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Distinct muscle regenerative capacity of human induced pluripotent stem cell-derived mesenchymal stromal cells in Ullrich congenital muscular dystrophy model mice

Stem Cell Research & Therapy volume 15, Article number: 340 (2024) Cite this article

Abstract

Background

Ullrich congenital muscular dystrophy (UCMD) is caused by a deficiency in type 6 collagen (COL6) due to mutations in COL6A1, COL6A2, or COL6A3. COL6 deficiency alters the extracellular matrix structure and biomechanical properties, leading to mitochondrial defects and impaired muscle regeneration. Therefore, mesenchymal stromal cells (MSCs) that secrete COL6 have attracted attention as potential therapeutic targets. Various tissue-derived MSCs exert therapeutic effects in various diseases. However, no reports have compared the effects of MSCs of different origins on UCMD pathology.

Methods

To evaluate which MSC population has the highest therapeutic efficacy for UCMD, in vivo (transplantation of MSCs to Col6a1-KO/NSG mice) and in vitro experiments (muscle stem cell [MuSCs] co-culture with MSCs) were conducted using adipose tissue-derived MSCs, bone marrow-derived MSCs, and xeno-free-induced iPSC-derived MSCs (XF-iMSCs).

Results

In transplantation experiments on Col6a1-KO/NSG mice, the group transplanted with XF-iMSCs showed significantly enhanced muscle fiber regeneration compared to the other groups 1 week after transplantation. At 12 weeks after transplantation, only the XF-iMSCs transplantation group showed a significantly larger muscle fiber diameter than the other groups without inducing fibrosis, which was observed in the other transplantation groups. Similarly, in co-culture experiments, XF-iMSCs were found to more effectively promote the fusion and differentiation of MuSCs derived from Col6a1-KO/NSG mice than the other primary MSCs investigated in this study. Additionally, in vitro knockdown and supplementation experiments suggested that the IGF2 secreted by XF-iMSCs promoted MuSC differentiation.

Conclusion

XF-iMSCs are promising candidates for promoting muscle regeneration while avoiding fibrosis, offering a safer and more effective therapeutic approach for UCMD than other potential therapies.

Background

Ullrich congenital muscular dystrophy (UCMD) is an early-onset, progressive disease characterized by muscle weakness, striking joint hyperlaxity, and progressive proximal joint contractures [1]. Patients with UCMD may be able to walk in early childhood; however, this ability is subsequently lost with the development of frequent respiratory failure [2]. Patients with UCMD have been confirmed to have autosomal mutations in the three major type 6 collagen (COL6) genes, COL6A1, COL6A2, orCOL6A3, which can cause deficient or dysfunctional microfibrillar COL6 in the extracellular matrix (ECM) of the muscle, resulting in the symptoms described above [2, 3]. Currently, no effective treatments for UCMD have been reported.

COL6 is widely distributed in the ECM of tissues throughout the body, including skeletal muscles, and interacts with many ECM and basement membrane proteins [4, 5]. One of the important roles of the COL6 protein is to connect the basement membrane to fibrous connective tissue; however, it has also been reported to interact with cytokines, integrins, and growth factors and is predicted to be involved in cell growth, differentiation, and regeneration [6,7,8,9,10]. A lack of COL6 has been reported to alter the structure and biomechanical properties of the ECM, induce mitochondrial defects [11,12,13,14,15], decrease autophagy [12, 14, 16], and impair muscle regeneration [17, 18].

In skeletal muscle, it has been reported that COL6 proteins are secreted from mesenchymal stromal cells (MSCs) [17]. MSCs typically maintain muscle homeostasis in healthy skeletal muscle tissues [19]. It has been demonstrated that the properties of MSCs change in pathological skeletal muscle tissues, leading to their differentiation into adipocytes and ectopic fat deposition [19, 20]. Specifically, Col6a1 GT/GT mice have been reported to possess MSCs with abnormal properties [21], which are believed to contribute to the progression of fibrosis [22]. Based on these findings, MSCs may serve as potential therapeutic targets for UCMD.

We have previously demonstrated that MSCs derived from induced pluripotent stem cells (iPS cells) (iMSCs) express COL6, and by local or intraperitoneal administration to Col6a1 knock out /NSG mice (Col6a1-KO/NSG mice; immunodeficient UCMD mouse models), were shown to have therapeutic effects, such as promoting muscle regeneration and maturation [23, 24]. Although iMSCs are still in the animal experimental stage and have not yet been clinically applied, there have been significant advancements in their development as potential therapeutics. Currently, a method to induce iMSCs in a xeno-free manner has been established [25]. Clinically applicable iPSCs, which edit HLAs to reduce the possibility of immune rejection at the time of allogeneic transplantation and enable transplantation into a wider range of recipients, have also been previously established [26]. We believed iMSCs generated by combining these approaches will become more suitable for clinical applications. Therefore, we used xeno-free induced MSCs derived from HLA-edited iPSCs (XF-iMSCs) in this study.

In contrast, Alexeev et al. transplanted adipose-derived MSCs (Ad-MSCs) into collagen VI-deficient mice and demonstrated their therapeutic effects, reporting adequate engraftment and continuous COL6 production [27]. In addition, Ad-MSCs have the advantage of being clinically applied in other conditions, such as knee osteoarthritis [28] and acute ischemic stroke [29].

Furthermore, MSCs derived from various tissues, such as the bone marrow (BM-MSCs) [30, 31], peripheral blood [32], umbilical cord blood [33, 34] and deciduous teeth [35] have recently been isolated from amniotic fluid [36, 37]. Some of them have also been clinically applied in other diseases, such as rheumatoid arthritis and lupus, among others.

However, no study has investigated how iMSCs (such as XF-iMSCs) differ from other MSCs (such as Ad-MSCs and BM-MSCs) that have the advantage of being used clinically. Therefore, the purpose of this study was to compare primary MSCs (Ad-MSCs and BM-MSCs), which have already been clinically applied in other diseases, with iMSCs to verify which MSCs are effective and safe in treating UCMD pathology and determine which cells are appropriate as sources of cell therapies for UCMD.

Methods

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).

Results

Characterization of MSCs

First, we confirmed whether primary MSCs, Ad-MSCs, BM-MSCs, and iPSC-derived XF-iMSCs possessed the characteristics of MSCs. The morphology of each type of MSCs was almost identical (Fig. 1a). Upon culture and immunocytochemistry examination, all MSCs robustly expressed COL6 (Fig. 1b). Additionally, qPCR revealed no significant differences in the expression levels of COL6A1 mRNA among the three types of MSCs (Fig. 1c). The protein levels of COL6A1 were also compared among the three types of MSCs using western blotting; however, no significant differences were observed (Figs. 1d, S1a). Furthermore, flow cytometry analysis confirmed that all types of MSCs used in this study uniformly expressed MSC-positive markers, including CD29, CD44, CD73, CD90, and CD105, but not the negative markers CD34 and CD45 (Figure S1b). All MSCs used in this study also expressed COL6, which is crucial for the treatment of UCMD, and retained their MSC properties, as verified via flow cytometry. Consequently, we performed in vivo and in vitro experiments using these cells.

Fig. 1
figure 1

Characteristics of Ad-MSCs, BM-MSCs, and XF-iMSCs. a Bright-field morphological images of Ad-MSCs, BM-MSCs, and XF-iMSCs. b Representative immunofluorescence images of Ad-MSCs, BM-MSCs, and XF-iMSCs stained with anti-COL6 antibodies. c mRNA expression of COL6A1. The mRNA expression level of each gene in Ad-MSCs, BM-MSCs, and XF-iMSCs was analyzed using RT-qPCR. Levels are shown relative to those in XF-iMSCs. Data are shown as the mean ± SD. d Signal intensity of the bands obtained from western blotting of the COL6A1 protein in Ad-MSCs, BM-MSCs, and XF-iMSCs. β-actin was used as the control. Relative band intensity is shown relative to that in XF-iMSCs. Data are shown as the mean ± SD. *p < 0.05; n = 3 (Ad-MSCs, BM-MSCs), n = 2 (XF-iMSCs)

Full size image

The most significant muscle fiber regeneration was observed in the XF-iMSCs transplantation group

Transplantation experiments were performed to compare the effects of MSC in vivo (Fig. 2a). One week after the transplantation, all MSC types (Ad-MSCs, BM-MSCs, and XF-iMSCs) resulted in MSC engraftment, leading to COL6 supplementation (Fig. 2b). The percentage of COL6-positive areas relative to the total area was significantly higher in samples transplanted with BM-MSCs than those transplanted with the other two cell types (Fig. 2c). The percentage of COL6-positive fibers (muscle fibers entirely surrounded by COL6) in the total muscle fiber count was also significantly higher in samples transplanted with BM-MSCs than in those transplanted with the other two types of MSCs (Fig. 2d). There was no difference between the Ad-MSC- and XF-iMSC-transplanted samples (Fig. 2c, d). The number of transplanted cells (hLamin A/C-positive nuclei) was significantly higher in the BM-MSC-transplanted group, followed by the Ad-MSC-transplanted group, and then the XF-iMSC-transplanted group (Fig. 2e).

Fig. 2
figure 2

Engraftment of cells and supplementation of COL6 in the TA muscles of Col6a1-KO/NSG mice one week after cell transplantation. a Schematic representation of MSC transplantation into the TA muscle of Col6a1-KO/NSG mice. b Representative sectional images of the entire TA muscle one week after cell transplantation. c–e Quantitative data of the percentage of the COL6-positive area (c), percentage of COL6-positive fibers (d), and the number of hLamin A/C-positive nuclei (e) one week after transplantation. Data are shown as the mean ± SD. f Cross-sectional images of the TA muscles one week after cell transplantation or medium injection. g, h Percentage of eMHC-positive TA muscle fibers with more than two nuclei (g) and average area of eMHC-positive single fibers (h) one week after transplantation. Data are shown as the mean ± SD. *p < 0.05; n = 11 (Ad-MSCs), n = 15 (BM-MSCs), n = 20 (XF-iMSCs), n = 4 (Control)

Full size image

A comparison of muscle fiber regeneration between groups one week after transplantation showed that eMHC, which is transiently expressed during skeletal muscle regeneration, was limited to very small-diameter fibers, as indicated by yellow arrowheads, in the control group (Fig. 2f), indicating incomplete muscle maturation. In the XF-iMSC transplantation group, there was an enhancement in the number of multinucleated regenerated muscle fibers (Fig. 2f, white arrowhead) and an increase in muscle fiber diameter, similar to previous reports [24]. In contrast, in the Ad-MSCs and BM-MSCs transplantation groups, although some multinucleated muscle fibers, indicated by white arrowheads, were observed, many regenerated muscle fibers remained mononuclear (Fig. 2f, yellow arrowhead). The percentage of regenerated myofibers (eMHC-positive) with two or more nuclei, which result from fusions of multiple myoblasts, in the total myofiber count was higher in the XF-iMSC-transplanted samples than in the BM-MSCs- or Ad-MSC-transplanted samples (Fig. 2g). In addition, the average cross-sectional area (CSA) of eMHC-positive single fibers was also significantly higher in the XF-iMSC-transplanted groups, whereas the BM-MSCs- and Ad-MSC-transplanted samples showed no significant difference compared to the control Col6a1 -KO/NSG mice muscle (Fig. 2h).

A decreased proportion of immature muscle fibers with small diameters was observed only in the XF-iMSC-transplanted group at 12 weeks

At 12 weeks after MSC transplantation, the transplanted cells (hLamin A/C-positive nuclei) remained engrafted and COL6-positive areas were observed in all transplantation groups (Fig. 3a). The percentage of the COL6-positive area relative to the total area was significantly higher in the BM-MSC-transplanted group, followed by the XF-iMSC-transplanted group and the Ad-MSC-transplanted group (Fig. 3b). The percentage of COL6-positive fibers relative to the total muscle fiber count was also significantly higher in samples transplanted with BM-MSCs than in those transplanted with Ad-MSCs or XF-iMSCs. No difference was observed between the Ad-MSC- and XF-iMSC-transplanted samples (Fig. 3c). The number of hLaminA/C-positive nuclei was significantly higher in the Ad-MSC- and BM-MSC-transplanted groups than in the XF-iMSC-transplanted group (Fig. 3d).

Fig. 3
figure 3

Cell engraftment and COL6 supplementation in the TA muscles of Col6a1-KO/NSG 12 weeks after cell transplantation. a Representative sectional images of the entire TA muscle at 12 weeks after cell transplantation. b–d Quantitative data on the percentage of COL6-positive area (b), percentage of COL6-positive fibers (c), and number of hLamin A/C (d) 12 weeks after transplantation. Data are shown as the mean ± SD. e Band graph representing the percentage of each muscle fiber size classified by diameter at 12 weeks after transplantation. Blue, 56 μm ≤ CSA short axis; yellow, 26 μm ≤ CSA short axis ≤ 55 μm; light green, CSA short axis ≤ 25 μm. f Average CSA of a single myofiber in the TA muscles. Data are shown as the mean ± SD. *p < 0.05; n = 12 (Ad-MSCs), n = 9 (BM-MSCs), n = 11 (XF-iMSCs), n = 27 (medium), n = 6 (WT)

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The percentage of muscle fibers with a short diameter (25 µm or less) was significantly decreased in the group transplanted with XF-iMSCs, reaching levels comparable to that of the WT. In contrast, no significant differences were observed between groups transplanted with Ad-MSCs or BM-MSCs and the negative control group (Fig. 3e). Similarly, comparable results were obtained for the average CSA of single muscle fibers, with a significant increase in muscle fiber diameter observed in the XF-iMSCs transplantation group. In contrast, the groups transplanted with Ad-MSCs or BM-MSCs showed almost no change from the medium-injected group, indicating no increase in muscle fiber CSA (Fig. 3f).

Significant fibrosis was observed in the BM-MSCs transplantation group, not in the XF-iMSCs transplantation group

To confirm whether the transplanted cells had an adverse effect on the pathological ectopic fibrosis in the muscle, Sirius Red staining was performed 12 weeks after transplantation. In the BM-MSCs transplantation group, significant ectopic fibrosis was evident, with enlargement of the interstitium (Figs. 4a). In the Ad-MSC transplantation group, some interstitial enlargement was also observed, although not as pronounced as that in the BM-MSCs transplantation group. Quantitative analysis revealed a significant increase in the percentage of the fibrotic area in the BM-MSCs transplantation group (Fig. 4b). The number of transplanted cells (hLamin A/C-positive nuclei) was significantly higher in the Ad-MSCs and BM-MSCs transplantation groups than in the XF-iMSCs transplantation group (Fig. 3d). Additionally, it was evident from the microscopy images that a large number of human nuclei accumulated in the interstitium in both Ad-MSCs and BM-MSCs transplantation groups (Fig. 4c).

Fig. 4
figure 4

Analysis of fibrosis in the TA muscles of Col6a1-KO/NSG mice after cell transplantation. a Sirius red-stained sectional images of the entire TA muscle 12 weeks after cell transplantation. b Quantitative data on the percentage of Sirius red-positive areas 12 weeks after transplantation. Data are shown as the mean ± SD. c Representative hLamin A/C- and laminin-stained sectional images of the TA muscle (enlarged) 12 weeks after cell transplantation. d Sirius red- and hLamin A/C-stained sectional images of the whole TA muscle 24 weeks after cell transplantation. e,f Quantitative data of the percentage of Sirius Red-positive areas (e) and the number of hLamin A/C-positive nuclei(compared to the number of hLamin A/C at 12 weeks after XF-iMSCs transplantation) (f) 24 weeks after cell transplantation. g Expression of fibrosis marker genes. The mRNA expression level of each gene was analyzed using RT-qPCR in Ad-MSCs, BM-MSCs, and XF-iMSCs. Levels are shown relative to those in XF-iMSCs. Data are shown as the mean ± SD. *p < 0.05, For tx12w, n = 12 (Ad-MSCs), n = 9 (BM-MSCs), n = 11 (XF-iMSCs, medium). For tx24w, n = 4 (XF-iMSCs, medium). qPCR experiment: n = 3 (Ad-MSCs, BM-MSCs), n = 2(XF-iMSCs)

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For the XF-iMSCs transplantation group, which did not show any particularly problematic findings at 12 weeks post-transplantation, Sirius Red staining was performed on the samples at 24 weeks after XF-iMSCs transplantation. No human nuclei accumulated in the interstitium, and the areas of ectopic fibrosis tended to be small and suppressed (Figures compared with medium injected group (Fig. 4d, e). In addition, no significant changes were observed in the number of human nuclei at 24 weeks after transplantation compared to that at 12 weeks after transplantation (Fig. 4f).

Furthermore, the mRNA expression levels of COL1A1 [42, 43] and POSTN [44, 45] were found to be significantly higher in BM-MSCs than in the groups transplanted with the other MSCs (Fig. 4g).

Co-culture with XF-iMSCs enhanced the differentiation of MuSCs derived from Col6a1-KO/NSG mice

To address the differences among the MSCs in terms of regeneration-promoting capacity, in vitro co-culture experiments were conducted to investigate how each MSC affects the primary MuSCs derived from COL6a1-KO/NSG mice. On the third day of co-culture (Fig. 5a), there was no significant difference in the number of total myogenic cells among the groups (Fig. 5b). However, when co-cultured with MSCs, the proportion of Pax7 + /MyoD- cells significantly decreased compared to the single culture (MuSCs alone), and the proportion of Pax7-/MyoD + cells significantly increased, indicating accelerated differentiation (Fig. 5c). Moreover, this phenomenon was particularly pronounced only in the group co-cultured with XF-iMSCs.

Fig. 5
figure 5

Effect of MSCs on myogenesis in co-culture experiments. a Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 3 days after single culture or co-culture with Ad-MSCs, BM-MSCs, or XF-iMSCs. b Total Number of DAPI + /hLamin A/C- mouse myogenic cells 3 days after co-culture. Data are expressed relative to XF-iMSCs and are presented as the mean ± SD of three independent experiments. c Percentage of Pax7 + /MyoD-, Pax7 + /MyoD + , and Pax7-/MyoD + cell populations 3 days after co-culture. Data from three independent experiments are shown as the mean ± SD. d Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 6 days after single culture or co-culture with Ad-MSCs, BM-MSCs, or XF-iMSCs. e,f Area of MHC + myotubes (e) and number of MHC + myotubes with four or more nuclei (f) 6 days after co-culture. Data are expressed relative to XF-iMSCs and are presented as the mean ± SD of three independent experiments. *p < 0.05. n = 6(Ad-MSCs, BM-MSCs and XF-iMSCs), n = 3 (single culture)

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On the sixth day of co-culture, differentiation progressed, and myotubes were detected in all groups. However, in the groups co-cultured with Ad-MSCs or BM-MSCs and the MuSC single-culture groups, fewer multinucleated, elongated muscle fibers were observed compared with the group co-cultured with XF-iMSCs (Fig. 5d). Analysis of the MHC-positive area (Fig. 5e) and the number of MHC-positive myotubes with four or more nuclei (Fig. 5f) revealed that co-culture with XF-iMSCs significantly promoted myotube differentiation compared with the other groups.

Insulin growth factor 2 (IGF2) knockdown in XF-iMSCs inhibited the differentiation of MuSCs derived from Col6a1-KO/NSG mice

To explore the properties of iMSCs and other MSCs aside from COL6 expression, we focused on IGF2 and peroxidasin (PXDN), factors for the enhancement of myogenic differentiation that are highly expressed in XF-iMSCs, as reported in previous studies [25]. First, qPCR analysis of IGF2 and PXDN expression was performed using Ad-MSCs, BM-MSCs, and XF-iMSCs. We found that XF-iMSCs have significantly higher IGF2 expression than Ad-MSCs or BM-MSCs (Fig. 6a). However, although PXDN tended to be more highly expressed in XF-iMSCs than other MSCs, there was no significant difference between primary MSCs (Ad-MSCs and BM-MSCs) and XF-iMSCs (Fig. 6a). Therefore, we decided to focus only on IGF2, which was highly expressed in the XF-iMSCs group and showed a considerable difference from the other groups. ELISA was also performed to detect IGF2 expression, and the results were similar to those of qPCR. The amount of IGF2 in the culture supernatant was significantly higher in the XF-iMSCs group and was almost undetectable in the Ad-MSC group (Fig. 6b). Based on these results, by knocking down IGF2 expression in XF-iMSCs, we verified whether the differentiation-promoting properties observed in the XF-iMSC co-culture experiments were impaired.

Fig. 6
figure 6

Effects of IGF2 knockdown in XF-iMSCs on Col6a1-KO/NSG MuSC differentiation. a mRNA expression of IGF2 and PXDN. The mRNA expression level of each gene was analyzed using RT-qPCR in Ad-MSCs, BM-MSCs, and XF-iMSCs. Levels are shown relative to those in XF-iMSCs. Data are shown as the mean ± SD. b Concentration of IGF2 in the culture supernatants of Ad-MSCs, BM-MSCs, and XF-iMSCs as obtained using ELISA. Data are shown as the mean ± SD. c mRNA expression level and concentration of IGF2 in the culture on co-culture day 3. Data are presented as the mean ± SD. mRNA expression levels are shown with levels relative to those in XF-iMSCs. d Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 3 days after single culture or co-culture with XF-iMSC, XF-iMSC_IGF2-KD, or XF-iMSC_mock. e Total number of DAPI + /hLamin A/C- mouse myogenic cells 3 days after co-culture. Data are expressed relative to XF-iMSCs and are presented as the mean ± SD of three independent experiments. f Percentage of Pax7 + /MyoD-, Pax7 + /MyoD + , and Pax7-/MyoD + cell populations 3 days after co-culture. Data from three independent experiments are shown as the mean ± SD. g mRNA expression level and concentration of IGF2 in the culture on co-culture day 6. Data are presented as the mean ± SD. mRNA expression levels are shown with levels relative to those in XF-iMSCs. h Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 6 days after single culture or co-culture with XF-iMSCs, XF-iMSC_ IGF2-KD, or XF-iMSC_mock. i, j Area of MHC + myotubes (i) and number of MHC + myotubes (j) with four or more nuclei on day 6 after co-culture. Data are expressed relative to XF-iMSCs and are presented as the mean ± SD of three independent experiments. co-cluture experiment: n = 6 (XF-iMSCs, XF-iMSC_ IGF2-KD and XF-iMSC_mock), n = 3 (single culture). qPCR and ELISA experiment: n = 2 (Ad-MSCs, BM-MSCs, XF-iMSCs)

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Co-culture experiments were conducted using four groups: XF-iMSCs, XF-iMSCs with IGF2 knockdown (XF-iMSC_IGF2-KD), XF-iMSCs with mock treatment (XF-iMSC_mock), and a MuSC single-culture group. On the third day of co-culture, we confirmed a significant decrease in IGF2 expression in XF-iMSC_IGF2-KD cells using qPCR and ELISA (Fig. 6c). Immunocytochemistry analysis showed that in the XF-iMSC and XF-iMSC_mock groups, only a few Pax7 + /MyoD- cells were detected. In contrast, many Pax7 + /MyoD- cells were observed in the XF-iMSC_IGF2-KD and MuSC single-culture groups (Fig. 6d). All groups did not show significant differences in the number of total myogenic cells (Fig. 6e). However, the XF-iMSC_IGF2-KD group showed a significant increase in the Pax7 + /MyoD- cell ratio compared to the XF-iMSC and XF-iMSC_mock groups, reaching a ratio similar to that of the MuSC single-culture group (Fig. 6f). Knocking down IGF2 in XF-iMSCs abolishes their differentiation-promoting properties in myoblasts.

On the sixth day of co-culture, we confirmed the knockdown of IGF2 using qPCR and ELISA, verifying that IGF2 in XF-iMSC_IGF2-KD cells was effectively knocked down (Fig. 6g). ICC analysis showed that fused, mature, and lengthened myotubes were observed in the XF-iMSC_IGF2-KD co-culture group compared to the MuSC single-culture group; however, the number of matured myotubes in the XF-iMSC_IGF2-KD co-culture group was significantly smaller than that in the XF-iMSC group. In the XF-iMSCs with mock treatment group, slightly fewer fused longer myotubes were observed than those in the normal XF-iMSCs group (Fig. 6h). Quantitative analysis revealed that the MHC-positive area (Fig. 6i) and the number of MHC-positive myotubes with four or more nuclei (Fig. 6j) were significantly lower in the XF-iMSC_IGF2-KD and MuSC single-culture groups than in the XF-iMSC and XF-iMSC_mock groups. These results suggest that IGF2 knockdown in XF-iMSCs inhibits myoblast fusion and myotube maturation.

IGF2 supplementation promoted myoblast fusion in Col6a1-KO/NSG mice

To analyze whether the supplementation of IGF2 is enough to promote MuSC differentiation, recombinant human IGF2 was added to MuSC single-culture conditions. On the third day of culture, a slight enhancement in the proliferation of MuSCs was observed in the IGF2-treated group (Fig. 7a), but no significant difference was observed between the IGF2-treated group and the IGF2-untreated group in terms of the number of total myogenic cells (Fig. 7b) or the proportion of myogenic cells population (Fig. 7c). However, on day 6, the group treated with IGF2 showed more multinucleated myotubes than the IGF2-untreated group (Fig. 7d). Analysis of the MHC-positive area (Fig. 7e) and the number of MHC-positive myotubes with two or more nuclei (Fig. 7f) indicated a significant increase in the IGF2-treated group compared to that in the IGF2-untreated group.

Fig. 7
figure 7

Effects of IGF2 supplementation on Col6a1-KO/NSG MuSC differentiation. a Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 3 days after IGF2 supplementation (IGF2-treated) or without IGF2 treatment (IGF2-untreated). b Total number of DAPI + /hLamin A/C- mouse myogenic cells after 3 days of culture. Data are expressed relative to IGF2-untreated and are presented as the mean ± SD of three independent experiments. c Percentage of Pax7 + /MyoD-, Pax7 + /MyoD + , and Pax7-/MyoD + cell populations after 3 days of culture. Data from three independent experiments are shown as the mean ± SD. d Representative immunofluorescence images of Col6a1-KO/NSG mouse-derived MuSCs 6 days after IGF2 supplementation (IGF2-treated) or without IGF2 treatment (IGF2-untreated). e, f Area of MHC + myotubes (e) and number of MHC + myotubes with two or more nuclei (f) 6 days after co-culture. Data are expressed relative to IGF2-untreated and are presented as the mean ± SD of three independent experiments. *p < 0.05. n = 3 (IGF2 treated, IGF2 untreated)

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Discussion

In this study, we compared the properties of various MSCs and verified their efficacy in treating UCMD. Our results demonstrated that XF-iMSCs exhibited the highest therapeutic efficacy in vivo and in vitro, promoting muscle regeneration in Col6a1-KO/NSG mice while displaying high safety by minimizing the risk of fibrosis exacerbation. Furthermore, our data suggest that IGF2 secreted by XF-iMSCs is one of the mechanisms underlying this therapeutic effect.

Ad-MSCs and BM-MSCs have been clinically applied to other diseases and are useful in regenerative medicine applications [28, 29, 46,47,48,49]. In contrast, although iMSCs have not yet been clinically applied, they have been reported to improve muscle regeneration and maturation in UCMD mouse models over a long period and have been shown to improve the functional aspects of the model mice in previous studies [23, 24]. Thus, iMSCs have been reported to exert good therapeutic effects, at least in UCMD mouse models, and their efficacy is expected in clinical practice. However, no study has investigated how they differ from other MSCs (such as Ad-MSCs and BM-MSCs) that have the advantage of being used clinically. In this study, we examined the therapeutic efficacy and risk of adverse reactions of the three types of MSCs from the perspective of their potential clinical application.

First, to investigate the effects of each MSC in vivo, transplantation experiments were conducted in Col6a1-KO/NSG mice, which yielded intriguing results. One week after transplantation, although the BM-MSCs group showed significantly higher levels of COL6 supplementation, which is considered crucial for UCMD therapy, the XF-iMSC-transplanted group exhibited significantly better muscle fiber regeneration than the other groups. This trend persisted even at 12 weeks after transplantation when the BM-MSC group still showed significantly higher levels of COL6 supplementation. However, a decrease in the proportion of small short-diameter muscle fibers and an overall increase in muscle fiber diameter were observed specifically in the XF-iMSCs transplantation group.

Next, we conducted co-culture experiments to verify how each MSC type affects MuSCs derived from a Col6a1-KO/NSG mice in vitro. Co-culture with each type of MSC showed faster differentiation and more multinucleated myotubes than the MuSC single-culture group, consistent with previous studies [23]. In particular, the group co-cultured with XF-iMSCs showed more rapid differentiation compared to the other co-culture groups. Considering the results of both in vivo transplantation and in vitro co-culture experiments, it was inferred that each MSC type had different properties. Moreover, these findings suggest that factors other than COL6 may be involved in muscle regeneration in Col6a1-KO/NSG mice.

Two reasons were considered for the lack of muscle fiber regeneration in the BM-MSCs group despite sufficient COL6 supplementation and the promotion of muscle fiber regeneration in the XF-iMSCs group, which did not have as much COL6 supplementation as the BM-MSCs group. First, some factors in BM-MSCs may inhibit muscle fiber regeneration. Significant fibrosis with an enlarged interstitium was observed in the BM-MSCs transplantation group at 12 weeks post-transplantation. Previous studies have reported that transplantation of BM-MSCs into skeletal muscle contusion model mice, not UCMD models, worsens fibrosis and inflammation [50]. The expression levels of fibrosis markers such as COL1 and POSTN were markedly higher in BM-MSCs than in other MSCs, and given that some previous studies indicated that fibrosis impedes muscle regeneration [44, 45, 50], it is plausible that these factors suppress muscle fiber regeneration.

Secondly, some other factors in XF-iMSCs may promote muscle fiber regeneration. In a previous study, we found that the transplantation of iMSCs into a muscle contusion model accelerated muscle regeneration [25]. In the study, we identified PXDN and IGF2 as factors contributing to skeletal muscle maturation. We also demonstrated that adding either factor during the muscle differentiation of primary myoblasts increased the number of twitching myotubes. The present study showed no statistically significant difference in PXDN expression levels between XF-iMSCs and primary MSCs. However, IGF2 expression was significantly increased in XF-iMSCs compared to the other groups. Therefore, we focused on IGF2 for further investigation.

IGF2 is a widely expressed 7.5-kDa mitogenic peptide hormone that regulates fetal development and differentiation; however, its role in adults is less well understood than that of IGF1. The liver is the main source of IGF2 in adults; however, it is also synthesized by many other tissues, from which it is released into the pericellular fluid [51]. However, the exact function of IGF2 remains unclear. Wilson et al. reported that IGF2 is required for the development and maintenance of the musculoskeletal system. It promotes the differentiation of C3H 10T1/2 fibroblasts, chick embryo cells, and myoblasts. Meanwhile, the transcription factor MyoD, which is required for the differentiation of fibroblasts into myoblasts, requires IGF2 expression; IGF2 blockage inhibits differentiation [52, 53]. In this study, the knockdown of IGF2 in XF-iMSCs and co-cultures with Col6a1-KO/NSG mice-derived primary MuSCs abrogated the promotion of MuSC differentiation, consistent with previous research [53]. Furthermore, in the rh-IGF2 supplementation experiment, a promoting effect on differentiation was observed, such as an increase in the number of myotubes with two or more nuclei on day 6 compared to the group without IGF2 supplementation. However, although a differentiation-promoting effect was observed with IGF2 supplementation, this effect was only observed when rh-IGF2 was added at approximately four times the amount secreted by XF-iMSCs (Figure S2). Furthermore, this differentiation-promoting effect was far from that observed in the co-culture with XF-iMSCs, as indicated by the fact that no differentiation-promoting effect was observed on day 3. It is also possible that just adding IGF2 to the culture medium was insufficient. In differentiating human iPSCs, heparan sulfate conjugation to the C-terminus of laminin E8 fragments brings fibroblast growth factors (FGFs) close to the human iPSC surface, enhancing FGF signaling pathways and initiating HOX gene expression, which triggers paraxial mesoderm differentiation with high efficiency [54]. Similarly, since human MSCs express heparan sulfate proteoglycans [55], IGF2 has a functional heparin-binding domain, and the IGF2/IGFBP2 complex acts by binding heparan sulfate proteoglycans [56], it is possible that IGF2 secreted by XF-iMSCs is similarly brought near the cell surface of MuSCs by these proteoglycans, thereby allowing IGF2 to efficiently act on MuSCs. Taken together, these findings suggest that, at least in the skeletal muscles of Col6a1-KO/NSG mice, the differentiation of MuSCs is promoted by IGF2 secreted from XF-iMSCs, which potentially contributes to muscle fiber regeneration.

It is thought that knocking down fibrosis markers in BM-MSCs or forcing the expression of IGF2 in BM-MSCs could produce cells that can replenish COL6 well and promote myofiber regeneration. However, the number of passages in primary MSCs is limited to 3–5 times before their differentiation and proliferation abilities will be altered. This makes it almost impossible to obtain stable cells after genetic manipulation and to expand them to the required quantity for transplantation. Moreover, primary MSCs derived from tissues, despite being invasive to collect, exhibit significant variability in their properties among donors [57, 58]. Additionally, fibrosis, particularly in patients with UCMD, which is characterized by symptoms such as increased connective tissue proliferation and degeneration of necrotic fibers, can lead to irreversible damage, raising concerns regarding its safety.

Conversely, the XF-iMSCs used in this study were obtained using minimally invasive methods, and selecting high-quality clones was not overly challenging. Furthermore, they can be passaged up to 15–20 times without problems [25, 59, 60], facilitating the realistic large-scale production of genetically manipulated cells.

In this study, the number of clones tested and the sample size were limited, potentially impacting the generalizability and robustness of the findings. Moreover, the research was confined to a single mouse model (Col6a1-KO/NSG mice), which may not fully capture the complexity and variability of UCMD in human patients. However, with low mRNA expression levels of fibrosis markers and the absence of fibrosis in transplantation experiments, XF-iMSCs exhibited superior safety profiles compared with other primary MSCs, suggesting that they could significantly contribute to the future development of UCMD therapy.

Conclusion

In summary, we investigated differential effects of Ad-MSCs, BM-MSCs, and XF-iMSCs on skeletal muscle regeneration and modulation of fibrosis in a mouse model of UCMD. Our study showed for the first time that XF-iMSCs specifically promoted muscle regeneration and increased myofiber diameter even 12 weeks after transplantation, a therapeutic effect not observed with BM-MSCs or Ad-MSCs. Furthermore, our data indicated that XF-iMSCs did not induce fibrosis, unlike Ad-MSCs and, particularly, BM-MSCs, which caused extensive fibrosis. Additionally, the finding that IGF2 secreted by XF-iMSCs promoted myotube maturation provides a valuable insight into the molecular mechanisms of muscle regeneration and may serve as a criterion for selecting the optimal clone of XF-iMSCs for the use in the treatment of patients with UCMD. Continued research in this field may lead to more refined and effective treatments for UCMD, ultimately improving patient outcomes and expanding our understanding of muscle regeneration and extracellular matrix dynamics.

Availability of data and materials

The data produced and analyzed in this study can be obtained from the lead contacts upon reasonable request.

Abbreviations

Ad-MSCs:

Adipose-derived mesenchymal stromal cells

BM-MSCs:

Bone marrow-derived mesenchymal stromal cells

COL1:

Type1 collagen

COL6:

Type 6 collagen

Col6a1-KO :

Col6a1 Knockout

CSA:

Cross-sectional area

eMHC:

Embryonic myosin heavy chain

ECM:

Extracellular matrix

FGFs:

Fibroblast growth factors

hLamin A/C:

Human lamin A/C

ICC:

Immunocytochemistry

iPSCs:

Induced pluripotent stem cells

IGF2:

Insulin growth factor 2

MSCs:

Mesenchymal stromal cells

NCCs:

Neural crest cells

iMSCs:

Mesenchymal stromal cells induced from induced pluripotent stem cells

MNCs:

Mononuclear cells

MuSCs:

Muscle stem cells

MHC:

Myosin heavy chain

PXDN:

Peroxidasin

TA muscles:

Tibialis anterior muscles

UCMD:

Ullrich congenital muscular dystrophy

XF-iMSCs:

Xeno-free induced MSCs derived from induced pluripotent stem cells

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Acknowledgements

We would like to thank Dr. A. Tanaka and Dr. A. Horinouchi for providing Ad-MSCs. We also express our gratitude to A. Tanaka and S. Tomita for supplying the model mouse littermates for in vitro fertilization. We would like to thank Editage (www.editage.jp) for English language editing. The authors declare that artificial intelligence was not used in this study.

Funding

This research was primarily funded by a Grant-in-Aid for Early-Career Scientists (# JP21K15418) from the Japan Society for the Promotion of Science awarded to N.T.-N. It was also partially supported by JSPS KAKENHI Grant number JP23K05780 to H.S. and grants from the Projects for Technological Development (#JP19bm0404044), the Core Center for iPS Cell Research (#JP13bm0104001), and Research Center Network for Realization of Regenerative Medicine, provided by the Japan Agency for Medical Research and Development (AMED) to H.S.

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Authors and Affiliations

  1. Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto, 606-8507, Japan

    Megumi Yokomizo-Goto, Nana Takenaka-Ninagawa, Chengzhu Zhao, Clémence Kiho Bourgeois Yoshioka, Mayuho Miki, Souta Motoike, Yoshiko Inada, Denise Zujur, William Theoputra, Makoto Ikeya & Hidetoshi Sakurai

  2. Department of Rehabilitation Medicine, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan

    Nana Takenaka-Ninagawa

  3. Department of Physical Therapy, Human Health Sciences, Graduate School of Medicine, Kyoto University, 53 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto, 606-8507, Japan

    Clémence Kiho Bourgeois Yoshioka & Mayuho Miki

  4. Department of Regeneration Science and Engineering, Institute for Life and Medical Sciences, Kyoto University, 53 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto, 606-8507, Japan

    Yonghui Jin & Junya Toguchida

  5. Department of Fundamental Cell Technology, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-Cho, Shogoin, Sakyo-Ku, Kyoto, 606-8507, Japan

    Junya Toguchida

Authors
  1. Megumi Yokomizo-Goto
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  2. Nana Takenaka-Ninagawa
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  3. Chengzhu Zhao
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  5. Mayuho Miki
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  13. Hidetoshi Sakurai
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Contributions

N.T.-N. and H.S. are the lead contacts in this study. M.G., H.S., and N.T.-N. were responsible for study conception, experimental design, data interpretation, manuscript preparation, and final validation. M.G. conducted most of the experiments. M.G., N.T.-N., C.K.B.Y., and M.M. performed histological analysis in the transplantation studies. N.T.-N. and C.K.B.Y. performed transplantation experiments. M.I., C.Z., S.M., D.Z., and W.T. generated xeno-free MSCs from HLA-edited iPSCs. S.M. and Y.I. conducted the FACS analysis of MSCs. Y.J. and J.T. generated the BM-MSCs. All authors discussed the results and contributed to the manuscript.

Corresponding authors

Correspondence to Nana Takenaka-Ninagawa or Hidetoshi Sakurai.

Ethics declarations

Ethics approval and consent to participate

This study was conducted in compliance with the guidelines of the Declaration of Helsinki, and written informed consent was obtained from all patients. The protocols for generating HLA-edited healthy donor-derived iPSCs were approved by the Ethics Committee of Center for iPS Cell Research and Application (approval number #CiRA19-03, Research on establishment of HLA-edited iPS cell stock for clinical application, approval date; 2019/June/4). Experiments involving human primary bone marrow were approved by the same committee (approval number #G1052, Establishment of evaluation criteria for regenerative medicine of musculoskeletal tissues, approval date; 2020/December/8). Experiments using human primary adipose tissue were approved by the Ethical Review Board for Clinical Studies at Nagoya University and the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (approval number #2005–0247-8, Research of adipose-derived stem cells for clinical application, approval date; 2022/January/24). Written informed consent was obtained from all donors of iPSCs, adipose tissue, and bone marrow used in this study. All animal experiments were conducted according to protocols approved by the Animal Research Committee of Kyoto University (approval number #21–146, Research on the induction of mesenchymal progenitor cells from human induced pluripotent stem cells and their transplantation therapy for refractory muscle disorders, approval date: 2021/April/1).

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The authors declare that they have no competing interests.

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Yokomizo-Goto, M., Takenaka-Ninagawa, N., Zhao, C. et al. Distinct muscle regenerative capacity of human induced pluripotent stem cell-derived mesenchymal stromal cells in Ullrich congenital muscular dystrophy model mice. Stem Cell Res Ther 15, 340 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-03951-6

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-03951-6

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512