Generation of immunodeficient Col6a1-KO/NSG
Compound heterozygous mice were produced by crossing NOD.CgPrkdcscidIl2rgtm1Wjl/SzJ (NSG; severely immunodeficient mice) with Col6a1GT/GT mice, as described previously [21]. Due to COL6 deficiency, these Col6a1GT/GT mice exhibit muscle weakness from a young age and display UCMD-specific pathology, including endomysial fibrosis caused by excessive activation of interstitial skeletal muscle mesenchymal progenitor cells [21].
Col6a1GT/GT/NSG mice produced via heterozygous crossing were identified by genotyping the resulting littermate population and used for experiments as Col6a1-KO/NSG (immunodeficient UCMD model) mice. Genomic DNA was extracted from the tail of each mouse for genotyping. To select homozygous Col6a1-KO/NSG (Col6a1GT/GT/Il2r−/−) mice, genomic PCR was performed by genotyping Fw-Rv primer pairs for Col6a1 and Il2r (the primer sequences are listed in Table S1). The facility where these animals were housed was managed under SPF conditions and certified by the Japanese Association for Laboratory Animal Science (JALAS) (No. 2020–1). The work has been reported in line with the ARRIVE guidelines 2.0
Induction of HLA-edited XF-iMSCs from iPSCs
MSCs were induced from HLAKO Ff-XT28s05 and HLAKO Ff-I14s04 iPSCs via neural crest cells (NCCs) (provided by CiRA Foundation), following previously established protocols [25]. Briefly, iPSCs were seeded onto iMatrix-511-coated culture plates at a density of 3.6 × 103 cells/cm2 in StemFit AK03N medium (Ajinomoto, Tokyo, Japan) and maintained in culture for 4 days. For NCC induction, the cells were cultured in StemFit Basic03 supplemented with 10 μM SB431542 (584–77,601, Sigma-Aldrich, St.Louis, MO, USA) and 1 μM CHIR99021(FUJIFILM, Wako, Tokyo, Japan) for 10 days, changing the spent medium every other day. CD271 high-expressing NCCs were sorted and seeded onto fibronectin-coated plates at a density of 1 × 104 cells/cm2 in Basic03 medium supplemented with 10 μM SB431542 and 20 ng/ml each of EGF and FGF2 (FUJIFILM Wako). The medium was changed every 3 days. For subculture, the cells were dissociated with Accutase (Innovative Cell Technologies, San Diego, CA, USA) and re-plated onto fibronectin-coated plates at a density of 1 × 104 cells/cm2. For MSC differentiation, expanded cells (passage number 2–4 [PN2-4]) were seeded onto fibronectin-coated plates at a density of 1 × 104 cells/cm2 in StemFit Basic03 medium supplemented with 10 μM SB431542 and 20 ng/ml each of EGF and FGF2. After 24 h, the spent medium was replaced with PRIME-XV MSC Expansion XSFM medium (FUJIFILM Irvine Scientific). Passages were performed every 3–4 days. At PN2, MSC differentiation was confirmed via FACS using human MSC markers (positive markers: CD29, CD44, CD73, CD90, and CD105; negative markers: CD34 and CD45).
Isolation of bone marrow-derived MSCs (BM-MSCs)
Healthy, non-dystrophic bone marrow fluid was obtained from all the subjects. The methods for dissociating cells and cultures have been described previously [38]. The bone marrow fluid was mixed with an equal volume of growth medium containing 10% fetal bovine serum (FBS; 556–33865, FUJIFILM Wako) in MEM-Glutamax (32571036, Gibco, MA, USA). The mixture was centrifuged at 1200 rpm for 5 min at room temperature (20–25 °C), and the top layer containing fatty components was discarded. The supernatant and intermediate layers were mixed in equal volumes of culture medium and centrifuged again at 1200 rpm for 5 min at room temperature. The supernatants and intermediate layers were transferred to new tubes. Erythrocytes were lysed by mixing an equal volume of 0.1 M citric acid/0.1% crystal violet solution with the cell suspension, and mononuclear cells (MNCs) with purple nuclei were counted. MNCs were seeded at a density of 2.5 × 105/cm2 and cultured at 37 °C with 5% CO2 and 3% O2.
Isolation of adipose-derived MSCs (Ad-MSCs)
Healthy, non-dystrophic adipose tissue was obtained from adipose tissue discarded around the donor renal capsule during kidney transplantation. The methods for dissociating cells and establishing cell cultures have been described previously [39, 40]. Briefly, the specimens were cut into 2 mm squares. The adipose tissue was then digested in Hank’s balanced salt solution containing 1 mg/ml collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37 °C for 1 h. The digested tissue was filtered through a 100- µm-pore filter to remove the undigested debris. The obtained stromal vascular fraction was then cultured in a medium comprising a 3:2 mixture of Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) and MCDB 201 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 1 ng/ml linoleic acid-albumin (Sigma-Aldrich), 1% v/v Insulin, Transferrin, Selenium (ITS) supplement (Sigma-Aldrich), 0.1 mM ascorbic acid phosphate ester magnesium salt (Wako Pure Chemical Industries, Osaka, Japan), 50 U/ml penicillin, 50 mg/ml streptomycin (Meiji Seika Ltd, Tokyo, Japan), and 2% FBS (Sigma-Aldrich).
Flow cytometry analysis
Flow cytometry was performed using a BD FACS Aria II_v1.87 (BD Biosciences, NJ, USA), following the manufacturer’s instructions. Cells were dissociated using Accutase, washed with FACS buffer (1% human serum albumin in PBS), and filtered through a 35- μm filter (Corning, NY, USA). The cells were then pelleted via centrifugation (1200 rpm, 3 min, 4 °C) and resuspended in FACS buffer with the appropriate antibodies, followed by a 60 min incubation at 4 °C. An isotype control was included in all experiments to eliminate nonspecific background signals. After the antibody reaction, the cells were washed with FACS buffer, pelleted, and washed again. Finally, the cells were resuspended in FACS buffer and collected using a cell strainer. Flow cytometry was performed using a BD FACS Aria II instrument. Results were collected using FACSDiva_v9.0.1 and analyzed using FlowJo_v10.8.1 software (BD Biosciences). Specific details regarding the antibodies used are listed in Table S2.
MSC transplantation into Col6a1-KO/NSG mice
Transplantation was performed according to the methods described in previous studies [23]. Male Col6a1-KO/NSG mice (5–8-week-old) were anesthetized with 3% veterinary inhalation anesthetic (Isoflurane [(2RS)-2-Chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane], 704239096, MSD, Tokyo, Japan). All types of MSCs were suspended in their respective expansion media (1 × 106 cells or 2 × 106 cells/50 μl) and injected into the center of the left tibialis anterior (TA) muscles with a 27G micro-syringe (Myjector syringe; Terumo, Tokyo, Japan). Simultaneously, the same amount of medium was injected into the right TA muscles and used for comparison during histological analysis. The work has been reported in line with the ARRIVE guidelines 2.0.
Tissue preparation and histological analysis
Sample preparation
Mice were euthanized using CO2 at 1, 12, or 24 weeks after cell transplantation. The TA muscles were mounted in Tragacanth Gum (FUJIFILM Wako) and frozen in 2-methylbutane (166–00615, Wako, Kyoto, Japan) with liquid nitrogen. Next, 10- μm-thick cryosections were made using Cryostat (Leica Biosystems, Nussloch, Germany), with two cryosections selected from 1 mm above the central portion and 1 mm below the central portion of one TA muscle. The selected sections were stained and used for analysis.
Fluorescence immunostaining and analysis
The selected sections were subjected to immunofluorescence staining using anti-COL6, anti-embryonic myosin heavy chain (eMHC), anti-human lamin A/C (hLamin A/C), and anti-laminin α2 antibodies and mounted with Aqua-PolyMount (18606–20, Polyscience, Niles, IL) with DAPI. Immunofluorescence images were obtained using a Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) and a BZ-X700 microscope (Keyence, Osaka, Japan). Area measurements and cell counts were performed using BZ-X700 software. The antibodies used in this experiment and their concentrations are listed in Table S2.
Sirius red staining
Of the two cryosection slides selected, the slide numbered next to the one with more COL6-positive areas by fluorescent immunostaining was selected, and Sirius Red staining was performed using a Picrosirius Red Stain Kit (24901–250, Polyscience, Niles, IL, USA) according to the manufacturer’s instructions. Images were captured using a BZ-X700 microscope (Keyence), and area measurements were performed using BZ-X700 software.
Isolation of muscle stem cells (MuSCs) from mice
MuSCs were isolated from 4 to 6-week-old female Col6a1-KO/NSG mice. Single cells were isolated from skeletal muscle tissue according to the protocol previously described [23]. Briefly, the mice were first euthanized with CO2 and then skeletal muscles from the lower and upper arms were collected and digested into single cells using 0.2% collagenase type 2 (LS004174, Worthington Biochemical, NJ, USA) and DMEM (08488–55, Nacalai Tesque, Kyoto, Japan). Subsequently, the samples were stirred in a stirrer at 37 °C for 1 h. Homogenization was then performed using an 18G needle, followed by stirring for 30 min. After stirring, homogenization was performed again using an 18G needle, then the sample was diluted in PBS and filtered through a 40- μm cell strainer (352340, Corning). The mixture was then centrifuged at 500 × g for 5 min at 4 °C. The supernatant was removed, and the cells were suspended in a MuSC expansion medium [DMEM supplemented with 20% FBS and 0.5% FGF2 (10 -µg/ml) (47107000, Oriental Yeast, Kyoto, Japan)] and seeded onto a collagen I-coated 10-cm dish (4020–010, IWAKI, Shizuoka, Japan). Subsequently, MuSCs were expanded for approximately 6 days using the pre-plating method to separate MuSCs from other types of non-myogenic cells, as previously described [41]. Briefly, the single-cell suspension was collected using the aforementioned method and seeded onto type 1 collagen (COL1)-coated dishes. The following day, cells that had not yet adhered to the dish were collected from the culture medium and re-seeded at a density of 5 × 105 cells in a new COL1-coated dish. Starting the day after re-seeding, the spent medium was changed approximately every two days to expand the MuSCs. The cells were used for the experiments before the monolayers became confluent (80–90% confluence).
Co-culture with MSCs and MuSCs derived from Col6a1-KO/NSG mice
Before seeding MuSCs, Ad-MSCs, BM-MSCs, and XF-iMSCs were each seeded with two clones at a density of 5 × 104 cells per well in 24-well plates coated with COL1 and cultured in their respective optimal expansion media. To stop MSC proliferation, mytomycin C (Wako, Kyoto, Japan) was added to the cells (final concentration: 8.9 μg/ml) 2 h before MuSC seeding, followed by two washes with PBS. Subsequently, a MuSC expansion medium containing DMEM supplemented with 20% FBS and0.5% FGF2 (10-µg/mL) (47107000, Oriental Yeast) was added to the cells. MuSCs derived from Col6a1-KO/NSG mice were seeded at a density of 5 × 103 cells/well on plates containing feeder cells (Ad-MSCs, BM-MSCs, or XF-iMSCs). After 3 d, the spent medium was changed to MuSC differentiation medium (DMEM supplemented with 10% FBS). Three and six days after MuSC seeding, the cells were subjected to immunofluorescence staining and analyzed.
Three independent experiments were performed using MuSCs from different mice to ensure the reliability and reproducibility of the results. In addition, to ensure more accurate comparisons between the three studies on the effects of each group on MuSCs, the results of the analysis were calculated as relative values.
Immunocytochemistry and analysis
Prior to immunostaining, cells on plates were fixed with 2% PFA and subjected to immunofluorescence staining using anti-Pax7, anti-MyoD, anti-hLamin A/C, anti-MHC, and anti-DAPI antibodies. The antibodies used in this study and their concentrations are listed in Table S2. Each clone was imaged in five pictures, and the myogenesis of MuSCs was analyzed using Keyence BZ-X700 software. The average value from these five images was used as the representative value for each clone of each MSC.
Relative values were calculated by dividing the average values obtained in each group by the average value obtained in the XF-iMSCs group.
The relative quantity of total myogenic cell count, MHC area, and myotube with four (two) or more nuclei analyses were calculated using this formula.
$$\begin{aligned} & Relative\,Quantity_{{total\,myogenic\,cell\,counts,MHC\,area\,or\,myotube\,with\,four\,(two)\,or\,more\,nuclei}} \hfill \\ & \quad = \frac{{Average\,Quantity_{{total\,myogenic\,cell\,counts,MHC\,area\,or\,myotube\,with\,four\,(two)\,or\,more\,nuclei}} }}{{Average\,Quantity_{{total\,myogenic\,cell\,counts,MHC\,area\,or\,myotube\,with\,four\,\left( {two} \right)\,or\,more\,nuclei – XF – iMSCs}} }} \hfill \\ \end{aligned}$$
Protein extraction and western blotting analysis
Cells were lysed in RIPA buffer (08714–04, Nacalai Tesque) containing a protease inhibitor cocktail (25955–11, Nacalai Tesque) and thoroughly sonicated (UCD-250, Bioruptor). Protein samples (approximately 4 µg) were mixed with a reducing agent (NP0004, Invitrogen, Carlsbad, CA, USA) and loaded onto 4–12% Bolt Bis–Tris Plus Gels (NM04122BOX, Thermo Fisher Scientific, Waltham, MA, USA). The gels were electrophoresed on an Invitrogen protein electrophoresis system to separate the proteins, which were then transferred to a PVDF membrane using an iBlot2 Gel Transfer Device (IB 2100, Thermo Fisher Scientific) with the P3 program. The membrane was blocked with Blocking One reagent (03953–95, Nacalai Tesque) and incubated overnight at 4 °C or 1 h at room temperature with the primary antibodies diluted in Can Get Signal Solution 1 (NKB-201, TOYOBO, Osaka, Japan). After three washes with TBS containing 0.05% Tween-20 (P7949, Sigma-Aldrich), the membrane was incubated for 1 h at room temperature with the corresponding secondary antibodies diluted in Can Get Signal Solution 2 (NKB-101, TOYOBO). If necessary, following three additional washes with 0.05% TBS-T, a third antibody, diluted in PBS, was applied for 5 min at 4 °C. Detailed antibody information is provided in Table S2. Detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (34094, Thermo Fisher Scientific). Images were visualized using an ImageQuant 8000 imaging system (Cytiva, Wilmington, DE, USA) and the bands were semi-quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Quantitative RT-PCR
Total RNA was purified using the ReliaPrep RNA Miniprep System (Z6012; Promega, Madison, WI, USA). Briefly, 200 ng of total RNA was reverse-transcribed to obtain single-stranded cDNA using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (FSQ301, TOYOBO) according to the manufacturer’s instructions. Quantitative PCR with a SYBRGreen mix (Applied Biosystems, Foster City, C) was performed in duplicate using a Step One Plus Real-Time PCR System (Applied Biosystems). The primer sequences are listed in Table S3.
Then, the relative expression was calculated by dividing the average values obtained in each group by the average value obtained in the XF-iMSCs group.
$${Relative\, Expression}_{group}=\frac{{Average\, Expression}_{group}}{{Average\, Expression}_{ XF-iMSCs}}$$
Here, the Average Expressiongroup refers to the average value obtained from each group, and Average the Expression XF-iMSCs refers to the average value obtained from the XF-iMSC group.
ELISA
Each cell line was seeded at a density of 5 × 104 cells/well into a 24-well plate. When the cells reached 90% confluency, the spent medium was changed, and the cells were cultured for another 24 h at 37 °C. For cells grown in serum-containing culture media, the corresponding medium without serum was used as the refreshing medium. The cell culture supernatants were centrifuged and stored at -80 °C until further analysis. Insulin growth factor 2 (IGF2) protein was quantified using a Human IGF-II/IGF2 Quantizing ELISA kit (DG200, R&D Systems, Minneapolis, MSP, USA). IGF2 levels were quantified based on a standard log/log curve fit, with the mean absorbance reading on the y-axis and the concentration on the x-axis. The optical density of each sample was obtained using an Envision multi-mode plate reader (PerkinElmer, Waltham, MA, USA).
siRNA-mediated IGF2 knockdown in XF-iMSCs
Briefly, 6 pmol IGF2 RNAi duplex (Silencer® Select siRNA s7216, Thermo Fisher Scientific) was diluted in 100 µl Opti-MEM™ I Reduced Serum Medium (31985062, Thermo Fisher Scientific) without serum in each well of a 24-well tissue culture plate. Next, 1 µl Lipofectamine RNAiMAX (13778150, Thermo Fisher Scientific) was added to each well containing the diluted RNAi molecules. The solutions were mixed gently and incubated for 10–20 min at room temperature. Next, 2 × 104 cells were suspended in 500 µl of complete growth medium without antibiotics to achieve 30–50% confluence 24 h after seeding. To each well containing the RNAi duplex/Lipofectamine RNAiMAX complexes, 500 µl of the diluted cell suspension was added (final volume: 600 µl; final RNA concentration: 10 nM). Finally, the cells were incubated for 72 h at 37 °C in a 5% CO2 incubator.
IGF2 supplementation experiments
The MuSCs obtained using the method mentioned above were seeded in COL1-coated 24-well plates. In the IGF2 supplementation group, Recombinant Human IGF-II (292-G2-050, R&D Systems) was added at a concentration of 3.6 ng/ml in MuSC expansion medium on days 1 to 3 and 2.4 ng/ml in MuSC differentiation medium from days 4 to 6, and then cultured. Three and six days after MuSC seeding, the cells were fixed with 2% PFA and subjected to immunofluorescence staining. Finally, the myogenesis of MuSCs was assessed using a KEYENCE BZ-X analyzer.
To ensure the reliability and reproducibility of the results, three independent experiments were performed using MuSCs from different mice. In addition, to ensure more accurate comparisons between the three studies on the effects of each condition on MuSCs, the results of the analysis were calculated as relative values.
Statistical analysis
Animals were excluded from the study only if they appeared to be in extremely poor health, such as injuries due to fighting. One-way analysis of variance (ANOVA) and Dunnett’s test were used to assess differences among groups. An unpaired two-group t-test was used to assess differences between two groups. Differences were considered significant at p-values < 0.05. All statistical analyses were performed using Excel (Microsoft) and MEPHAS software (Osaka University, Japan).
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