Efficient engraftment of pluripotent stem cell-derived myogenic progenitors in a novel immunodeficient mouse model of limb girdle muscular dystrophy 2I

Defects in α-dystroglycan (DG) glycosylation characterize a group of muscular dystrophies known as dystroglycanopathies. One of the key effectors in the α-DG glycosylation pathway is the glycosyltransferase fukutin-related protein (FKRP). Mutations in FKRP lead to a large spectrum of muscular dystrophies, including limb girdle muscular dystrophy 2I (LGMD2I). It remains unknown whether stem cell transplantation can promote muscle regeneration and ameliorate the muscle wasting phenotype associated with FKRP mutations. Here we transplanted murine and human pluripotent stem cell-derived myogenic progenitors into a novel immunodeficient FKRP-mutant mouse model by intra-muscular injection. Upon both mouse and human cell transplantation, we observe the presence of donor-derived myofibers even in absence of pre-injury, and the rescue of α-DG functional glycosylation, as shown by IIH6 immunoreactivity. The presence of donor-derived cells expressing Pax7 under the basal lamina is indicative of satellite cell engraftment, and therefore, long-term repopulation potential. Functional assays performed in the mouse-to-mouse cohort revealed enhanced specific force in transplanted muscles compared to PBS-injected controls. Altogether, our data demonstrate for the first time the suitability of a cell-based therapeutic approach to improve the muscle phenotype of dystrophic FKRP-mutant mice.


Background
Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive degeneration and muscle weakness. Among them, dystroglycanopathies represent a significant subgroup, which is characterized by hypoglycosylation of α-dystroglycan (DG; OMIM 128239) [1]. α-DG is a key effector of the dystrophin glycoprotein complex as it ensures the binding of the actin cytoskeleton to the extracellular matrix (ECM) [2], however, this binding requires functionally glycosylated α-DG. α-DG glycosylation is composed of both N-linked and O-linked glycans, with the latter mediating the binding of α-DG to ECM proteins, such as laminin, agrin, perlecan, neurexin, and pikachurin [3][4][5][6][7][8]. Therefore, hypoglycosylation of α-DG in muscle leads to reduced α-DG binding to the ECM, fragile sarcolemma, and ultimately to the dystrophic phenotype [9].
At present, 18 genes involved in the α-DG glycosylation pathway have been linked to dystroglycanopathies [10], including the fukutin-related protein (FKRP; OMIM 606596). FKRP is a ribitol-phosphate transferase that utilizes CDPribitol as a substrate to add a ribitol-phosphate into the glycosylation chain, a critical step to generate functionally glycosylated α-DG [11,12]. FKRP mutations are associated with a large spectrum of dystroglycanopathies from severe forms, such as congenital muscular dystrophy and Walker-Warburg syndrome, to limb girdle muscular dystrophy type 2I (LGMD2I) [13][14][15]. As in other types of MDs, only palliative treatments are currently available for these patients.
Several promising approaches are being investigated to restore functional glycosylation of α-DG using available FKRP-mutant mouse models [16][17][18][19][20][21][22]. One strategy is to increase the levels of metabolites involved in the FKRPmediated α-DG glycosylation process, such as ribitol treatment [16,23], which has been shown to rescue α-DG functional glycosylation in FKRP P448L mice. Nevertheless, this promising result needs further investigation to determine if this beneficial effect can be extended to other FKRP mutations, and if ribitol treatment presents any detrimental side effects. In any case, most efforts to date have focused on gene therapy by delivering fully functional FKRP via adenoassociated virus (AAV) to muscle cells of FKRP-mutant mouse models [17][18][19][20]. These studies have shown improvement of the dystrophic phenotype upon injections of AAV expressing FKRP under the control of systemic or muscle specific promoters. However, this approach has raised two major concerns, the efficacy of FKRP delivery, which was shown to decrease with age [20], and potential dose-dependent toxicity, suggesting that FKRP expression levels may need to be controlled [19]. In a combined gene/ cell therapy study, Frattini and colleagues overexpressed FKRP in satellite cells isolated from FKRP-mutant mice. Transplantation of these engineered satellite cells into FKRP dystrophic mice led to the rescue of α-DG functional glycosylation, which the authors hypothesized to be due to the diffusion of FKRP via exosomes from the injected cells [21]. Even though the mechanism of rescue focused on the exosome delivery of FKRP, it remains to be determined the nature of engraftment, meaning clear identification and characterization of engrafted donor-derived myofibers.
In the context of cell therapy, pluripotent stem cells are particularly attractive due to their unlimited proliferative capacity and ability to differentiate into all cell types, allowing for the generation of large numbers of myogenic progenitors endowed with in vivo regenerative potential, as shown by transplantation studies using several mouse models of MD [24][25][26][27]. To determine whether LGMD2I associated with FKRP mutations could benefit from the transplantation of pluripotent stem cell-derived myogenic progenitors, here we generated an immunodeficient FKRP-mutant mouse model and validated this through the transplantation of mouse and human pluripotent stem cell-derived myogenic progenitors. Our data show robust engraftment that is accompanied by restoration of α-DG functional glycosylation and amelioration of disease phenotypes, thus supporting the therapeutic benefit of cell transplantation for LGMD2I and potentially other FKRPassociated muscle disorders.
For the transplantation of human cells, we used PAX7induced myogenic progenitors since these have been extensively characterized in our laboratory for their in vivo regenerative potential [27,[29][30][31]. For this, a human wildtype induced pluripotent stem (iPS) cell line was transduced with doxycycline-inducible iPAX7-IRES-GFP lentivirus, as previously described [29]. Transduced iPS cells were cultured in suspension for 2 days to derive embryoid bodies, which were further cultured in medium supplemented with 10 μM GSK3β inhibitor (CHIR99021; Tocris) for 2 days, followed by treatment with 200 nM BMP inhibitor (LDN193189; Cayman Chemical) and 10 μM TGFβ inhibitor (SB431542, Cayman Chemical) for 2 days to derive somitic mesoderm-like cells. At day 5, cells were treated with 1 μg/ml doxycycline (dox) to induce PAX7 expression. Embryoid bodies were then plated onto gelatin-coated dishes in the presence of dox and bFGF (5 ng/ml) to derive a monolayer of cells. Four days later, these were dissociated and purified by FACS based on GFP expression to purify for PAX7+ myogenic progenitors, which were maintained in culture for up to 3-4 passages in the presence of dox and bFGF (Fig. S2d) [32].
Generation and characterization of FKRP P448L -NSG mice All animal studies were performed according to protocols approved by the University of Minnesota Institutional Animal Care and Use Committee. The FKRP P448L mouse model [33] was obtained from Jackson Laboratories, where this strain was backcrossed to generate a congenic C57BL/ 6 (B6). To generate an immunodeficient FKRP P448L mouse model, we have crossed the FKRP P448L mutant onto the NSG (NOD/SCID; IL2 receptor gamma) background. These mice lack all functional classes of lymphocytes. F1 males (carrying gamma-c, which is X-linked) were backcrossed, and N1 pups carrying FKRP mutations and homozygous for NOD/SCID and IL2Rg were identified by PCR. To confirm immunodeficiency, peripheral blood was collected from the facial vein of B6, FKRP P448L , and FKRP P448L -NSG mice, stained with anti-mouse CD3e PE (145-2C11), anti-mouse CD19 PE-Cy7 (1D3), anti-mouse NK1.1 FITC (PK136), and anti-mouse pan-NK cells (DX5) antibodies, and analyzed by flow cytometry.

Cell transplantation and muscle collection
Prior to transplantation, mice were anesthetized with ketamine/xylazine at 80 mg/kg by intraperitoneal (IP) injection. Cell transplantation was performed in tibialis anterior (TA) muscles of 3-5 weeks FKRP P448L -NSG or FKRP P448L mice that had been pre-injured or not with cardiotoxin (CTX, 15 μl of 10 μM stock; Latoxan). Myogenic progenitors were injected at 1 × 10 6 (resuspended in 15 μl of PBS) using a 22 g Hamilton syringe. As control, the contralateral leg was injected with 15 μL of PBS. For the transplantation of immunocompetent FKRP P448L mice, recipients received intraperitoneal (IP) injections of the immunosuppressant agent tacrolimus (MedChemExpress) at a dose of 5 mg/kg. Treatment began 2 days before transplantation and ended by the day of euthanasia [29]. Engraftment of mouse cells was assessed at 4 weeks (short-term) and 5 months (long-term) post-transplantation. Engraftment of human cells was assessed at 6 weeks post-transplantation (short-term).
Merge images of DAPI, IIH6, and RFP or LAMIN A/C were used to quantify donor-derived fibers. A total of 10-12 cryosections, separated by approximately 460 μm, were analyzed for the quantification of donor-derived myofibers. For the quantification of donor-derived satellite cells, merge images of laminin α-2, RFP, Pax7, and DAPI were used to quantify the proportion of Pax7+/RFP-and Pax7+/ RFP+ cells. For the quantification of centrally nucleated myofibers, we used merge images of dystrophin, IIH6, RFP, and DAPI.

Western blot
For biochemical analysis, TA muscles were snap frozen in liquid nitrogen, pulverized in a liquid nitrogen cooled mortar and pestle, and resuspended in lysis buffer Tris-buffer saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 1% Triton X-100 (Sigma) and a cocktail of protease inhibitors (Complete, Millipore). Lysates were placed on an end over rotator for 30 min at 4°C and then centrifuged at 13000 rpm for 30 min at 4°C. The supernatant containing the protein extract was collected, and protein concentration was determined using Bradford assay (Sigma). Briefly, 100 μg of protein lysates were loaded in each lane, resolved in SDS-PAGE, transferred to PVDF membranes (Immobilon-P; Millipore), and blocked 1 h in PBS, 0.1% Tween®20 (Sigma), and 5% milk (RPI). Membranes were incubated overnight at 4°C with primary antibodies: IIH6 (1:1000, 05-593 Millipore) and β-DG (1:1000; MANDAG2 DSHB). After incubation with infrared fluorescent secondary antibodies (Li-cor and Thermo Fischer Scientific), membranes were visualized with Licor's Odyssey® Infrared Imaging System. Image processing and quantification were performed with the Image studio software.

Muscle preparation for mechanical studies
For the measurements of contractile forces, mice were anesthetized with avertin (225-250 mg/kg by IP injection) and intact TA muscles were dissected and placed in an experimental organ bath filled with mammalian Ringer solution containing 120.5 mM NaCl, 20.4 mM NaHCO 3 , 10 mM dextrose, 4.8 mM KCl, 1.6 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM NaH 2 PO 4 , and 1.0 mM pyruvate. Each chamber was perfused continuously with 95% O 2 -5% CO 2 and maintained at a temperature of 25°C. Muscles were stimulated by an electric field generated between two platinum electrodes placed longitudinally on either side of the muscle (Square wave pulses 25 V, 0.2 ms in duration, 150 Hz). Three minutes of recovery period were allowed between stimulations. Specific force (sFo) was determined by normalizing maximum isometric tetanic force to cross section area (CSA) which were obtained by dividing the muscle mass (mg) by the product of muscle length (mm) and 1.06 mg/mm 3 the density of skeletal muscle [24].

Statistical analysis
Differences between two groups were assessed by using the Student's t test or the Mann-Whitney U test (force measurement). Differences between multiple groups were assessed by ANOVA. Statistical analyses were performed using the Prism Software (GraphPad). p values < 0.05 were considered significant.

Generation of an immunodeficient FKRP-mutant mouse model for LGMD2I
To provide a receptive environment for the transplantation of mouse and human cells, we generated an immunodeficient FKRP-mutant mouse model by crossing FKRP P448L mice [33] with the NSG strain (NOD/SCID; IL2 receptor common gamma chain) [36,37]. Once we obtained FKRP P448L -NSG mice homozygous at all loci, the peripheral blood of these mice was analyzed by FACS, which confirmed the depletion of B, T, and NK cells in the FKRP P448L -NSG mouse model, whereas samples from control B6 and FKRP P448L mice contained all these three lymphocyte subsets (Fig. 1a). Western blot analysis using the IIH6 antibody, which is specific to the α-DG laminin binding domain [3,4], confirmed the lack of functionally glycosylated α-DG in FKRP P448L -NSG mice, similarly to their immunocompetent counterparts (Fig. 1b).
Previous literature [38] and our own findings (data not shown) indicate that background staining with the IIH6 antibody may become an issue as the FKRP-mutant mice get older (> 2-3 months), thus potentially interfering with proper engraftment assessment. An alternative to circumvent this hurdle would be to transplant younger FKRP P448L -NSG mice at 3 weeks of age, which would allow for engraftment assessment under 2 months of age. Of note, histological assessment of TA muscles from 7-week-old FKRP P448L -NSG mice confirmed dystrophic phenotype, as shown by the presence of centrally nucleated myofibers (Fig. S1a, b). To verify the feasibility of this transplantation timing, we assessed IIH6 immunoreactivity in several muscles of 7-week-old FKRP P448L -NSG mice by western blot and immunofluorescence staining. As shown in Fig. 1 c and d, relatively low levels of α-DG functional glycosylation were detected, and therefore 3-week-old FKRP P448L -NSG mice were used for the transplantation studies described here.

Transplantation into pre-injured muscles of FKRP P448L -NSG mice
To facilitate in vivo tracking, we began transplantation studies using mouse ES cell-derived myogenic progenitors expressing the fusion protein H2B-RFP (Fig. S2a, b). Importantly, H2B-RFP genetic manipulation did not interfere with the capacity of these cells to express functionally glycosylated α-DG, as shown by IIH6 immunoreactivity in H2B-RFP-labeled ES cell-derived myotubes (Fig. S2c). H2B-RFP-labeled ES cell-derived myogenic progenitors were injected directly into the TA muscles of FKRP P448L -NSG mice. In this cohort, TA muscles were pre-injured with CTX 24 h prior to cell transplantation. As control, we also transplanted immunocompetent FKRP P448L mice, which were treated daily with the immunosuppressive agent tacrolimus. Engraftment was Western blot for IIH6 in TA muscle lysates from 10-week-old B6, FKRP P448L , NSG, and FKRP P448L -NSG mice. β-DG was used as loading control. c Western blot for IIH6 in TA, triceps (Tri), gastrocnemius (Gas), quadriceps (Quad), diaphragm (Dia), and heart lysates from 7-week-old B6 and FKRP P448L -NSG mice. β-DG was used as loading control. d Representative images show IIH6 immunostaining in TA, Tri, Gas, Quad, and Dia muscles from 7-week-old B6 and FKRP P448L -NSG mice. DAPI stained nuclei. Scale bar is 100 μm assessed 4 weeks later by immunostaining with IIH6 and RFP antibodies, which clearly revealed the presence of donor-derived RFP+ myofibers that were also positive for IIH6 (Fig. 2a). Similar levels of engraftment were observed in both FKRP P448L -NSG and tacrolimus-treated FKRP P448L mice (Fig. 2b).
Having validated the novel immunodeficient FKRP P448L -NSG model, we next assessed the ability of human iPS cell-derived myogenic progenitors to engraft in the FKRP P448L -NSG model. Six weeks after transplantation, human engraftment was determined by immunostaining using IIH6 in combination with an antibody specific to human LAMIN A/C. As expected, we did not detect myofibers positive for human LAMIN A/C and IIH6 in PBSinjected muscles (Fig. 2c, upper panels). On the other hand, evident human donor-derived myofiber contribution was observed in muscles that had been transplanted with human iPS cell-derived myogenic progenitors, as shown by the presence of myofibers positive for both LAMIN A/C and IIH6 (Fig. 2c, lower panels). Quantification showed approximately 200 double-positive myofibers (Fig. 2d). Human specific DYSTROPHIN and PAX7 immunostaining confirmed the human origin of the LAMIN A/C+ fibers and their regenerative capacity (Fig. S3). These results confirm the usefulness of this model for human cell transplantation.

Pre-injury is not required to enable efficient engraftment in FKRP P448L -NSG mice
To determine whether pre-injury is required for muscle engraftment in the FKRP P448L -NSG mouse model, next we transplanted mouse ES cell-derived myogenic progenitors into non-injured TA muscles. An average of 350 donorderived RFP+/IIH6+ myofibers were quantified per TA muscle among 17 FKRP P448L -NSG recipient mice from 3 different cohorts (Fig. 3a-c). To examine the distribution of donor-derived myofibers, in another cohort of 7 recipient mice, we quantified engraftment along the length of transplanted TA muscles (approximately 4500 μm). Consistent engraftment was detected along the length of the muscle (Fig. 3d), showing the ability of myogenic progenitors to distribute well within transplanted muscles. Quantification of centrally nucleated myofibers revealed that approximately 60% of donor-derived myofibers were centrally nucleated at 4 weeks post-transplantation (Fig. 3e). Similar engraftment was observed following the transplantation of these cells into non-injured muscles of tacrolimus-treated immunocompetent FKRP P448L mice (Fig. S4). In addition, to better assess the overall rescue of α-DG functional glycosylation in the whole TA muscle, we normalized the area positive for both RFP and IIH6 to the total section area (Fig. S5a). This analysis revealed about 15% restoration in the levels of α-DG functional glycosylation in transplanted TA muscles (Fig. S5b). This rescue was consistent along the muscle, as shown by the quantification of the area containing functionally glycosylated α-DG (Fig. S5b). To verify whether human myogenic progenitors are also able to promote regeneration in the absence of pre-injury, we transplanted human iPAX7 iPS cell-derived myogenic progenitors in non-injured TA muscles of 3-week-old FKRP P448L -NSG mice. As shown in Fig. S6a-d, human donor-derived myofibers are also observed in the absence of pre-injury but at lower levels when compared to mouse transplanted counterparts (127.83 ± 19.94 vs. 346.76 ± 35.39, respectively; Fig. S6b, c), probably due to the xeno nature of this transplantation.
To further validate the rescue of α-DG functional glycosylation, we performed western blot analysis in mice that had been transplanted with mouse ES cell-derived myogenic progenitors. We determined the linear range of detection for IIH6 and β-DG antibodies with different amounts of total protein and observed that 100 ug of total protein from FKRP P448L -NSG cells injected TA lysates would be adequate for quantification (Fig. S7a-c). As shown in Fig. 3 f and g, high levels of α-DG functional glycosylation were detected in TA muscles that had been transplanted with myogenic progenitors. This finding was further confirmed with two additional independent transplantation cohorts (Fig. S7d), and corroborated by the laminin-overlay assay. Consistently with IIH6 data, superior laminin binding was observed in transplanted muscles (Fig. S7e, f). Importantly, using isolated muscle force measurements, we observed improvement in muscle strength in transplanted TA muscles when compared to PBS-injected controls (Fig. 3h).

Donor-derived satellite cell engraftment and long-term regeneration
Next, we investigated whether pluripotent stem cellderived myogenic progenitors have the ability to seed the satellite cell compartment following their transplantation into FKRP P448L -NSG mice. This is critical to ensure the long-term repopulation potential of transplanted cells. To address this question, we stained muscle sections with antibodies to Pax7 (to identify satellite cells), RFP (to distinguish donor contribution), and laminin-α2 (to confirm sublaminal localization). Our results show that transplanted myogenic progenitors are able to populate the satellite pool, as shown by the presence of Pax7+/RFP+ nuclei identified under the basal lamina (Fig. 4a, b). Quantification of engrafted areas revealed that approximately 20% of Pax7+ cells were also positive for RFP, denoting significant donor contribution to the satellite cell compartment (Fig. 4c).
The high percentage of donor-derived satellite cells suggests that transplanted cells may be endowed with long-term regenerative capacity. To test this, we assessed long-term engraftment in a cohort of FKRP P448L -NSG mice that had been injected with cells at 3 weeks of age in the absence of injury. Immunohistological analysis and subsequent quantification confirmed the presence of donor-derived myofibers at 4 months post-transplantation (Fig. 5a-c). Of note, we observed that persistence of engraftment was accompanied by reduction in the percentage of centrally nucleated myofibers (~20%; Fig. 5d) when compared to results from short-term transplantation (6 0%; Fig. 3e). Quantification of RFP+ satellite cells in this long-term cohort showed the persistence of donorderived RFP+/Pax7+ cells, thus suggesting maintenance of satellite cell engraftment (Fig. 5e). These results confirm the long-term regenerative potential of mouse ES cell- Fig. 2 Validation of FKRP P448L -NSG mice as a model for transplantation studies. a Representative images show immunostaining for IIH6 (in green) and RFP (in red) in muscle sections from FKRP P448L (upper panel) and FKRP P448L -NSG (lower panel) mice that had been pre-injured with CTX and injected with mouse ES cell-derived myogenic progenitors in the right leg and PBS in the contralateral leg. DAPI stained nuclei (in blue). Scale bar is 50 μm. b Engraftment quantification based on the number of RFP+/IIH6+ myofibers (from a). Data are shown as mean + SEM (n = 4; 4 females for FKRP P448L , and 2 males and 2 females for FKRP P448L -NSG). c Representative images show immunostaining for IIH6 (in green) and human LAMIN A/C (in red) in muscle sections from FKRP P448L -NSG that had been injected with human iPS cell-derived myogenic progenitors (lower panel) or PBS (upper panel). DAPI stained nuclei (in blue). Scale bar is 50 μm. d Engraftment quantification based on the number of IIH6+/LAMIN A/C+ myofibers (from c). Data are shown as mean + SEM from 2 independent cohorts (n = 8; 6 males and 2 females) Fig. 3 Engraftment analysis upon cell transplantation in non-injured muscles. a Representative images, capturing the whole engraftment area, show immunostaining for IIH6 (in green) and RFP (in red) in TA muscles from non-injured FKRP P448L -NSG that had been injected with PBS (upper panel) or mouse ES cell-derived myogenic progenitors (lower panel). DAPI stained nuclei (in blue). Scale bar is 200 μm. b High magnification image of donorderived engrafted myofibers (from a, white square). Scale bar is 100 μm. c Engraftment quantification based on the number of RFP+/IIH6+ myofibers. Data are shown as mean + SEM from three different cohorts (n = 17; 9 males and 8 females). d Distribution of the number of RFP+/IIH6+ myofibers along the TA muscle (n = 7). e Quantification of the percentage of centrally nucleated myofibers in the PBS injected TA muscles, RFP-and RFP+ area of the cell injected TA muscles. Data are shown as mean + SEM (n = 4; 2 males and 2 females). **p < 0.01. f Representative western blot for IIH6 in TA lysates from 7-week-old FKRP P448L -NSG mice that have been injected at 3-weeks of age with mouse ES cell-derived myogenic progenitors or PBS (contralateral muscle as negative control) (n = 5; 2 males and 3 females). B6 and NSG muscles were used as reference. β-DG was used as a loading control. g Respective quantification of IIH6 band intensity. e Normalized with β-DG. Data are shown as mean + SEM. **p < 0.01. h Effect of cell transplantation on specific (sF 0 : F 0 normalized to CSA) force. Data are shown as mean + SEM (n = 6; 3 males and 3 females). B6 mouse TA muscles were used as a reference (n = 8, 2 males and 6 females). *p < 0.01 derived myogenic progenitors in the LGMD2I mouse model, and the amelioration of dystrophic pathology.

Discussion
Here we generated an immunodeficient FKRP-mutant mouse model, which allowed us to test the effectiveness of mouse and human cell transplantation to rescue α-DG functional glycosylation in the context of LGMD2I. We show that pluripotent stem cell-derived myogenic progenitors engraft robustly in this mouse model, and that engraftment results in restoration of functionally glycosylated α-DG. Extensive characterization of engraftment levels revealed a maximum of 600 donor-derived myofibers, contributing to up to 30% rescue of α-DG functional glycosylation. Biochemical assessment, including western blot to IIH6 and laminin binding assay, corroborated functional rescue of α-DG functional glycosylation. Importantly, muscle strength was improved in engrafted muscles, and long-term studies demonstrated persistence of myofiber and satellite cell engraftment.
An interesting and unexpected finding of the present study was the robust and widespread engraftment observed in the FKRP P448L mouse model in the absence of muscle pre-injury (in both immunodeficient and immunocompetent background). Due to the relatively low turnover in skeletal muscle, strategies to induce muscle injury prior to transplantation, such as CTX, barium chloride, cryoinjury, or irradiation, are commonly used to enhance the muscle regenerative response and therefore, better assessment of the repopulation potential of a given cell population [24,39,40]. This applies not only to non-disease mice but also to mouse models of MDs, such as the mdx for Duchenne MD [24,[41][42][43] and the α-sarcoglycan null for LGMD2D [44], among others. Attesting this, Vallese and colleagues have shown superior muscle engraftment following the transplantation of human myoblasts in cryoinjured recipient muscles when compared to non-injured counterparts [45]. In the context of pluripotent stem cell-derived myogenic progenitors, we have documented that their transplantation into non-injured muscles of mdx mice results in more limited engraftment, which was also more restricted to the injection site, opposed to CTX-injured muscles [24]. Of note, previous studies have shown that pre-injury is not required for the engraftment of these myogenic progenitors in dystrophin/utrophin double knockout mice [26,46], probably due to the much more severe dystrophic phenotype characteristic of this Duchenne MD mouse model [47].
Even though further studies would be required to understand the mechanisms underlining the enhanced regenerative response of transplanted cells in the absence of pre-injury in FKRP P448L -NSG and immunocompetent FKRP P448L dystrophic mice. These findings are highly relevant for pre-clinical studies since they better represent the scenario of a clinical trial aimed at cellbased therapy for MD patients, which evidently would not make use of such pre-injury procedures.

Conclusions
Using a newly generated immunodeficient FKRP-mutant mouse model, we have shown that transplanted pluripotent stem cell-derived myogenic progenitors are able to engraft, rescue α-DG functional glycosylation, and improve muscle strength, providing proof-of-concept for the potential therapeutic application of stem cell therapy for LGMD2I associated with FKRP mutations.

Supplementary information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s13395-020-00228-3. Figure S1. Histological characterization of FKRP P448L -NSG mice. a) Representative images show H&E staining in TA muscle cryosections from 7-week-old B6 (control) and FKRP P448L -NSG mice. Arrows indicate centrally located nuclei and asterisks denote the presence of infiltrating mononuclear cells. Scale bar is 50 μm. b) Quantification of the percentage of centrally nucleated myofibers in the TA muscles of 7-weel-old B6 and FKRP P448L -NSG mice. Data are shown as mean + SEM (TA muscles from 4 mice). ***p < 0.001. Supplementary Figure S2. Mouse ES cell labelling/differentiation and human iPS cell differentiation. a) Outline representing the labeling of iPax3-GFP mouse ES cells with the H2B-RFP encoding lentivirus and subsequent myogenic differentiation. b) Representative FACS plots show percentage of RFP+ cells at different stages of differentiation: left: ES cells, center: embryoid bodies (EBs) before sorting, and right: myogenic progenitors used for transplantation (P4). c) Representative images show immunostaining for IIH6 and RFP in myotubes resulting from the in vitro differentiation of ES cells. IIH6, RFP, and nuclei are shown in green, red and blue, respectively. Scale bar 50 μm. d) Outline representing the timeline of myogenic differentiation of human iPAX7 iPS cells. Supplementary Figure S3.

Additional file 1: Supplementary
Characterization of human engraftment. a) Representative images show immunostaining for human DYSTROPHIN (in gray) and human LAMIN A/ C (in red) in muscle sections from CTX-injured FKRP P448L -NSG mouse TA muscles that had been injected with human iPS cell-derived myogenic progenitors or PBS (from Fig. 2c). DAPI stained nuclei (in blue). Scale bar is 100 μm. b) Representative images show satellite cell staining in the TA muscles described in (a). Circles show cells double-positive for PAX7 (green) and LAMIN A/C (red) under the basal lamina (Lam in gray) indicating donor-derived satellite cells. Nuclei in blue. Scale bar is 50 μm. c) High magnification image of donor-derived satellite cell. Scale bar is 20 μm. Supplementary Figure S4. Engraftment analysis in non-injured muscles of FKRP P448L immunocompetent mice. a) Representative images show immunostaining for IIH6 (in green) and RFP (in red) in non-injured TA muscles from FKRP P448L mice that had been injected with PBS (upper panel) or mouse ES cell-derived myogenic progenitors (lower panel). DAPI stained nuclei (in blue). Scale bar is 100 μm. b) Engraftment quantification based on the number of RFP+/IIH6+ myofibers (from a). Data are shown as mean + SEM (n = 5; 2 males and 3 females). c) Distribution of the number of RFP+/IIH6+ myofibers along the TA muscle (n = 5; 2 males and 3 females). Supplementary Figure S5. Engrafted area quantification in non-injured muscles of FKRP P448L -NSG mice. a) Representative image used to assess the size of the engrafted area (marked in red) compared to the total cryosection area (marked in blue). IIH6 (gray) and RFP (red) allow the delimitation of the area of engraftment. Scale bar is 500 μm. b) Distribution along the length of TA muscle of the percent engraftment (RFP+/IIH6+) area. Data are shown as mean + SEM (n = 7; 4 males and 3 females). Supplementary Figure S6. Engraftment analysis in non-injured muscles transplanted with human iPS cells. a) Representative images show immunostaining for IIH6 (in green) and human LAMIN A/C (in red) in muscle sections from non-injured FKRP P448L -NSG mouse TA muscles that had been injected with human iPS cell-derived myogenic progenitors (lower panel) or PBS (upper panel). DAPI stained nuclei (in blue). Scale bar is 50 μm. b) Engraftment quantification based on the number of IIH6+/LAMIN A/C+ myofibers (from a). Data are shown as mean + SEM (n = 6, 4 males and 2 females). c) Distribution of the number of IIH6+/LAMIN A/C+ myofibers along the TA muscle (n = 6; 4 males and 2 females). d) Representative images show immunostaining for human DYSTROPHIN (in gray) and human LAMIN A/C (in red) in muscle sections from non-injured FKRP P448L -NSG mouse TA muscles injected with iPS cell-derived myogenic progenitors or PBS (from a). DAPI stained nuclei (in blue). Scale bar is 50 μm. Supplementary Figure S7. Additional western blot analysis and Laminin overlay assay. a) Western blot for IIH6 and β-DG in TA lysates from 7-week-old FKRP P448L -NSG mice (2 TA muscles pooled) that had been injected at 3-weeks of age with mouse ES cell-derived myogenic progenitors. To determine the linear range of detection for IIH6 and β-DG antibodies, an increasing amount of protein (0, 25, 50, 100, 125, 150, 200 μg) was loaded. b) Quantification of IIH6 band intensity according to the amount of protein loaded. c) Quantification of the β-DG band intensity related to the amount of protein loaded. d) Western blot for IIH6 in TA lysates from 7-week-old FKRP P448L -NSG mice that had been injected at 3-weeks of age with mouse ES cell-derived myogenic progenitors or PBS (contralateral muscle as negative control). Data from two independent experiments (n = 5 for each), and their respective quantification of IIH6 band intensity normalized with β-DG. Data are shown as mean + SEM. *p < 0.01. e) Detection of laminin binding. No calcium served as negative control. f) Quantification of laminin band intensity normalized with β-DG. Data are shown as mean + SEM (n = 4; 2 males and 2 females). *p < 0.01.