-/- genotyping was performed with polymerase chain reaction (PCR) with oligonucleotides forward: 5'-TCACATGGAGTGAATGCTCTG-3' and reverse: 5'-AGTGAACACCAGGCCAGTTT-3' . All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh.
MDSPCs were isolated from 8-week-old Zmpste24
-/- mice and WT littermates by using a modified preplate technique according to a previously established protocol [16, 17]. MDSPCs were cultured in proliferation medium (PM) containing Dulbecco's modified Eagles medium (DMEM, high glucose) supplemented with 10% horse serum, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (all from Invitrogen, Grand Island, NY, USA), and 0.5% chick embryo extract (Accurate Chemical, Westbury, NY, USA), on collagen type I-coated flasks (Sigma-Aldrich, St. Louis, MO, USA). Cells were used between passages 20 and 30.
Proliferation in vitro
The population doubling time (PDT) over a 72-hour period was measured from time-lapse images acquired through a live-cell imaging (LCI) system (Automated Cell Technologies, Inc., Pittsburgh, PA, USA), as previously described [25, 26]. In brief, cells were plated at an initial density of 2,000 cells/well in collagen type I-coated 24-well plates, and images were acquired at 15-minute intervals over a period of 3 days by using an LCI system equipped with a 10× objective. Five images at each time point were randomly selected and analyzed, by using ImageJ software (v. 1.44p; National Institutes of Health, Bethesda, MD, USA), from three independent experiments on three distinct cell lines in each group.
Myogenic differentiation in vitro
MDSPCs were seeded onto collagen type I-coated six-well plates (1 × 105 cells/well) and cultured for 2 days in PM. Myogenic differentiation was induced by replacing the PM with differentiation media (DMEM, 2% FBS, 1% penicillin-streptomycin) and cultured for 3 days. Immunostaining for fast myosin heavy chain (f-MyHC), a marker of terminal myogenic differentiation, was performed to stain differentiated muscle cells. Cells were fixed with -20°C methanol, blocked with 5% goat serum, and incubated at room temperature with primary mouse anti-MyHC fast (1:250; Sigma-Aldrich) and secondary biotinylated IgG (1:250; Vector Laboratories, Burlingame, CA, USA) each for 1 hour, and streptavidin-594 (1:500; Sigma-Aldrich) for 15 minutes. The nuclei were visualized by staining with DAPI (4', 6' diamidino-2-phenylindole, 100 ng/ml; Sigma-Aldrich) for 10 minutes. Fluorescent images were taken by using a Leica DMIRB inverted microscope equipped with a QImaging Retiga digital camera with Northern Eclipse software (v. 6.0; Empix Imaging, Inc., Cheektowaga, NY, USA). Total cell nuclei and nuclei within f-MyHC-positive myofibers, from at least 15 fields, each from three replica platings, were counted by using ImageJ. The myogenic differentiation was calculated as percentage of cells expressing f-MyHC (red) per total nuclei (DAPI, blue).
MDSPCs from WT and Zmpste24
-/- were lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (Sigma-Aldrich). Protein concentration was measured with the Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from each sample were loaded on 10% SDS-polyacrylamide gels and run for 2 hours at 100 volts. Proteins were then transferred for 60 minutes by 100 volts to a nitrocellulose membrane (Millipore, Billerica, MA, USA) and blocked by 5% nonfat dry milk (Bio-rad) in Tris-buffered saline Tween-20 (TBST) for 1 hour. Samples were probed with goat N-terminal anti-lamin A/C (1:200, sc-6215), goat C-terminal anti-prelamin A (1:200, sc-6214), and rabbit anti-β-actin (1:200, all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), overnight at 4°C. After washing 3 times with TBST, the membrane was incubated with secondary antibody rabbit anti-goat IgG (H&L) coupled to horseradish peroxidase (HRP; Santa Cruz Biotechnology, Inc.) and mouse anti-rabbit IgG-HRP for 1 hour at room temperature. Blots were developed by using a SuperSignal West Femto with enhanced chemiluminescent substrate (1:100,000; Thermo Scientific, Rockford, IL, USA), and the bands were detected on x-ray film.
Reverse transcriptase polymerase chain reaction (RT-PCR)
RNA was extracted from cultured cells by using TRIzol (Invitrogen) and a RNeasy Mini kit (Qiagen Inc., Hilden, Germany). The amount of RNA yield was quantified by using a Nano-Quant (Tecan, San Jose, CA, USA), and a total of 1 μg of RNA was reverse-transcribed into cDNA by using the Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. Then, cDNA was mixed with GoTaq polymerase, dNTPs, and green GoTag reaction buffer (Promega, Madison, WI, USA) and amplified on a thermal cycler (Eppendorf Mastercycler proS), according to the manufacturer's instructions for 30 cycles at 58°C annealing temperature. PCR products were separated by using 2% agarose gels with 1% ethidium bromide. The gel images were captured with a BioRad Gel Doc system. The following primers were used: Stem cell antigen-1 (Sca-1), forward 5'-CCTACTGTGTGCAGAAAGAGC-3' and reverse 5'-CAGGAAGTCTTCACGTTGACC-3'; CD34, forward 5'-GCAGCTTTGAGATGACATCACC-3' and reverse 5'-CTCAGCCTCCTCCTTTTCACA-3'; myogenin, forward 5'-CTACAGGCCTTGCTCAGCTC-3' and 5'-TTGTGGGCGTCTGTAGG-3'; desmin, forward 5'-AACCTGATAGACGACCTGCAG-3' and reverse 5'-GCTTGGACATGTCCATCTCCA-3'. β-actin, forward 5'-GGGTCAGAAGGACTCCTATG-3' and reverse 5'-CTTTGATGTCACGCAGCACGATT-3' was used as a loading control.
-/- MDSPCs were plated in the lower compartment of Costar Transwell Permeable Supports (Corning, Tewksbury, MA, USA) in PM at a density of 3,000 cells/well in a 24-well collagen type I-coated plate. WT MDSPCs were seeded into 6.5-mm transwell membrane inserts at the same density in PM and placed above the Zmpste24
-/- MDSPCs. The 24-well plates containing the transwells were then placed in the LCI system for 72 hours to measure proliferation of the Zmpste24
-/- MDSPCs, as described earlier. As a control, each plate contained wells of Zmpste24
-/- MDSPCs with empty transwell membrane inserts. To measure the differentiation potential of Zmpste24
-/- MDSPCs after coculture, transwell inserts were removed after 72 hours, and the PM media was switched to differentiation media. After 2 to 3 days, myogenic differentiation of the cells was tested by immunostaining for f-MyHC, as described earlier. Changes in differentiation potential of Zmpste24
-/- MDSPCs was also tested by using conditioned media from the WT MDSPCs. WT MDSPCs were cultured for 2 days in 25 cm2 collagen-coated flasks and were then treated with differentiation media for 3 days. The supernatant was collected and used as conditioned media for 8-week-old Zmpste24
-/- MDSPCs, whereas unconditioned differentiation media was used as a control.
The 3 × 105 donor MDSPCs isolated from WT or Zmpste24
-/- mice were intramuscularly transplanted into the gastrocnemius muscles of 8- to 9-week-old mdx/SCID mice (C57BL/10ScSn DMDmdx/J/CB17-Prkdcscid/J; The Jackson Laboratory), an animal model of Duchenne muscular dystrophy that is also immunocompromised. Mice were killed 2 weeks after transplantation, and the gastrocnemius muscles (n = 3) were harvested, frozen in 2-methylbutane pre-cooled in liquid nitrogen, and cryosectioned (10 μm). Sections were immunostained for dystrophin, the cross-sectional area of dystrophin-positive myofibers was measured, and the distribution of the fiber areas was plotted as previously described [27, 28].
Statistical analyses were carried out by using Sigmastat (Jandel Scientific, v. 2.0, San Rafael, CA, USA) software package. The Student's t test or Mann-Whitney Rank Sum test was used for direct comparisons between groups. For multiple comparisons, the one-way ANOVA or the Kruskal-Wallis one-way ANOVA on ranks was applied. Pairwise multiple comparisons were performed by using the Tukey's Test after rank-based ANOVA. All values are expressed as the mean ± SD, and P < 0.05 was considered significant.