Induction of myogenic cells from hiPSCs
201B7, 409B2 and 454E2 iPSCs were provided by S. Yamanaka at Kyoto University. 201B7 was generated from a healthy donor using retroviral vectors (20). 409B2 and 454E2 were integration-free hiPS clones generated from healthy donors using episomal vectors (21). hiPSCs were cultured on iMatrix-511 (Nippi)-coated 6-well plates in StemFit AK02N medium (Ajinomoto) supplemented with penicillin/streptomycin/amphotericin B (1% v/v) (FujiFilm) as described previously (36, 37). Myogenic cells were induced from hiPSCs as described (36, 37). After floating culture, cells were induced to differentiate into myogenic cells on collagen type I-coated 10-cm dishes (Iwaki) in 10% FBS (Gibco)/DMEM (FujiFilm) in the presence of 1 μM SB431542 (Wako) for one week. The induced cells were then collected and incubated with ERBB3-APC (1B4C3, BioLegend), CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), and CD271-BB515 (C40-1457, BD Pharmingen). Myogenic cells were sorted as the CD57(-) CD271(+) ERBB3(+) fraction (36, 37).
Derivation of mesenchymal stem cells from hiPSCs (iMSCs)
MSCs were induced from hiPSCs using the STEMdiff Mesenchymal Progenitor kit (Stemcell Technologies) according to the manufacturer’s protocol. In brief, hiPSCs were first induced into early mesoderm progenitor cells with STEMdiff Mesenchymal Induction Medium. Four days later the medium was changed to MesenCult-ACF medium. The cells were then passaged into 6-well plates coated with MesenCult-ACF Attachment Substrate. At day 24, the induced cells were analyzed for cell surface markers, proliferative potential, and differentiation potential. In this study, iMSCs were used between passages 4 and 8.
Human bone marrow MSCs (BM-MSCs)
BM-MSCs were purchased from Lonza (PT-2501), and cultured on gelatin-coated 6-well plates (Iwaki) using MSCGMTM Mesenchymal Stem Cell Growth Medium BulletKitTM (Lonza). Cells were used between passages 4 and 8.
Human myogenic cells
Hu5/KD3 cells are a human myoblast cell line (38). The cells were cultured on collagen-coated dishes (Iwaki) in high-glucose DMEM (FujiFilm) supplemented with 20% FBS (Gibco) and 2% Ultroser G (Biosepta, Pall) as previously described (38). Adult human skeletal muscle myoblasts (hSKMM) were obtained from Lonza (#CC-2580) and cultured on collagen-coated dishes (Iwaki) in 10% FBS/high glucose DMEM. Recombinant human uPAR protein (R&D systems), recombinant BDNF (Peprotech), or recombinant IGF-BP2 (Peprotech) was added to the culture at different concentrations (2.5, 5.0, 10, 20, or 50 ng/ml).
MTT assay
The cells were plated in 24-well collagen-coated plates at different cell densities: 5x104, 7.5x104, and 1.0x105 cells per well (n=3 for each group). The next day, 0.1 ml of 0.5% MTT solution was added per well and incubated for 3 h in a CO2 incubator. After aspiration of the medium, 1.0 ml of acid isopropanol was added to each well, and then the well contents were transferred to 1 ml tubes and centrifuged. The absorbance intensity of the supernatant at OD590 nm was measured in a plate reader (BioTek).
Wound healing migration assay
Hu5/KD3 cells were seeded at a density of 1.0x106 cells/well in 6-well collagen-coated plates (Iwaki) in 10% FBS/DMEM supplemented with cytokines at different concentrations (0–50 ng/ml). The next day, a straight scratch was made in individual wells with a sterile 200 μl pipette tip and photographed with an Olympus DP21 attached to an Olympus CKX41 inverted light microscope. After 6 h of culture in a CO2 incubator, the cells were again photographed (6 views/condition), and the gaps were measured with ImageJ software.
Trilineage differentiation of MSCs
The trilineage differentiation potential of induced MSCs was examined using a human mesenchymal stem cell functional identification kit (R & D Systems) according to the manufacturer’s protocols.
Osteogenic differentiation: Cells were seeded at a density of 4.2 x 103 cells/cm2 in αMEM basal medium in collagen-coated 24-well plates, and were cultured in a 5% CO2 incubator at 37°C. When the cells reached 50–70% confluency, the medium was replaced with osteogenic differentiation medium. After 21 d, the cells were fixed for immunostaining or Alizarin red S staining (Sigma).
Adipogenic differentiation: Cells were seeded at a density of 2.1 x 104 cells/cm2 in αMEM basal medium in collagen-coated 24-well plates and cultured in a 5% CO2 incubator at 37°C. When the cells had grown to 100% confluence, the medium was replaced with 0.5 mL of adipogenic differentiation medium. After 21 d, the cells were fixed for immunostaining or Oil red O staining (Sigma). Nuclei were counterstained with Harris’ hematoxylin.
Chondrogenic differentiation: Cells (2.5 x 105) were centrifuged at 200 x g for 5 min at RT in a 15 mL conical tube and resuspended in 1.0 mL of D-MEM/F-12 basal medium. The cells were again centrifuged, resuspended in 0.5 mL of chondrogenic differentiation medium, and centrifuged to form a cell pellet. The pellets were incubated upright in a 5% CO2 incubator at 37°C. After 21 d, the chondrocyte pellets were fixed and sectioned.
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 10 min and treated with 0.1% Triton X-100 for 10 min at RT. After blocking with 5% goat serum (Cedarlane)/2% bovine serum albumin (BSA; Sigma) in PBS, the cells were reacted with primary antibodies: a rabbit polyclonal myogenin antibody (Santa Cruz Biotechnology) and an anti-pan myosin heavy chain (MHC) antibody, MF20 (R&D Systems) at a 1:200 dilution overnight at 4°C. To detect the differentiation of MSCs, an anti-human osteocalcin antibody (R&D Systems, #967801), an anti-mouse FABP4 antibody (R&D Systems, #967799), or an anti-human aggrecan antibody (R&D Systems, #967800) was used. For detection of undifferentiated human iPS cells, an anti-hSOX2 antibody (Cell Signaling, #3579, anti-hNANOG antibody (R&D Systems, AF1997), or an anti-hOCT-3/4 antibody (R&D Systems, AF1759) was used. The next day, after washing with PBS (−), the cells were incubated with Alexa 568 goat anti-mouse IgG2b, Alexa 488 goat anti-rabbit IgG, Alexa 568 donkey anti-goat IgG, or Alexa 488 donkey anti-mouse IgG (Molecular Probes) for 2 h. The nuclei were then stained with DAPI (Tokyo Chemical Industry). The images were recorded with an all-in-one microscope (BZ-X810) and analyzed with hybrid cell count software (Keyence).
RNA isolation, cDNA synthesis, and qPCR
RT-qPCR was performed as previously described (36, 37). RNA was isolated from cells with TRIzol (Invitrogen) or an RNeasy Mini Kit (Qiagen) and reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Perfect Real Time, Takara). cDNA was amplified with SYBR Premix EX Taq II (Til RNaseH Plus, Takara) and the primer sets listed in Supplementary Table 1. The signal was monitored with a CFX Connect Real-Time System (Bio-Rad), and ΔCt (1/2^ (Cq of the gene-median Cq)) was calculated. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal was used for normalization.
FACS analysis
Cells were incubated with antibodies diluted as suggested by the suppliers in 200 μl of PBS containing 2% FBS (wash buffer) for 30 min on ice, washed in wash buffer, and analyzed using a FACSAria Fusion cell sorter (BD Biosciences). For analysis of iMSCs, CD73-PE (clone AD2, BD Pharmingen), CD105-FITC (clone 266, BD Pharmingen), CD90-PE-Cy7 (clone 5E10, BD Pharmingen), CD34-APC (clone 581, BD Pharmingen), and CD45-APC (clone HI30, BD Pharmingen) were used. The results were analyzed using BD FlowJo software (v.10).
Coculture in a Transwell system
MSCs were plated on Transwell inserts (3.0-µm pore polycarbonate; Corning). These inserts were placed in collagen-coated 12-well plates (Iwaki) already containing cultured human myogenic cells at a ratio of 1:1 for 4 d or 1 wk using 10% FBS/DMEM. To evaluate cell proliferation, nuclei were stained with DAPI and counted using Keyence hybrid cell count software. For evaluation of differentiation, the fusion index was calculated: myogenin-positive nuclei inside the myotubes/total nuclei x 100%.
Mice and cell cotransplantation
NSG-mdx4cv mice (23) were kindly provided by M. Kyba of the University of Minnesota. C57BL/6-background mdx mice were a generous gift from T. Sasaoka (Niigata University, Japan). NOD/Scid mice were purchased from CLEA Japan, Inc.
Under general anesthesia with isoflurane, both right and left TA muscles of NSG-mdx4Cvmdx or NOD/Scid mice were injured by direct injection of a solution of 1.2% BaCl2 in PBS (50 μLl/TA). Myogenic cells were injected into the left TA muscles one day later. First, the myogenic cell cultures were dissociated with 0.05% trypsin-EDTA (Gibco) and resuspended in 10% Matrigel (Corning)/PBS. Under general anesthesia, 60 μL of a cell suspension containing 5x105 myogenic cells was injected into the left TA muscle of 6-month-old NSG-mdx4cvmale mice or NOD/Scid male mice (3–5 months old) using a 27 G syringe (Terumo). For a negative control, 60 μL of 10% Matrigel/PBS was injected into the right TA muscle. At the same time, 300 μL of a cell suspension containing 1x106 MSCs (iMSCs or BM-MSCs) was injected into the intraperitoneal cavity of the mice using a 29 G syringe. For a negative control, PBS vehicle was injected intraperitoneally. The intraperitoneal MSC injections were repeated once per week for the next 3 wks (4 times in total). To examine the effects of uPAR on cell transplantation, Hu5/KD3 cells were cultured for 3 D in 10% FBS/DMEM supplemented with 20 ng/ml recombinant uPAR. Recombinant uPA (1310-SE, R&D Systems) was also added to the culture 24 h before transplantation at a concentration of 20 ng/ml. The cells (1.5x106 cells/TA) were then suspended in 50 μl of PBS buffer containing 5% Matrigel and 5 μg/ml uPAR with or without 5 μg/ml uPA and injected into the TA muscles of NOD/Scid mice that had been preinjured one day earlier with 1.2% BaCl2 solution.
Histopathological and immunohistochemical analysis
TA muscles and diaphragms were frozen in liquid nitrogen-cooled isopentane, cut into 10 μm sections with a cryostat, and analyzed as previously described (36, 37). For immunohistochemical staining, the tissue sections were fixed for 10 min in chilled acetone and air-dried. After blocking with 5% goat serum/2% BSA in PBS, the sections were incubated overnight at 4°C with primary antibodies: anti-human lamin A/C (Santa Cruz), anti-human spectrin (Leica), anti-laminin α 2 chain (clone 4H8-2, Enzo Life Sciences), anti-human lamin B1 (rabbit polyclonal, HistoSure), or anti-dystrophin antibody (rabbit polyclonal, Abcam) (1:100–1:400 dilution). The next day, after washing with PBS, the sections were incubated with fluorescence-labeled secondary antibodies (Alexa 488 goat anti-mouse IgG2b and Alexa568 goat anti-rabbit IgG (Molecular Probes) or Alexa568 goat anti-rat IgG (Molecular Probes)) for 2 h, and mounted in Vectashield with DAPI (Vector Laboratories, Inc). Images were recorded with a Keyence microscope BZ-X810. For evaluation of cell engraftment into dystrophin-deficient mdx muscles, human lamin A/C (+) dystrophin (+) double-positive myofibers were counted. For CSA measurement, 6 images (200X original magnification) stained with anti-laminin α2 chain antibody from a transverse section of one TA muscle (3 TA muscles/group) were randomly selected. To evaluate fibrosis, the Sirius red-stained area was calculated from three random images/TA (100X original magnification) and analyzed using hybrid cell count software (Keyence).
Cytokine array
Cytokine levels in culture media were examined using a Human Cytokine Antibody Array (120 targets, Abcam, Cambridge, UK) according to the manufacturers’ instructions. Culture media were harvested from 80–90% confluent cultures of iMSCs derived from 201B7 or 409B2 iPSCs and BM-MSCs. Unused medium (10% FBS/DMEM) was used as a background control. Signals were detected using a ChemiDoc MP Imaging System (Bio-Rad). Data were analyzed with Image Lab 6.0 (Bio-Rad).
Functional assessment of mdx muscle
The grip strength of mice was measured using a force transducer (Model MK-380M, Muromachi Kikai). The treadmill running test was performed on a Muromachi Model MK-680 (Muromachi Kikai). After 3 wks of acclimatization, the mice (n=3/group) were subjected to exhaustion tests at 5° inclination with the following protocol: 5 min at 5 m/min followed by incremental speed increases of 1 m/min every 3 min until the mice were exhausted. Exhaustion was defined as the fourth time a mouse spent 5 s on the shocker plate without attempting to re-engage the treadmill.
Statistical analysis
Data were analyzed and plotted using Prism 8 software (ver. 8.4.0, GraphPad). Means ± SDs or means ± SEMs are shown. Two experimental groups were compared by an unpaired two-tailed Student’s t-test. Multiple groups were compared by one-way ANOVA with Sidak’s multiple comparisons test or Dunnett’s test. *, **, ***, and **** indicate p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.