Takenaka-Ninagawa et al. Stem Cell Research & Therapy
https://doi.org/10.1186/s13287-021-02514-3(2021) 12:446

RESEARCH

Open Access

Collagen-VI supplementation by celltransplantation improves muscle
regeneration in Ullrich congenital muscular
dystrophy model mice
Nana Takenaka-Ninagawa1*, Jinsol Kim1, Mingming Zhao1, Masae Sato1, Tatsuya Jonouchi1, Megumi Goto1,
Clémence Kiho Bourgeois Yoshioka1, Rukia Ikeda1, Aya Harada1, Takahiko Sato2, Makoto Ikeya1, Akiyoshi Uezumi3,
Masashi Nakatani4, Satoru Noguchi5 and Hidetoshi Sakurai1*

Abstract
Background: Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis throughseveral secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich
congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic
phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known
about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of thisstudy was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation.
Methods: To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments
were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived fromhealthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patientderived iMSCs (UCMD-iMSCs).
Results: All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of
immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs
were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were
promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal
muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed
improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not.
Conclusions: These findings indicate that COL6 supplementation improves muscle regeneration and maturation in
UCMD model mice.
Keywords: Induced pluripotent stem cells, Mesenchymal stromal cells, Skeletal muscle regeneration, Ullrich congenital
muscular dystrophy, COL6 related disease* Correspondence: nana.takenaka@cira.kyoto-u.ac.jp; hsakurai@cira.kyotou.ac.jp
1
Department of Clinical Application, Center for iPS Cell Research andApplication (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku,
Kyoto 606-8507, Japan
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Takenaka-Ninagawa et al. Stem Cell Research & Therapy(2021) 12:446

Background
Ullrich congenital muscular dystrophy (UCMD) is a slowly
progressive disease characterized by muscle weakness frombirth, muscle atrophy, proximal joint contracture, distal joint
hyperextension, and ultimately respiratory failure (1, 2). At
present, no effective cure has been found. Patient biopsies revealed a deficiency of type 6 collagen (COL6) in the muscles,
and later it was found that COL6A1, COL6A2, and COL6A3
mutations cause defective extracellular microfibril assembly(3–5).
COL6 is a fibril molecule with a molecular weight ofabout 500 kDa and is widely distributed in the extracellular matrix (ECM) of systemic tissues including skeletal
muscle (6–9). It interacts with fibrous collagen such as
collagen 1 and many kinds of ECM and basement membrane proteins (10–12). Although one of the major functions of COL6 protein is to connect the basement
membrane to fibrous connective tissue, it has also beenreported to interact with integrin, cytokines, and growth
factors and is thought to be involved in cell proliferation,differentiation, and regeneration (13–24). Moreover,
COL6 deficiency alters the ECM structure and biomechanical properties and leads to mitochondrial defects(25–30), decreased autophagy (31–33), and impaired
muscle regeneration (34, 35).
Previous case reports on UCMD patients have described histological phenotypes (36–39). The pathological findings of UCMD are dystrophic changes thatconsist of connective tissue proliferation, degenerated
necrotic fibers, marked variation in fiber size, and type 1
fiber atrophy and pre-dominance (36). In 2017, a UCMD
model mouse (Col6a1GT/GT mouse) completely deficientof COL6 was established (40). In the skeletal muscle ofthis model and another UCMD model, the number of
medium- and large-diameter muscle fibers approximatedthat in wild-type (WT) mice, but the number of muscle
fibers with extremely small diameters was much larger(40, 41), a phenotype consistent with the pathology of
UCMD patients.
In previous studies, fibroblast (35) and human adipose
tissue-derived MSC (ADSC) (34) transplantations weredone to examine if COL6 supplementation can regulate
the regeneration of toxin-injured skeletal muscle in
UCMD model mice. Although short-term effects, such as
the enhancement of satellite cell proliferation and regenerating myofibers, were demonstrated on intact and damaged muscles, long-term effects, such as the enhancementof myofiber maturation and enlargement of fiber size, arestill unknown in fibroblast transplantations (35). In thecase of ADSC transplantation, while long-term engraftment was demonstrated on injured muscle, the therapeutic effects for muscle regeneration and maturation
were not (34). In addition to COL6 secretion, other ECM
and soluble factors produced by MSCs in skeletal muscle

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play important roles in the differentiation, regeneration,and homeostasis of myogenic cells and the maintenanceof skeletal muscle fibers (42–45). Additionally, MSCs
show abnormal properties in UCMD mouse models (40).
Therefore, it remains unclear whether the therapeutic effect of cell transplantation is due to COL6 supplementation or healthy MSC supplementation.
The aims of this study are (1) to clarify the long-term
effects of COL6 supplementation by cell transplantationand (2) to clarify whether the therapeutic effects of MSC
transplantation are due to COL6 supplementation orother factors. Therefore, in the present study, using induced pluripotent stem cell (iPSC) technology (46), theshort-term and long-term effects of COL6 on the intact
muscle of UCMD model mice in cell transplantation
therapy were directly examined by comparing fully functional MSCs with MSCs deficient in COL6 secretion(COL6-deficient MSCs). As a result, the transplantationof functional MSCs promotes the regeneration and maturation of muscle fibers by supplementing COL6, but
the transplantation of COL6-deficient MSCs does not.
Similarly, co-culture experiments with healthy MSCs
promoted the proliferation, fusion, and maturation ofskeletal muscle satellite cells (MuSCs) derived from
Col6a1KO mice, whereas co-culture experiments with
COL6-deficient MSCs did not.

Methods
Generation of Col6a1KO mice

Heterozygous mice were produced by crossing NOD.CgPrkdcscidIl2rgtm1Wjl/SzJ (NSG mouse; severe immunodeficient) with Col6a1GT/GT mice (40). Subsequently, byheterozygous multiplication, Col6a1GT/GT/NSG mice
were identified by genotyping the resulting litter population and used in the experiments as Col6a1KO (immunodeficient UCMD model) mice. For the genotyping,genome DNA was extracted from the tail of each mouse.
To select homozygous Col6a1GT/GT/Il2r-/- mice, genomic PCR was done by genotyping Fw-Rv primer pairs
for Col6a1 and Il2r (the primers are listed in Table S4).
Induction of iMSCs from iPSCs

iMSCs (induced mesenchymal stromal cells) were derived from iPSCs through neural crest cells (NCCs), as
reported previously(47). Even though cells in the stromaof skeletal muscle tissue are called MSCs, fibro adipogenic progenitor cells and mesenchymal progenitor cells,
the cells used in this study were defined as MSCs according to (47). Human iPSCs were cultured on an
iMatrix-511 (Nippi, Tokyo, Japan)-coated cell culture
plate or dish in StemFit AK03N (Ajinomoto, Tokyo,
Japan) and then induced to differentiate into NCCs as
described previously (47). Briefly, the induction andmaintenance of NCCs were performed using previously

Takenaka-Ninagawa et al. Stem Cell Research & Therapy(2021) 12:446

reported CDMi medium (48), which contains Iscove's
modified Dulbecco’s medium/Ham’s F-12 1∶1, 1x
chemically defined lipid concentrate (GIBCO, Grand Island, NY, USA), 15 μg/mL apo-transferrin (Sigma-Aldrich, St. Louis, MO, USA), 450 μM monothioglycerol(Sigma-Aldrich), 5 mg/mL purified BSA (99% purified by
crystallization; Sigma-Aldrich), 7 μg/mL Insulin (FUJIFILM Wako, Osaka, Japan), and penicillin/streptomycin(Invitrogen, Carlsbad, CA, USA). To induce NCCs,
10 μM SB431542 (SB) (584-77601, Sigma-Aldrich) and1 μM CHIR99021 (CHIR) (FUJIFILM Wako) were added
to the CDMi medium. The cells were trypsinized, andp75+ (CD271+) NCCs were then sorted by FACS AriaII(BD Biosciences, San Jose, CA, USA) according to the
manufacturer’s protocol (the antibodies used are listed
in Table S3). EGF (059-07873, FUJIFILM Wako) andbasic FGF (47079000, Oriental Yeast CO., LTD, NIB,Tokyo, Japan) were added to CDMi medium to maintain
NCCs.
The expanded NCCs (passage number, 3-10) wereseeded onto fibronectin-coated plates at a density of 1 ×
104 cells/cm2 in CDMi medium supplemented with
10 μM SB, 20 ng/mL EGF, and 20 ng/mL basic FGF. The
medium was replaced the next day with α-MEM (nacalaitesque, Kyoto, Japan) supplemented with 5 ng/mL basic
FGF and 10% fetal bovine serum (FBS) (556-33865, FUJIFILM Wako). The morphology of the cells started tochange approximately 4 days after the induction. Passages
were performed every 3 days using Accutase (nacalai tesque) at a density 5 × 105 cells in a 10-cm dish (Corning,Corning, NY, USA) coated with fibronectin and cultured
with αMEM supplemented with 5 ng/mL basic FGF and
10% FBS and maintained at 37 °C in 5% CO2.

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listed in Table S3). The collected pMSCs were cultured
in GM and maintained at 37 °C in 5% CO2 and 3% O2.
MSC transplantation into Col6a1KO mice

Col6a1KO mice (4–6-weeks old) were anesthetized forsurgery with 3% Forane inhalant liquid (AbbVie, North
Chicago, IL, USA). Human iMSCs or pMSCs were suspended in αMEM (2 × 106 cells/50 μL) and injected into
the mice using a 27G micro-syringe (Myjector syringe;
Terumo, Tokyo, Japan) at the center of the tibialis anterior (TA) muscles. TSG6 (R&D systems, Minneapolis,
MN, USA) was co-injected at a concentration of 2 μg/L
to improve the cell engraftment efficiency (50). The
same amount of αMEM was injected into the TA muscleof the limb opposite the limb into which the cells weretransplanted and used as a comparison target for histological analysis.
Statistical analysis

Animals were excluded from the study only if theirhealth status was compromised; for instance, if they had
visible wounds due to fighting. The investigators wereblinded to the outcome assessments. A one-way analysis
of variance (ANOVA), Welch’s ANOVA, and the
Kruskal-Wallis test were conducted to assess differencesamong three groups or more. Unpaired Student’s t tests
were calculated to assess differences among two groups.
Differences were considered significant for p values ≤0.05. All statistical analyses were performed using EZR
version 1.37 (Jichi Medical University Saitama Medical
Center) (51).
Other materials and methods

Other materials and methods are described in the Supplemental Methods.
Isolation of primary (p)MSCs

Non-dystrophic healthy muscle samples were obtained
from the gluteus medius muscles of female subjects
undergoing total hip arthroplasty. The methods for dissociating cells from the muscle samples and sorting
pMSCs (PDGFRα+) are described elsewhere (49). Briefly,
muscle samples were transferred to PBS and digested
with 0.2% type II collagenase (Worthington, Columbus,
OH, USA). Muscle slurries were filtered through a cellstrainer (BD Biosciences). Cells were resuspended in
growth medium (GM) consisting of DMEM (Sigma-Aldrich) supplemented with 20% FBS, 1% penicillinstreptomycin (Katayama Chemical, Osaka, Japan), and
2.5 ng/mL basic FGF, seeded onto a collagen I-coateddish (Iwaki, Shizuoka, Japan), and maintained at 37 °C in
5% CO2 and 3% O2. The cells were trypsinized, andPDGFRα+ pMSCs were sorted using FACS Vantage SE(BD Biosciences) or MoFlo Astrios (Beckman Coulter,
Brea, CA, USA) (the antibodies and reagents used are

Results
Comparison of healthy iMSCs, KO-iMSCs, and pMSCs

To elucidate whether the supplementation of COL6 isindispensable for the improvement of muscle regeneration and maturation in UCMD model mice, COL6A1
knockout iPSCs were created using the CRISPR-Cas9
System (Figure S1) and differentiated into KO-iMSCs.
Properties of the iMSCs, KO-iMSCs, and pMSCs werecompared. Unlike iPSCs, iMSCs had a low expression ofpluripotency marker genes (Figure 1a). The expressionof PDGFR-A, an MSC marker gene, and its protein werehighly expressed in all three MSC types (Figure 1a, b).
COL6A1 mRNA was highly expressed in pMSCs andiMSCs, but barely in iPSCs or KO-iMSCs (Figure 1a).
COL6 was expressed intracellularly and further secreted
after forming a fibril structure outside the cell in pMSCs
and iMSCs, but not in KO-iMSCs or iPSCs (Figure 1b,bottom row). Western blotting analysis verified that

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Fig. 1 Characteristics of pMSCs, iMSCs, and KO-iMSCs. a The expression of marker genes. The mRNA expression level of each gene was analyzed
by RT-qPCR in pMSCs derived from skeletal muscle, iMSCs, KO-iMSCs, undifferentiated healthy iPSCs, and COL6A1KO-iPSCs. Levels are shown
relative to the level in healthy iPSCs for the pluripotency markers NANOG, SOX2, and OCT3/4 or in pMSCs for the MSC markers COL6A1 and PDGF
R-A. Data are shown as the mean ± SD. n = 3. b Immunofluorescence images of pMSCs, iMSCs, KO-iMSCs, and healthy iPSCs. The inserts in thelower panels are higher magnifications and show the fibril structure. Scale bars, 100 μm, and 10 μm (inserts). c Western blots of COL6A1 protein
expression in pMSCs, iMSCs, and KO-iMSCs. β-Actin was used as the control. Average fold changes between iMSCs, KO-iMSCs, and pMSCs areindicated below. d PCA plot showing the clustering of cell populations based on the transcriptomes. Proportion of variance: PC1 = 43.764%, PC2= 19.746%, PC3 = 17.682%. e Heatmap of MSC markers, myogenic markers, and pluripotency markers

COL6 protein was expressed in pMSCs and iMSCs, but
not in KO-iMSCs (Figure 1c). Flow cytometer analysis
confirmed pMSCs and both types of iMSCs uniformly
expressed MSC-positive markers including CD105,
CD73, CD44, and CD201 (49) but not the negative
marker CD45 (Figure S2). A comprehensive gene expression analysis by RNA-seq showed that iMSCs and
KO-iMSCs have properties closer to pMSCs than to undifferentiated iPSCs or MuSCs derived from healthy human skeletal muscle tissue (Figure 1d). In addition,
iMSCs, KO-iMSCs, and pMSCs showed a high expression of MSC markers, but a low expression of pluripotency markers and myogenic markers (Figure 1e). These
results confirmed that iMSCs are a comparable cell
population with properties similar to skeletal muscle
derived-MSCs, and KO-iMSCs have similar character
except for COL6 expression.

of TA muscles that received iMSC or pMSC transplantation expressed COL6 protein (Figure 2d, e). However,after 12 weeks, the COL6 expression was only maintained in the pMSC-transplanted limbs, whereas in theiMSC transplanted limb, the size of the COL6-positive
area had decreased (Figure 2d, e). In addition, the transplanted cells in all cases did not remain at the transplanted site but migrated to disperse (Figure S3a). No
pathological fibrosis was seen in all transplanted limbs(Figure S3b). The number of human Lamin A/C-positivetransplanted pMSCs, iMSCs, and KO-iMSCs greatly decreased between 1 and 2 weeks after the transplantation(Figure S4a). A time-course histological analyses showedthat the COL6 expression level was reduced at 4 weeks
after the transplantation of pMSCs or iMSCs and maintained until 24 weeks (Figure S4b, c).

COL6 was supplemented by the local injection of iMSCs/
pMSCs into Col6a1KO mouse muscle

COL6 supplementation by iMSCs/pMSCs transplantation
improves the impairment of muscle maturation in
Col6a1KO micepMSCs, iMSCs, and KO-iMSCs were transplanted into
the TA muscles of Col6a1KO mice intramuscularly (Figure 2a). Two or 12 weeks after the transplantation, histological analysis of the TA muscles was performed.
Without cell transplantation, PDGFRα-positive MSCs
were found in the interstitium of skeletal muscle tissue,
but COL6 protein was deficient (Figure 2b), consistent
with a previous report (40). On the other hand, in theskeletal muscle tissue of WT mice of the same age,
COL6 protein wrapped each muscle fiber (Figure 2b).
Two weeks after the transplantation, human Lamin A/
C-positive transplanted cells were engrafted in the interstitium, and PDGFRα expression was maintained in the
TA muscle sections of Col6a1KO mice (Figure 2c). Furthermore, COL6 protein was detected around human
Lamin A/C-positive transplanted cells in the TA muscle
tissues transplanted with pMSCs and iMSCs at 2 and 12
weeks after the transplantation (Figure 2d). COL6 protein secreted from the transplanted cells wrappedaround the host muscle fibers in a manner consistent
with skeletal muscle tissue in WT mice. On the other
hand, in the muscle tissues transplanted with KOiMSCs, COL6 protein was not detected at all (Figure2d). At 2 weeks after the transplantation, 5% of the area

It has been reported that the proportion of smalldiameter skeletal muscle fibers is greater in Col6a1GT/GT
mice than in WT mice (40). This phenotype was true for
the Col6a1KO mice used in this study (Figure 3a, b).
However, at 2 weeks after the transplantation, the proportion of small-diameter fibers decreased in muscle tissue transplanted with iMSCs and pMSCs (Figure 3a, b).
This trend continued at 12 weeks after the transplantation, and the proportion of large-diameter fibers increased. The average cross-sectional area (CSA) of single
muscle fibers was increased in TA muscles transplantedwith pMSCs and iMSCs at 12 weeks (Figure 3c). The
number of TA muscle fibers tended to increase with
iMSC or KO-iMSC transplantation at 2 weeks (FigureS5a), and at 12 weeks, the number of myofibers in
pMSC- and iMSC-transplanted TA muscles reached thatof WT mice (Figure S5a). The whole CSA and muscle
wet weight of iMSC- and pMSC-transplanted TA muscles increased compared with Col6a1KO muscles,
whereas KO-iMSC-transplanted TA muscles showed nodifference with Col6a1KO muscle (Figure S5b, c). These
results indicated that COL6 supplementation by COL6producing pMSCs/iMSCs increased the muscle fiber
diameter in Col6a1KO TA muscle.

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Fig. 2 Cell engraftment and COL6 supplementation in TA muscles of Col6a1KO mice after cell transplantation. a Schematic representation of the celltransplantation into the TA muscle of Col6a1KO mice. b Sectional images of TA muscles in a Col6a1KO mouse and a WT mouse. Arrowheads indicate
PDGFRα+ cells. c Sectional images of TA muscles 2 weeks after the cell transplantation. Boxed areas in the upper panels are shown with higher
magnification in the lower panels. Scale bars, 25 μm (upper) and 12 μm (lower). d Sectional images of TA muscles at 2 weeks (upper) and 12 weeks (lower)
after medium injection (WT and Col61KO) or cell transplantation. e Quantitative data of the COL6 positive area per whole TA CSA 2 weeks (tx2w) or 12
weeks (tx12w) after the transplantation. Data are shown as the mean ± SD. For tx2w, n = 17 (WT), n = 10 (medium; Col6a1KO), n = 9 (pMSCs), n = 12
(iMSCs), and n = 7 (KO-iMSCs); for tx12w, n = 17 (WT), n = 11 (medium; Col6a1KO), n = 6 (pMSCs, iMSCs, and KO-iMSCs each). *P < 0.05. **P < 0.01

Fig. 3 Histological changes in skeletal muscle tissue of Col6a1KO mice by COL6 supplementation. a Quantification of myofiber CSAs in the TA muscles ofCol6a1KO mice 2 weeks (tx2w) and 12 weeks (tx12w) after medium injection or cell transplantation. Control WT mice are the same age as Col6a1KO mice.
The x-axis shows the short axis of the muscle fiber CSA in μm. b The short axis of the myofiber CSAs in a shown as band graphs (blue, 56 μm ≤ CSA shortaxis; yellow, 26 μm ≤ CSA short axis ≤ 55 μm; red, CSA short axis ≤ 25 μm). *P < 0.05 vs WT, #P < 0.05 vs Col6a1KO, §P < 0.05 vs pMSC, †P < 0.05 vs iMSC. c
CSA per single myofiber in TA muscles. Data are shown as the mean ± SD. For tx2w, n = 15 (WT), n = 10 (medium; Col6a1KO), n = 9 (pMSCs), n = 12
(iMSCs), and n = 7 (KO-iMSCs); for tw12w, n = 17 (WT), n = 11 (medium; Col6a1KO), n = 6 (pMSCs, iMSCs, and KO-iMSCs each). *P < 0.05. **P < 0.01

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Fig. 4 Small-diameter regenerating muscle fibers (eMHC+) in Col6a1KO mice were matured by COL6 supplementation. a Sectional images of TA muscles
1 week after medium injection (Col6a1KO) or cell transplantation. Yellow arrows indicate multi nuclei eMHC+ fibers. b Number of eMHC-positive fibers 1
week after the transplantation. c CSA per single eMHC+ TA muscle fiber 1 week after the transplantation. d Number of nuclei in eMHC+ TA muscle fibers
1 week after the transplantation. e % of eMHC+ TA muscle fibers with more than three nuclei 1 week after the transplantation. f Sectional images of whole
TA muscle 1 week after medium injection (Col6a1KO) or cell transplantation. Two serial sections were stained. g Western blots for COL6A1 protein
expression in pMSC-, iMSC-, and KO-iMSC-transplanted TA muscle tissue samples of Col6a1KO mice and TA muscle tissue samples of WT mice. GAPDH wasused as the control. Data are the mean ± SD. n = 20 (medium; Col6a1KO), n = 10 (iMSCs), and 6 (pMSCs and KO-iMSCs each). *P < 0.05. **P < 0.01

COL6 supplementation by iMSCs/pMSCs transplantation
promotes muscle regeneration in Col6a1KO mice

In a previous study using muscle biopsies from UCMD
patients, regenerating muscle fibers labeled with an antibody for embryonic myosin heavy chain (eMHC), which
is transiently upregulated in regenerating fibers, werelimited to only small diameters (36). Consistently, alleMHC-positive fibers were small in the muscles ofCol6a1KO mice, suggesting that the fibers remained immature. In all transplanted TA muscles, eMHC-positive
fibers gathered in the areas of the transplanted cells (Figure 4a). The number of eMHC-positive fibers significantly increased in MSC-transplanted TA muscles(Figure 4b). The CSA of eMHC-positive fibers was increased in COL6-producing pMSC/iMSC-transplanted
muscles (Figure 4c). In iMSC-transplanted TA muscles,
regenerating muscle fibers were seen to be multinucleated (Figure 4a, arrows). The number of nuclei per
muscle fiber was increased in iMSC-transplanted TA
muscle (Figure 4d), and more than 40% of regeneratingmuscles had more than three nuclei, but the percentage
was only 5% in Col6a1KO mice (Figure 4e). An analysis
using serial sections showed that eMHC-positive fibers
were greatly matured in the region supplemented with
COL6 protein (Figure 4f). A western blotting analysis
showed pMSC- or iMSC-transplanted Col6a1KO muscles restored COL6 protein to the same or higher levels
than in WT muscle (Figure 4g). These results indicatethat COL6-producing pMSCs/iMSCs promote the regeneration of eMHC-positive small-diameter muscle fibers in Col6a1KO TA muscles.
The therapeutic effect of the transplantation in adultmice with advanced symptoms was also examined. Transplantation of iMSCs into the TA muscles of 30-week-old
Col6a1KO mice showed that COL6 supplementation andmuscle regeneration were correlated (Figure S6).
To analyze whether the supplementation of COL6 protein is enough to promote muscle regeneration, recombinant human COL6 (rh-COL6) protein was injected alone
into Col6a1KO muscles (Figure S7a). The rh-COL6 protein was slightly detected in muscle tissue 1 week after the
injection (Figure S7b), but almost all of it had disappearedat 2 weeks. In addition, eMHC-positive regenerating myofibers at 1 week after the administration did not increase
and remained small (Figure S7c). As a result, the injectionof rh-COL6 protein did not affect either the number of
eMHC-positive fibers or CSA.
COL6 supplementation by iMSCs/pMSCs transplantation
promotes the activation of MuSCs and the proliferation ofmyoblasts

Subsequently, the effects of COL6 supplementation on
MuSC proliferation and differentiation were examined.
Previous studies have reported that COL6 acts on quiescent (Pax7+/MyoD-) MuSC self-replication in intact
skeletal muscle tissue and increases the number anddiameter of regenerating central nuclei fibers during regeneration in response to damage caused by cardiotoxin
injection (35). Since COL6 supplementation in intact
Col6a1KO skeletal muscle enhances regeneration (Figure 4a–e), how COL6 contributes to MuSCs in the uninjured situation was analyzed. In TA muscles
transplanted with pMSCs, iMSCs, or KO-iMSCs, thenumber of Pax7+ MuSCs tended to increase compared
to untreated Col6a1KO TA muscles but not significantly(Figure 5a, b). The abundance ratio of quiescent (Pax7+/
MyoD-) MuSCs and activated (Pax7+/MyoD+) MuSCs
was examined. In skeletal muscle supplemented with
COL6 by the cell transplantation, the ratio of activated
MuSCs increased (Figure 5c). Furthermore, the number
of MyoD+ myoblasts increased with either pMSC or
iMSC transplantation, but not with KO-iMSC transplantation (Figure 5d, e). More than 60% of MyoD+ cells
were Ki67-positive proliferative myoblasts for both thepMSC and iMSC transplantation, but less than 40% for
the KO-iMSC transplantation (Figure 5d, f). The increased CSA per eMHC-positive fiber, the activation of
MuSCs, and the proliferation of myoblasts all occurredin the areas supplemented with COL6 protein secreted
from the transplanted cells (Figure 5 g). Importantly, thebenefits of the pMSC and iMSC transplantations were
muted if COL6 was not secreted. These results suggestthat COL6 supplementation by the engrafted cells promotes muscle regeneration via MuSC activation andmyoblast proliferation.
rh-COL6 does not promoteCol6a1KO MuSCs proliferationor maturation in vitro

To confirm in vitro the promotion of skeletal muscle regeneration and maturation shown in the MSC

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Fig. 5 Verification of MuSC proliferation and differentiation and regenerative muscle maturation by COL6 supplementation. a Sectional images of TA muscle 1
week after medium injection (Col6a1KO) or cell transplantation. Yellow arrows indicate Pax7+ nuclei. The inserts are higher magnifications. b Number of Pax7+
cells 1 week after the transplantation. n = 20 (medium; Col6a1KO), n = 6 (pMSCs), n = 10 (iMSCs), and n = 6 (KO-iMSCs). c Quantification of Pax7 + MyoD–, Pax7 +MyoD+, and Pax7–MyoD+ cell populations 1 week after the transplantation. Data are shown as the mean ± SD. n = 20 (medium; Col6a1KO), n = 6 (pMSCs), n =10 (iMSCs), and n = 6 (KO-iMSCs). *P < 0.05 vs. KO, **P < 0.01 vs. KO, §P < 0.05 vs. iMSCs. d Sectional images of TA muscle 1 week after medium injection(COL6a1KO) or cell transplantation. Yellow arrows indicate MyoD+/Ki67+ nuclei. White arrow heads indicate MyoD+/Ki67- nuclei. Boxed areas indicated in theupper panels are shown with higher magnification in the lower panels. e Number of MyoD+ cells 1 week after the transplantation. n = 20 (medium; Col6a1KO), n= 6 (pMSCs), n = 10 (iMSCs), and n = 6 (KO-iMSCs). f % of Ki67+ cells per MyoD+ cells in the TA muscles of Col6a1KO mice 1 week after medium injection(Col6a1KO) or cell transplantation. n = 20 (medium; Col6a1KO), n = 6 (pMSCs), n = 10 (iMSCs), and n = 6 (KO-iMSCs). g Sectional images of TA muscles 1 week
after iMSC transplantation. Yellow arrows indicate Pax7+ cells or MyoD+ cells. Data in b,e,f are shown as the mean ± SD. *P < 0.05. **P < 0.01. n.s., not significant

transplantation experiments, primary MuSCs were collected from the skeletal muscle tissue of Col6a1KO miceand used in culture experiments. First, to verify the direct effect of COL6, MuSCs were cultured in a rh-COL6
protein-coated dish. Since COL1-coated dishes are usually used to cultivate MuSCs, a COL1-coated dish wasused for comparison.
The number of cells on day 3 culture was not significantly different between COL1-coated and COL6-coateddishes (Figure S8a, b). Myogenesis progressed similarly,
and the ratio of Pax7+/MyoD+ cells was high in bothcases (Figure S8c, d).
There was no significant difference in the number of
MHC-positive myotubes, the fusion index, or the CSAof MHC-positive myotubes between the two coats on
day 6 culture (Figure S8e, f).
COL6 produced by co-cultured cells promotes theproliferation of Col6a1KO-MuSCs and enhanced thematuration of differentiated myotubes

Next, Col6a1KO-MuSCs were co-cultured with pMSCs,
iMSCs, or KO-iMSCs. After culturing in growth medium
for 3 days, the number of human Lamin A/C-negative nuclei was counted to verify the expansion of MuSCs.
MuSCs proliferated significantly more when co-cultured
with either pMSCs or iMSCs than with KO-iMSCs (Figure
6a, b), but the activation status of the MuSCs was similar
among the three groups (Figure 6c). In all three cases,
however, compared to the case without co-culture withfeeder cells, the proportion of Pax7+/MyoD- quiescentsatellite cells decreased and the proportion of Pax7-/
MyoD+ myoblasts increased (compare Figure 6a, c andFigure S8c, d). These results indicate that the differentiation of satellite cells was promoted by the co-culture.
pMSCs and iMSCs were found near the formed myotubes, and the myotubes appeared to be wrapped with secreted COL6 on day 10 of the co-culture (Figure 6d).
Also, MuSCs in these co-cultures showed an expanded
MHC positive area, indicating that the differentiation andmaturation of skeletal muscle was promoted by COL6
protein secreted by pMSCs and iMSCs (Figure 6d, e).
These effects were significantly dampened in KO-iMSC
co-cultures. Furthermore, the number of nuclei containedin the myotubes was significantly increased in MHCpositive cells differentiated from MuSCs co-cultured with
pMSCs and iMSCs, but not with KO-iMSCs or no coculture (Figure 6f). These observations indicate that COL6
protein secreted from pMSCs and iMSCs promoted myoblast fusion and myotube maturation. Interestingly, myotubes derived from MuSCs co-cultured with either
pMSCs or iMSCs showed sarcomere structures and spontaneous contractions on day 10 of the co-culture (Supplemental movie 1). On the other hand, while MuSCs cocultured with KO-iMSCs could form myotubes, they did
not show sarcomere structures or spontaneous contractions on day 10 (Supplemental movie 2).
Three days after replacing the co-culture with differentiation medium, the expression levels of genes related toskeletal muscle differentiation and maturation were analyzed by qPCR using specific murine gene primers (Figure6g). As a result, the expressions of all myogenic differentiation markers (Myf5, Myod1, Myogenin), myotube maturation markers (Myh8, Myh1), and markers related toskeletal myoblast fusion (Myomaker, Myomerger) weresignificantly increased in MuSCs co-cultured with iMSCs
for 6 days, but the expression level of Pax7 was low in
MuSCs co-cultured with iMSCs or KO-iMSCs with nosignificant difference (Figure 6g). These results indicatethat COL6 secreted from iMSCs promotes myogenic differentiation, myoblast fusion, and myotube maturation.
To validate the potential for myogenesis by COL6,
Col6a1,2 knockdown (KD) experiments were conductedin another fibroblastic cell type. Using MyoD-RFP mice,
myogenic cells were isolated by RFP expression and cocultured with mouse embryonic fibroblasts (MEFs) that
expressed COL6 (Figure S9a). The MEFs were treatedwith siRNA for Col6a1 and Col6a2 (Figure S9b, Table
S6). RFP-positive myogenic cells proliferated better when
co-cultured with control MEFs than with KD MEFs (Figure S9c). These results indicate that skeletal muscle differentiation and maturation are delayed by Col6a1,2 KD.
UCMD patient-derived iMSCs showed defective potential
for promoting muscle regeneration

Next, a validation experiment using patient-derived
iMSCs was conducted. The donor of these patient-Takenaka-Ninagawa et al. Stem Cell Research & Therapy

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Fig. 6 Effect of COL6 protein on myogenesis in co-culture experiments. a Immunofluorescence images of Col6a1KO mouse-derived MuSCs 3 days
after co-culture with pMSCs, iMSCs, or KO-iMSCs. Yellow arrowheads indicate MyoD+/Pax7- cells. b Number of DAPI+/human Lamin A/C- mouse
MuSCs 3 days after co-culture. Data of three independent experiments are shown as the mean ± SD. **P < 0.01. c Percentage of Pax7 + MyoD–,
Pax7 + MyoD+ and Pax7–MyoD+ cell populations 3 days after co-culture. Data of three independent experiments are shown as the mean ± SD.
d Immunofluorescence images of Col6a1KO mouse-derived MuSCs 10 days after co-culture with pMSCs, iMSCs, or KO-iMSCs. Nuclei were stainedwith DAPI. e The area of MHC+ myotubes derived from mouse MuSCs 10 days after co-culture. Data of three independent experiments areshown as the mean ± SD. **P < 0.01. f Number of nuclei in MHC+ myotubes derived from mouse MuSCs 10 days after co-culture. Data of three
independent experiments are shown as the mean ± SD. **P < 0.01. g The expression of marker genes. The mRNA expression of each gene wasanalyzed by RT-qPCR in primary MuSCs derived from Col6a1KO mice 6 days after co-culture with iMSCs or KO-iMSCs. Expressions are shown
relative to values in MuSCs co-cultured with iMSCs for 6 days. MuSC marker: Pax7; myogenic markers: Myf5, Myod1, and Myogenin; myoblast
fusion-related genes: Myomaker and Myomerger; muscle fiber-maturation markers: Myh8 and Myh1. Data of three independent experiments areshown as the mean ± SD. **P < 0.01

derived iMSCs has a missense mutation in the COL6A1
gene, which creates protein misfolding and prevents thecreation of COL6 with the correct tertiary structure inskeletal muscle tissue. As a result, the misfolded COL6
in this patient is unable to function and develops a
UCMD pathology. The UCMD-iMSCs expressed MSC
markers similar to MSCs and iMSCs (Figure S10). In
addition, to confirm that the secreted COL6 protein had
a normal three-dimensional structure, immunofluorescence staining was performed using an antibody thatrecognizes only the triple helical domain of the protein(t-COL6 antibody). iMSCs derived from healthy subjects
were stained not only with the pan COL6 antibody but
also with t-COL6 antibody (Figure S10b). On the other
hand, in UCMD-iMSCs, the COL6 protein was not detected by the t-COL6 antibody, but it was recognized by
the pan COL6 antibody. These results indicate that
UCMD-iMSCs have the ability to produce COL6 but
not with the proper three-dimensional structure.
Then, UCMD-iMSCs were transplanted into Col6a1KO
mice. Two and 12 weeks after the transplantation, the secretion of COL6 protein around the transplanted cells was
detected by the pan COL6 antibody but not by the tCOL6 antibody (Figure 7a). COL6 was secreted and expanded at almost the same level as that seen in the iMSC
transplantation experiments (Figure 7b). The CSA andnumber of muscle fibers were measured in the muscle tissue samples 2 and 12 weeks after the transplantation. No
significant difference was detected between the control
limb (no transplantation) and the UCMD-iMSC transplanted limb (Figure 7c, d).
Further analysis of eMHC-positive regenerative muscle
fibers in the tissue sample 1 week after the transplantation revealed no hypertrophy (Figure 7e). Again, no significant difference was detected between the control
limb and the UCMD-iMSC transplanted limb (Figure 7f,g). These results indicated that COL6 produced by
iMSCs derived from dominant negative type UCMD patient cells had no therapeutic effect on Col6a1KO mousemuscle.

Discussion
In the present study, iMSCs promoted the regenerationand maturation of skeletal muscles in Col6a1KO UCMD
model mice in vitro and in vivo. On the other hand,
iMSCs derived from two types of COL6-deficient iPSCs(KO-iMSCs and UCMD-iMSCs) had no effect on improving the phenotype. Overall, these results demonstrated
that MSC therapies depend on the secretion of COL6 fortheir therapeutic effect on UCMD model mice.
The pathogenesis mechanism of COL6-related diseases
has been roughly classified into the following: autophagy
abnormality (31–33), apoptosis enhancement due tomitochondrial dysfunction (25–30), and skeletal muscle
regeneration/maturation deficiency (34, 35). The current
study focused on the mechanism for skeletal muscle regeneration/maturation deficiency in UCMD and the development of new therapeutic methods.
Skeletal muscle regeneration and maturation deficiency has been reported as a characteristic phenotype
in muscle biopsy samples of human UCMD patients (36)and UCMD model mice (40, 41). However, little is
known about the COL6-dependent mechanism for theoccurrence and progression of the deficiency. Previous
reports about cell transplantation treatment in UCMD
model mice have only given short-term observations (34,35), and no mention has been made as to whether thetherapeutic effect of the transplantation is temporary or
long-lasting. In the present study, the course up to 24
weeks after the transplantation was followed. At 12
weeks, no benefit was seen with COL6-deficient iMSCs,
but significant muscle fiber hypertrophy was inducedwith COL6-producing pMSCs/iMSCs. These results suggest a long-term effect that promotes muscle maturation. The transplanted pMSCs/iMSCs and secreted
COL6 were still present at 24 weeks, and the therapeutic
effect was maintained.
To directly demonstrate that the pathological phenotype depended on COL6, two types of COL6-deficient
MSCs: KO-iMSCs and UCMD-iMSCs were created. Previous studies have reported that factors secreted from

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Takenaka-Ninagawa et al. Stem Cell Research & Therapy(2021) 12:446

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Fig. 7 UCMD-iMSC transplantation experiments. a Sectional images of TA muscle 2 weeks (tx2w, left 2 columns) and 12 weeks (tx12w, right 2 columns) after
UCMD-iMSC transplantation. b Quantitative data of the COL6-positive area per whole TA muscle CSA 2 and 12 weeks after the transplantation. Data are shownas the mean ± SD. n = 6 (2 weeks), n = 6 (12 weeks). **P < 0.01. c CSA per muscle fiber in the TA muscles of Col6a1KO mice 2 and 12 weeks after medium
injection or UCMD-iMSC transplantation. Data are shown as the mean ± SD. n = 3. n.s., not significant. d Number of muscle fibers in the TA muscles ofCol6a1KO mice 2 and 12 weeks after medium injection or UCMD-iMSC transplantation. Data are shown as the mean ± SD. n = 3. n.s., not significant. e Sectional
images of TA muscles 1 week after medium injection or UCMD-iMSC transplantation. f Number of eMHC-positive fibers 1 week after medium injection or
UCMD-iMSC transplantation. Data are shown as the mean ± SD. n = 3. n.s., not significant. g CSA per eMHC+ muscle fiber in the TA muscles of Col6a1KO mice
1 week after medium injection or UCMD-iMSC transplantation. Data are shown as the mean ± SD. n = 3. n.s., not significant

healthy MSCs can promote muscle regeneration (myogenesis)(42, 43). On the other hand, MSCs in UCMD
muscle may have reduced or lost this muscle regeneration ability(40). Thus, MSC supplementation, regardlessof COL6 expression, may temporarily improve thephenotype. In the present study, the transplantation of
KO-iMSCs slightly activated myoblasts, but far less sothan when pMSCs/iMSCs were transplanted. Moreover,
COL6 supplementation by the transplantation ofpMSCs/iMSCs significantly promoted the number of nuclei in eMHC-positive regenerating myofibers and the
CSA of the myofibers, but these observations not madewith KO-iMSC transplantation. This finding is consistent with the co-culture experiments in this study: coculture with iMSCs promoted the growth, fusion, andmaturation of Col6a1KO-MuSCs, and the expression ofgenes related to MuSC activation and myotube fusion
increased, but not when KO-iMSCs were used for the
co-culture. These results indicate that the effect of MSC
transplantation on myoblast fusion and muscle fiber
maturation is synergized by COL6 secretion.
The in vitro experiments in this study showed that
iMSC co-culture promoted myoblast proliferation andmuscle maturation, consistent with the in vivo experiments. Considering that this effect was not observed
when using COL6-coated dishes, we hypothesized that
the COL6 effect requires three-dimensional interactionsof the protein with the surrounding myoblasts and myofibers. Consistently, transplanted pMSCs/iMSCs tightly
surrounded and wrapped the myotubes with secreted
COL6 protein. As for why rh-COL6 did not have this effect, one possibility is that the protein cannot diffuse,
thus remaining in only a small portion of the injectedmuscle and vanishing in less than 2 weeks. Without
COL6-producing cells (i.e., pMSCs/iMSCs), sufficient
COL6 protein may not be delivered and sustained in
UCMD muscle.
There have been two previous reports on cell transplantation for UCMD. The primary cells used in thosestudies showed a certain therapeutic effect on UCMD
muscles (34, 35). However, the practical application ofprimary cell-based therapy is difficult, because primarycells have limited growth (52). In fact, the pMSCs derived from adult skeletal muscle tissue used in this study

as a positive control have an upper limit for subculture
in vitro, and changes in their properties such as differentiation and proliferation ability occurred after about 3–5
passages (data not shown). Furthermore, tissue collection from donors always involves invasion, and the variability in properties between donors is large (53, 54).
Finally, primary cells from patients have mutations in
COL6A1-3, such that autologous transplants cannot be
used therapeutically. In contrast, iPSCs, which are thesource of iMSCs, can be propagated indefinitely while
maintaining stemness. Especially, iMSCs created from
HLA-edited iPSCs may be a universal therapeutic product (55).
This study did not confirm the improvement ofmuscle motor function by iMSC transplantation. However, in UCMD model mouse neonates transplanted
iMSCs intraperitoneally and systemically, COL6 protein
was expressed in skeletal muscle tissue of the wholebody and could improve motor function (Harada et al.,in revision). Therefore, there is a high possibility that
iMSCs can be used as a cell source for transplantation inplace of pMSCs.

Conclusions
Two main key findings were demonstrated in this study.
First is that iMSCs have equivalent therapeutic effects topMSCs in UCMD model mice, such as the enhancementof muscle regeneration and maturation. Second is thatthese therapeutic effects are mediated by COL6 supplementation. In the future, it is expected that iMSCs derived from HLA-edited iPSCs will be used for clinicalapplication in patients. These results not only raise thepossibility of cell transplantation treatment as a new
therapeutic means but could also help to select treatment targets when developing new therapeutics for
UCMD.
Abbreviations
COL6: Type 6 collagen; MSCs: Mesenchymal stromal cells; UCMD: Ullrich
congenital muscular dystrophy; iPSC: Induced pluripotent stem cell;
iMSCs: iPSC-derived MSCs; MuSCs: Skeletal muscle satellite cells;
ECM: Extracellular matrix; KO-iPSCs: COL6A1 knocked out iPSC; UCMDiPSCs: iPSCs generated from UCMD patients; KO-iMSCs: KO-iPSC-derived
MSCs; UCMD-iMSCs: UCMD-iPSC-derived MSCs; pMSCs: Primary MSCs derived
from human skeletal muscle tissues; NSG mouse; severe
immunodeficient: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; Col6a1KO

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Page 16 of 18

mouse: Col6a1GT/GT/NSG mouse; NCCs: Neural crest cells; TA muscle: Tibialis
anterior muscle; rh-COL6: Recombinant human COL6; CSA: Cross-sectional
area

the iPSCs and pMSCs used in this study. All animal experiments were carriedout according to protocols approved by the Animal Research Committee of
Kyoto University.

Supplementary Information

Consent for publication
Not applicable

The online version contains supplementary material available at https://doi.
org/10.1186/s13287-021-02514-3.
Additional file 1: Supplemental Tables. Tables S1-S6
Additional file 2: Supplemental Methods
Additional file 3: Supplemental Figures and Legends. Figures S1-S10
Additional file 4: Supplemental movie 1
Additional file 5: Supplemental movie 2
Acknowledgements
We thank P. Karagiannis for advice and critical reading of the manuscript, T.
Yamamoto for advice on analyzing the RNA-seq data, A. Hotta for advice on
generating the KO-iPSCs by CRISPR-Cas9, S. Fukada for the SM/C-2.6 antibody, and H. Takenaka for technical support of the statistical analysis. We also
thank A. Tanaka for providing littermates of the model mice by in vitro
fertilization, and Dr. Morita for the preparation of human muscle samples.

Competing interests
The authors declare that they have no competing interests.
Author details
Department of Clinical Application, Center for iPS Cell Research andApplication (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku,
Kyoto 606-8507, Japan. 2Department of Anatomy, Fujita Health University,
Toyoake, Aichi 470-1192, Japan. 3Muscle Aging and Regenerative Medicine,Research Team for Geriatric Medicine, Tokyo Metropolitan Institute ofGerontology, Tokyo 173-0015, Japan. 4Division for Therapies against
Intractable Diseases, Institute for Comprehensive Medical Science (ICMS),
Fujita Health University, Toyoake, Aichi 470-1192, Japan. 5Department of
Neuromuscular Research, National Institute of Neuroscience, Department ofClinical Development, Translational Medical Center, National Center of
Neurology and Psychiatry, Kodaira, Tokyo 187-8551, Japan.
1

Received: 6 May 2021 Accepted: 13 July 2021
Authors’ contributions
As for corresponding authorship, N.T-N. is specifically responsible for thein vivo experiments and is the lead contact of this manuscript. H.S. is
specifically responsible for the in vitro experiments.
N.T-N. and H.S. conceived the study, designed the experiments, interpreted
the data, and wrote and approved the final manuscript. N.T-N. performed
most of the experiments. J.K., M.G., and R.I. carried out the histologicalanalysis of the transplantation studies. M.Z. generated and characterized the
KO-iPSCs. N.T-N., C.K.B.Y., and M.Z. carried out the western blotting analysis.
M.S. carried out the FACS sorting of MuSCs from Col6a1KO mice. T.J. generated the UCMD-iPSCs and carried out the RNA-seq analysis. M.G., R.I., and
A.H. performed the mouse genotyping and treatments. T.S. carried out theanalysis of the co-culture experiments of COL6A1,2KD-MEFs and MyoD-RFP
mouse-derived myoblasts and provided conceptual advice. M.I. induced theiNCCs, KO-iNCCs, and UCMD-iNCCs and provided conceptual advice. A.U.
and M.N. isolated MuSCs and MSCs from human skeletal muscles. S.N. generated the Col6a1GT/GT mice and provided conceptual advice. All authors discussed the results and commented on the manuscript.
Funding
This work was mainly supported by a Grant-in-Aid for Young Scientists (#JP18K15119) from the Japan Society for the Promotion of Science to
N.T-N. and partially supported by grants from the Projects for Technological
Development (#JP20bm0404044) and the Core Center for iPS Cell Research(#JP20bm0104001), Research Center Network for Realization of Regenerative
Medicine from the Japan Agency for Medical Research and Development(AMED) to H.S.
Availability of data and materials
All raw data files for the RNA-sequencing are available to the public throughthe DDBJ submission portal D-way (DRA009935/ftp://ftp.ddbj.nig.ac.jp/ddbj_
database/dra/fastq/DRA009/DRA009935/). KO-iPSC and UCMD-iPSC will bedeposited in the RIKEN BRC.

Declarations
Ethics approval and consent to participate
This study was performed in accordance with the guidelines of the
Declaration of Helsinki and conducted after obtaining written informed
consent. The patient-derived iPSC generation protocols were approved by
the Kyoto University Graduate School and Faculty of Medicine, Ethics Committee (approval numbers #R0091 and #G259). Experiments using human primary skeletal muscle cells were approved by both the Ethical Review Board
for Clinical Studies at Fujita Health University and the Kyoto University Graduate School and Faculty of Medicine, Ethics Committee (approval number#Rin55). The written informed consents were obtained from the donors ofReferences
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