Isolation and characterization of hAFS cells
Human amniotic fluid (16 to 18 weeks of gestation) was obtained from donors at Kyungpook National University Hospital who provided informed consent. The amniotic fluid was transferred to the Joint Institute for Regenerative Medicine (JIRM): Kyungpook National University Hospital-Wake Forest Institute for Regenerative Medicine for isolation of hAFS cells. Isolation of hAFS cells and experimental procedures were approved by the Institutional Research Board of Kyungpook National University Hospital (KNUHBIO_09-1008). Briefly, amniotic fluid was centrifuged and cultured in (D)MEM high-glucose containing 10% FBS, and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) for one week, as previously described . For maintenance of human AFS cells, the cells were cultured in α-MEM medium containing 15% ES-FBS, 1% glutamine, and 1% penicillin/streptomycin (Invitrogen), supplemented with 18% Chang B and 2% Chang C (Irvine Scientific, Santa Ana, CA, USA) at 37°C in a 5% CO2 atmosphere. Confluent hAFS cells were harvested by trypsinization for further expansion.
Expression of pluripotent markers was identified by RT-PCR using specific primers for OCT4, SCF, GATA4, VIM, CK18 and FGF5. Cell surface mesenchymal stem cell markers such as CD44, CD73, CD90 and CD105 were analyzed using flow cytometry (FACS Aria, BD bioscience, San Jose, CA, USA), as described previously . PE-conjugated primary antibodies were obtained from BD bioscience.
Preparation of lentiviruses and transduction
The human MYOD or empty DNA vector was inserted into the lentiviral vector pHJ-1. To make the virus particle solution, human MYOD or empty pHJ-1 lentiviral vector was co-transfected with lentiviral packaging vectors (pHDM-Hgpm2, pRC/CMV-Rev1b, and pHDM.G) into 6 × 104 cells/cm2 293FT cells. The culture supernatant was collected 48 hours later and centrifuged for removal of 293FT cells and debris. They were frozen for storage in a deep-freezer. Lentivirus supernatant was thawed with water at room temperature and mixed with an equal volume of culture media. For lentiviral transduction, the viral supernatants were added to 6 × 103 cells/cm2 hAFS cells in the presence of 8 μg/ml polybrene for 12 hours. After 12 hours, the cells were washed with PBS and changed with fresh culture media. First, hAFS cells were infected with eGFP lentivirus, and approximately more than 80% of the hAFS cells were eGFP-positive. The infected cells were then sorted using FACS Aria. The eGFP-positive hAFS cells were cultured for three days and then transduced again with MyoD or EV lentivirus. During this time period, the cells did not show any sign of cell death (data not shown).
Reverse transcription PCR
Total RNA was extracted from cultured hAFS cells using Tri-solution (Bioscience, Gyeongsan, Korea). First-strand complementary DNA was synthesized with 1 μg total RNA using the Super Script II First-Strand Synthesis System (Invitrogen). From the 20 μl complementary DNA reaction volume, 1 to 2 μl were used for each PCR assay. The primers used in this study were as follows: MYH: forward, 5′-TGTGAATGCCAAATGTGCTT -3′, and reverse, 5′-GCCTTTATTTTGATCACC-3′; MYOG: forward, 5′-CAGCGAATGCAGCTCTCACA-3′, and reverse, 5′-AGTTGGGCATGGTTTCATCTG-3′; MYOD: forward, 5′-AGCACTACAGCGGCGACT-3′, and reverse, 5′-GCGACTCAGAAGGCACGTC-3′; DES: forward, 5′-CCTAC TCTGCCCTCAACTTC-3′, and reverse, 5′-AGTATCCCAACACCCTGCTC-3′; and MEF2C: forward, 5′-GGGTGGAGACCTCACGTCTG-3′, and reverse, 5′-TTATTTATCCTTTGATTCA CTGATG-3′. PAX3: forward, 5′-GTCAACCAGCTCGGCGGTGTTT-3′, and reverse, 5′-ATGGCACCAGGACGTATGGGT-3′, MEOX1: forward, 5′-AAAGGACCGAGGCGTGCAGC-3′, and reverse, 5′-CTCCTCCTGGGGCAGGCTGT-3′, SIX1: forward, 5′-CTTAAAGGCTACT GAGTGCGCCG-3′, and reverse, 5′-TGCGTAAAGCCAAACGACGGCA-3′, EYA2: forward, 5′-CGC TGCTGTGTGGACTCTGAGT-3′, and reverse, 5′-AGTGGGTGA GGTGCTGAAGGAAGGG-3′, GLI2: forward, 5′-TGGAATTTGGAACTGGCTTC-3′, and reverse, 5′-CCTCATTAAGGCC AAGGTC A-3′, FOXC1: forward, 5′-AGGAAGGCGAGAGGAGCAGAACAT -3′, and reverse, 5′-GATTGGCAGGGCAGATCACCC-3′, FOXC2: forward, 5′-TCCACGCCGCCTCTCTATCGC-3′, and reverse, 5′-TTGCGTCTCTGCAGCCCCTTAAT-3′. A primer set of the housekeeping gene GAPDH was used as an internal control. Complementary DNA was amplified using a LA Taq™ polymerase with GC buffer (Takara, Tokyo, Japan) with a total of 25 to 40 cycles. PCR products were resolved by agarose gel electrophoresis.
hAFS cells were detached physically from culture dishes using a cell scrapper and sonicated in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)). Protein concentration was determined using a BCA protein assay kit (Interchim, Montlucon, France). Protein samples were separated in SDS-PAGE and transferred to Protran membranes (Whatman, Florham Park, NJ, USA). The membrane was blocked with 3% non-fat dry milk in TBS-T and each primary and corresponding secondary antibody was incubated for one hour. Primary antibodies and dilutions used were as follows: mouse monoclonal anti-MyoD (BD biosciences) at 1:500; rabbit polyclonal anti-Myf5 (C-20) (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) at 1:200; mouse monoclonal anti-desmin (BD biosciences) at 1:500; rabbit polyclonal anti-dystrophin (Abcam Inc., Cambridge, MA) at 1:200 and mouse monoclonal anti-FLAG M2 (Sigma-Aldrich Co. St. Louis, MO, USA). Secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Invitrogen. The signal was detected using WesternBright ECL (Advensta, Menlo Park, CA, USA).
Nucleus and cytoplasm were fractionated as described previously . Briefly, collected cells were re-suspended with buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.05% NP40), placed on ice for 10 minutes and centrifuged at 4°C at 3,000 rpm for 10 minutes. Supernatant was kept as a cytoplasmic fraction. The pellets were resuspended in 374 μl of buffer B (5 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 26% (v/v) glycerol) and 26 μl of 4.6 M NaCl (300 mM NaCl). The re-suspended pellets were homogenized with full strokes in a Dounce or glass homogenizer and placed on ice for 30 minutes, followed by centrifugation (14,000 rpm) at 4°C for 30 minutes. The supernatant was used as nuclear fractions.
Immunostaining and H&E staining
Cells plated on cover slips were fixed with 4% paraformaldehyde-PBS, and permeabilized with 0.25% Triton X-100 for MYOD, desmin, α-actinin staining. Nonspecific reactions were blocked with 3% normal goat serum. Cells were then incubated with mouse monoclonal anti-MyoD (BD Bioscience), mouse monoclonal anti-desmin (BD Bioscience) and mouse monoclonal anti-α-actinin (BD Bioscience) primary antibodies, at the dilutions recommended by the manufacturer, overnight at 4°C, followed by incubation with secondary antibodies for one hour at room temperature. Anti-mouse Alexa Fluor 488-conjugated secondary antibodies (Invitrogen) and 0.1 μg/ml of DAPI (Santa Cruz Biotechnology, Inc.) were used for immunofluorescence. Cover slips were mounted on slides using fluorescent mounting medium (Dako, Carpinteria, CA, USA).
Muscle tissues were fixed with 4% paraformaldehyde-PBS for 30 minutes at 4°C. The tissues were cryostat sectioned (10 μm thick) and permeabilized with PBS containing 0.25% Triton X-100 for five minutes. The sections were then blocked with 3% normal goat serum in PBS, and incubated rabbit polyclonal anti-dystrophin (Abcam, Cambridge, UK) at 1:50 to 1:100 dilutions overnight at 4°C. The tissue sections were washed with PBS and then incubated with TRITC-conjugated anti-rabbit immunoglobulin G (IgG) antibodies (Sigma-Aldrich) at a 1:500 dilution for one hour at room temperature. The sections were washed with PBS and mounted with fluorescent medium (Dako) with DAPI. Immunofluorescence was visualized using an LSM 5 fluorescence microscope (Zeiss, Jena, Germany).
For hematoxylin/eosin (H&E) staining, the muscle samples were fixed with 4% paraformaldehyde-PBS at 4°C for two days. The tissues were embedded with paraffin and sectioned with 5 μm thickness. The sections were stained with H&E staining using a standard protocol.
Human AFS cells transplantation in mice
All animal experiments were performed under the guidance of the Institutional Ethics Committee of Kyungpook National University. Immunodeficient BALB/cSlc-nu mice were maintained under conventional housing conditions using a chamber system. Male mice aged 12 weeks were used. hAFS cells at passage 11 were used for in vivo injection. Before injection, hAFS cells were transduced with eGFP-lentivirus, and then eGFP-positive hAFS cells were sorted by fluorescence activated cell sorting (FACS). The eGFP-positive hAFS cells were cultured for three days and transduced again with empty vector- or MYOD-lentivirus. The cells were trypsinized with 0.25% trypsin/EDTA (Invitrogen) and washed with DPBS (Wellgene, Daegu, Korea). MYOD transduced hAFS cells were injected into the left tibialis anterior (TA) muscles of mice two days after lentivirus infection. Efficiency of lentivirus infection was confirmed with Western blot using MYOD antibody (Ab) as well as eGFP transduction rate. The transduction rate was approximately 80% of infected hAFS cells. BALB/cSlc-nu mice were anesthetized with intramuscular injection of 80 μl of a solution (33.8% Zoletil and 6.5% Rompun in PBS). At the point of transplantation, the left TA muscle was injected with 40 μl of 10 μM cardiotoxin, and approximately 500,000 MYOD- or EV-hAFS cells were injected using a 300 μl insulin syringe to the center of the muscle. The mice were euthanized at 7 and 21 days. The TA muscles were cross cryostat sectioned and stained with H&E and immunofluorescence. To analyze morphological changes in MYOD-hAFS cell-injected TA muscle, we examined three mice in each group. A total of 144 sections per each group (48 per mouse) were stained with H&E. For immunohistochemistry (IHC) with dystrophin, forty eight sections from each group around the center area were used, and the IHC images of dystrophin were used to calculate an average area of muscle fibers and percentage of centrally nucleated myofibers. The average size and percentage of centrally nucleated muscle fibers were measured with i-solution image software (Daejeon, Korea).