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

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.

Keywords: Insulin growth factor 2; Mesenchymal stromal cells; Skeletal muscle regeneration; Type 6 collagen; Ullrich congenital muscular dystrophy.

Fig. 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)

Fig. 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)

Fig. 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)

Fig. 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)

Fig. 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)

Fig. 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)

Fig. 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)