Stratagene, CA, USA) according to the manufacturer’s instructions. The 1.5-kb
human GDF5 core DNA sequence (Open Biosystems, AL, USA) was cloned into the
pAdtrack-cytomegalovirus (CMV) vector (which contained a gene for GFP). The
resultant pAdtrack-CMV-GDF5 was used to generate the adenoviral GDF5 vector
through homologous recombination with the adenoviral backbone vector, pAdEasy-1,
in BJ5183 bacterial cells. The correct colony for Ad-GDF5 plasmid was tested by
digestion with Pac I restriction enzyme. The right plasmid was then linearized with
Pac I and then used to produce adenoviruses in AD293 packaging cells, resulting in
an Ad-GDF5 vector. Virus stocks were identified by a reverse transcription
polymerase chain reaction (RT-PCR) method, amplified in AD293 cells, purified by
Adeno-Ⅹ™ virus purification kit (Clontech-Takara Bio, CA, USA), and titered by
tissue culture infective dose (TCID50). Ad-GFP, which has only the gene for GFP,
was generated using a similar method as for Ad-GDF5.
HMSCs source and culture
Iliac crest bone marrow aspirate was preferred for the isolation and expansion of
hMSCs. The hMSCs were isolated using a Percoll (TBD Science, Beijing, China)
density gradient centrifuge method. These cells were cultured in a 10-cm cell culture
plate with Dulbecco’s Modified Eagle Medium (DMEM, Gibco, CA, USA)
supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA) at 37°C in a
humidified incubator under 5% CO2. After 8 days, nonadherent cells were discarded
and adherent cells were cultured to confluence, with medium changed every 3 days.
Cells were maintained at subconfluent level and passaged using trypsin/EDTA
(Thermo Scientific HyClone, MA, USA). Flow cytometry method was used for the
characterization of hMSCs with the antibodies CD34-FITC (BD pharmingen, CA,
USA), CD166-PE (AbD Serotec, Kidlington, UK), and CD105-PE (AbD Serotec,
Kidlington, UK) (Supplementary Fig. S1).
For differentiation studies, there are four groups: (1) hMSCs infected with
Ad-GDF5 cultured in standard medium consisted of DMEM supplemented with 10%
FBS; (2) hMSCs cultured in osteogenic medium consisted of DMEM supplemented
with 10% FBS, 5 mM β-glycerol phosphate (Sigma-Aldrich, MO, USA), 50 μg/mL
ascorbate-2-phosphate (Sigma-Aldrich, MO, USA), and 1 nM dexamethasone
(Sigma-Aldrich, MO, USA); (3) hMSCs infected with Ad-GFP cultured in standard
medium, and (4) hMSCs cultured in standard medium.
Adenovirus-mediated gene transfer and determination of the optimal
multiplicity of infection (OMI)
At confluence, MSCs from passage 2 were trypsinized and seeded into a six-well
plate at a density of 1 × 104 cells/well. Following overnight culture, cells were
washed with PBS and Ad-GDF5 was added at a multiplicity of infection (MOI) of 50,
100, 200, and 300 in 1 mL of SM per well. 2 hours later, the mediums containing
adenovirus were changed with SM, and then the mediums were changed every 3 days.
Because of the GFP existing in Ad-GDF5, hMSCs infected with Ad-GDF5 were
viewed daily by fluorescence microscopy (Olympus IX71, Tokyo, Japan). For
accurate quantification of GFP-positive cells, cells were trypsinized and analyzed by
fluorescence-activated cell sorting every 2 days. For control, SM without virus was
added. The infection of hMSCs with Ad-GFP was conducted in a similar way.
RNA extraction, complementary DNA synthesis, and real-time PCR
Gene expressions of GDF5 and genes related to osteogenesis (type I collagen
[COL1A1], alkaline phosphatase [ALP], and osteocalcin [OC]) of the four groups
were determined at days 1, 7, and 14. At each time point, total RNA of the cells was
extracted using Trizol reagent (Invitrogen, CA, USA) and stored at −20°C. To obtain
complementary DNA (cDNA), reverse transcriptase was performed using the
PrimeScriptTM RT-PCR Kit (Takara, Otsu, Japan) according to the manufacturer’s
instructions.
Gene expression was assayed by real-time PCR using the Brilliant SYBR Green
QPCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturer’s
instructions. Primers for GDF5, COL1A1, ALP, and OC were designed using Primer
Premier version 5.0 software (Supplementary Table S1). All the primers including
housekeeping genes GAPDH were purchased from Invitrogen Biotechnology Co, Ltd.
A 20-μL PCR reaction solution (SYBR Green Real-time PCR Master Mix 10μL,
forward primer 0.5 μL, reverse primer 0.5 μL, cDNA 1 μL, DEPC-H2O 8 μL) was
amplified in a Roche Lightcycler (Roche, Basel, Switzerland). Samples were initially
denatured at 94°C for 1 minute, followed by 50 cycles of denaturation at 94°C for 10
seconds, annealing at 55°C (ALP and OC), 57°C (COL1A1), or 59.8°C (GDF5) for
10 seconds, and extension at 72°C for 10 seconds, while the fluorescence sign was
collected during the extension step. The cycle threshold (Ct) was calculated for
further statistical analysis, and the standard curves were established with serial
dilution of sample RNA. Finally, the expression ratios of the genes were obtained by
analyzing with standard curve using Roche Lightcycler software version 4.05, and the
relative expression of target genes was normalized to GAPDH expression.
Supplementary figure legends
Fig. S1 Passage 3 hMSCs were characterized using flow cytometric method.
Approximately 98.5% cells were negative for CD34 (A), 89.9% cells were positive
for CD105 (B), and 91.0 cells were positive for CD166 (C).
Fig. S2 Continuous change of GFP expression in Ad-GDF5-infected hMSCs (A) and
the images of GFP expression (B). The cells were transfected with Ad-GDF5 (MOI
50, 100, 200, and 300), and incubated in SM for 1, 3, 5, 7, 9, 11, and 13 days
respectively, and then analyzed by fluorescence-activated cell sorter at each time
point (A). Error bars represent mean±SD (n=3). A small part of cells was dead and
detached from the culture plate (arrows) at MOI 300 (a) 5 days after infected with
Ad-GDF5, while there were nearly no dead cells at MOI 200 (b). Bar is 200µm
Supplementary Table S1 the primer of the gene of interest and the gene related to
osteogenic differentiation for real-time PCR
Gene
ALP
Sequence
Gene Bank number
5’-CATCCTGTATGGCAATGGG-3’
GI:116734716
5’-GCGGCAGACTTTGGTTTC-3’
COL1A1 5’-TTCCTGCGCCTGATGTCC-3’
GI:110349771
5’-TTTGGGTTGCTTGTCTGTTT-3’
OC
5’-CAGCCACCGAGACACCAT-3’
GI:158517828
5’-CAGCAGAGCGACACCCTA-3’
GDF5 5’-GCTCTAGAGCTACCTGCAGCCACACG-3’ GI:22749746
5’-GCTCTAGAGCTACCTGCAGCCACACG-3’
Size
466bp
393bp
316bp
1505bp