Research ArticleMuscular Dystrophy

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

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Science Translational Medicine  27 Jun 2012:
Vol. 4, Issue 140, pp. 140ra89
DOI: 10.1126/scitranslmed.3003541


Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.


Induced pluripotent stem cells (iPSCs) are the product of reprogramming adult somatic cells to an embryonic stem cell (ESC)–like state using specific transcription factors (1, 2). They show extensive self-renewal and generate differentiated progeny representing all three germ layers. Deriving patient-specific iPSCs to study diseases in vitro is already under way (3). Genetic correction of patient-specific iPSCs for autologous cell transplantation may be a promising strategy for treating a variety of diseases including the muscular dystrophies (4). A critical step in designing iPSC-based protocols for treating skeletal muscle disorders is the development of techniques for inducing iPSCs to be committed to a muscle-specific progenitor cell fate. Recent studies have described the generation of satellite cells (the main progenitor cells resident in skeletal muscle that are responsible for muscle regeneration) and their in vitro–activated progeny (myoblasts) from murine iPSCs and from murine and human ESCs (57). However, these progenitor cells have the same limitations as satellite cells in adult muscle for the purposes of cell therapy; that is, they cannot be delivered to muscle systemically and, in addition, they have poor survival and limited migration capabilities (8). Other mesoderm cell types have been shown to contribute to muscle regeneration. Some of these (principally Pax3/7-positive cells) can also be generated from mouse and, very recently, human ESC- and iPSC-derived embryoid bodies (814). Moreover, mesenchymal stem cells and vasculogenic pericytes derived from human ESCs and iPSCs have been shown to ameliorate limb ischemia after transplantation into mice with a ligated femoral artery (15, 16).

Human pericytes have been shown to contribute to regeneration of mesodermal tissues (17, 18). Mesoangioblasts (MABs), which are derived from alkaline phosphatase–positive (AP+) human skeletal muscle pericytes, are a valuable cell population because, when they are delivered systemically in the arterial circulation they colonize and contribute to muscle regeneration of the dystrophic muscle (19). Moreover, lineage-tracing experiments in the mouse demonstrated that they naturally contribute to skeletal muscle growth and regeneration (20). However, human MABs have a limited life span, and the need to obtain billions of cells to treat all of the skeletal muscles of patients with muscular dystrophy challenges the proliferative capabilities of these cells. The possibility of deriving MABs from iPSCs offers the advantage of producing unlimited numbers of myogenic progenitor cells that can be delivered systemically.

On the basis of evidence of safety and efficacy in mouse models of limb-girdle muscular dystrophy 2D (LGMD2D) and Duchenne’s muscular dystrophy (DMD) (α-sarcoglycan–null mice and mdx mice, respectively) and a dog model of DMD (19, 2127), allogeneic human leukocyte antigen (HLA)–matched MABs have been expanded under clinical-grade conditions and are currently being transplanted into DMD patients in a phase 1/2 clinical trial at San Raffaele Hospital (Milan, Italy; EudraCT no. 2011-000176-33).

To develop an autologous cell therapy for LGMD2D (28, 29), we set out to isolate human MABs from several patients but invariably failed to derive and/or expand cell populations with a MAB phenotype. Further analysis showed that these patients have a reduced number of AP+ pericytes. To overcome this problem, we developed a new protocol to derive MAB-like cells initially from iPSCs derived from healthy patients and subsequently from iPSCs derived from myoblasts and fibroblasts from LGMD2D patients. We derived iPSCs from the skeletal muscle cells of LGMD2D patients, generated MABs from these iPSCs, and expanded the MABs in vitro (Fig. 1). We then transduced the MABs with a lentiviral vector carrying the wild-type human α-sarcoglycan gene (SGCA) and transplanted the corrected cells into α-sarcoglycan–null immunodeficient mice. We then measured expression of human α-sarcoglycan and showed functional amelioration of some of the motor and force deficits of the α-sarcoglycan–null immunodeficient mice after transplantation with MABs derived from mouse iPSCs.

Fig. 1

iPSC-based cell therapy. (A) Fibroblasts and myoblasts were first isolated from muscle biopsies of LGMD2D patients, and then iPSCs were generated using the reprogramming factors OCT3/4 (O), KLF4 (K), and SOX2 (S) ± cMYC delivered by retroviral vectors. (B) A specific protocol was developed to induce mesodermal commitment of iPSCs and their differentiation into MAB-like cells (HIDEMs). (C) The HIDEMs were transduced with lentiviral vectors carrying a therapeutic gene (to genetically correct the SGCA gene defect) that also carried an inducible version of the myogenic regulator MyoD (MyoD-ER) to enhance their myogenic differentiation. (D) Finally, HIDEMs were transplanted into an immune-deficient mouse model of LGMD2D (Sgca-null/scid/beige). The figure was produced using Servier Medical Art (


LGMD2D patients have reduced numbers of pericytes

To test the therapeutic potential of using human MABs for LGMD2D cell therapy, we first attempted to isolate them from muscle biopsies from LGMD2D patients (table S1). Unfortunately, the isolation of AP+ pericyte-derived MABs was not successful, and the vast majority of cells growing in culture from the biopsy were CD56+ (NCAM1) myoblasts (Fig. 2A), which proliferated very slowly (30). Next, we tried to purify MABs from LGMD2D skeletal muscle cell preparations from biobanks (Fig. 2A and table S1) based on expression of the markers AP and CD56 (AP and CD56 are expressed by 20 to 40% and 3 to 8% of human adult MABs, respectively, in donor samples from healthy individuals after initial expansion; n = ~30 samples analyzed for the preclinical studies of the clinical trial mentioned above). In four LGMD2D patients, the AP+ cells either were greatly reduced in number or could not differentiate into myotubes in vitro (Fig. 2B). Furthermore, the muscle biopsy from patient 5 contained mainly AP and CD56 cells (presumably fibroblasts).

Fig. 2

Reduction of AP+ pericytes in LGMD2D. (A) The histograms show FACS analysis for AP and CD56 staining of six skeletal muscle cell preparations from one healthy donor and five LGMD2D patients (Pt. 1 to 5). The first two histograms are of cells isolated and cultured from muscle biopsies, whereas the remaining four histograms are of cells obtained from tissue banks (see Materials and Methods). (B) The images depict in vitro skeletal muscle differentiation of the samples in (A). MyHC, myosin heavy chain. Scale bar, 80 μm. (C) Hematoxylin and eosin (H&E) staining (pink-purple) combined with AP staining (blue) of skeletal muscle sections from the patients shown in (A) and (B), demonstrating reduced numbers of AP+ pericytes in sections from LGMD2D patients. Black arrows indicate AP staining for the control and LGMD2D patient 1. Scale bar, 100 μm. Images in the lower row contain magnifications of the fields within the white rectangles. (D) Bar graph quantifying the reduction of AP in LGMD2D patients (Pt., black bars) shown in (C) (plus three additional patients) versus AP in matched healthy controls (CT, white bars). ***P < 0.0005, unpaired t test. (E) Histology and quantification of AP+ pericyte reduction in Sgca-null mice (n = 6) compared with matched wild-type (WT) control mice (n = 6) at two different ages (2 and 8 months; right-hand images are magnifications of the fields contained in the white rectangles). Scale bar, 100 μm. ***P < 0.0005, unpaired t test.

To explain this finding, we quantified the number of AP+ pericytes in sections from seven different LGMD2D skeletal muscle biopsies (five of which were obtained from the muscles used to generate the cells described above; see tables S1 and S2). The results showed a strong reduction in the number of AP+ cells in comparison with age-matched healthy controls (54.7%; Fig. 2, C and D), suggesting a possible disease-specific cellular depletion or functional alteration in AP+ pericytes from LGMD2D patients. Notably, a similar reduction in AP+ pericytes was observed in α-sarcoglycan–null (Sgca-null) mice (31) (Fig. 2E).

Generation of MAB-like progenitor cells from human iPSCs

One strategy to overcome the limited availability of MABs from LGMD2D patients is to derive iPSCs from the patients and then generate MABs from the iPSCs in vitro. To prove the feasibility of this strategy, we developed a method that allows easy, robust, and relatively fast derivation (<3 weeks) of mesodermal progenitor cells similar to MABs from healthy donor human iPSCs, which we refer to as HIDEMs (human iPSC-derived MAB-like stem/progenitor cells; detailed in Fig. 3A). This protocol results in a homogeneous population of clonogenic (15.03 ± 7.38% SEM of expandable clones derived from single cells using limiting dilution; n = 6 HIDEM lines) nontumorigenic cells (0 of 27 immunodeficient mice transplanted with these cells developed tumors). This method avoided having to purify [by fluorescence-activated cell sorting (FACS)] progeny from iPSC-derived embryoid bodies.

Fig. 3

Generation and characterization of HIDEMs derived from healthy donors. (A) Scheme of the differentiation protocol from the original fibroblast (or myoblast) donor cells to the generation of HIDEMs. (B and C) Phase-contrast morphology (B) and AP staining (C) of HIDEMs derived from healthy donors 1, 3, and 4 and human adult MABs from a healthy individual at the same passage number (p7 or 8) in culture showing comparable features. Scale bars, 50 μm. (D) Growth curves of two HIDEM lines (one of which was derived from VIF iPSCs) and control human MABs showing comparable proliferation rates. (E) Representative (n = 3) gel containing a ladder of PCR products showing telomerase activity of donor fibroblasts before reprogramming (f), iPSCs (i), and HIDEMs (h) assayed by a telomeric repeat amplification protocol (TRAP). VIF HIDEMs do not have a fibroblast lane because they were purchased as iPSCs. Control primary mouse embryonic fibroblasts (MEFs) and a negative control (CT) are shown. (F) Karyotype analysis showing correct ploidy in HIDEMs, which were generated from iPSCs derived from two representative healthy donors (donor 1: 46,XX; donor 3: 46,XY) after >20 population doublings. (G) Immunofluorescence analysis for the reprogramming factors (SOX2, cMYC, and OCT4) and for Nanog showing their absence in HIDEMs. Scale bar, 30 μm. Insets show positive control cells: iPSC colonies for SOX2, OCT4, and Nanog, and HeLa cells for cMYC. (H) Bar graph depicting a representative example of a quantitative real-time PCR analysis of total and exogenous SOX2, OCT4, and KLF4 transcripts from iPSCs (black bar), immature HIDEMs (red bar), and mature HIDEMs (green bar).

HIDEMs resembled human MABs in morphology, AP expression, and proliferative capacity (Fig. 3, B to E). Karyotype analysis demonstrated correct maintenance of ploidy in the HIDEMs after extensive passaging in culture (>20 population doublings; Fig. 3F). Immunofluorescence and quantitative real-time polymerase chain reaction (PCR) analyses revealed the absence of expression of the reprogramming factors, showing that they had been silenced and therefore could not interfere with differentiation and/or tumorigenesis (Fig. 3, G and H; Supplementary Materials).

Surface marker analysis (Fig. 4A) revealed up-regulation of MAB markers during the derivation process, in particular CD13, CD44, CD49b, and CD146 (a perivascular marker), and down-regulation of pluripotency markers such as SSEA4. HIDEMs, like MABs taken directly from healthy human muscle, are CD56-negative, negative, or weakly positive for endothelial markers such as Flk1, and also show variable positivity for AP (19) after a transient down-regulation during early differentiation (an enzyme assay confirmed AP detection in samples showing a reduced AP signal by FACS analysis; fig. S1).

Fig. 4

Molecular signature and skeletal muscle differentiation of HIDEMs. (A) FACS analysis of undifferentiated iPSCs, partially differentiated (immature) HIDEMs, differentiated (mature) HIDEMs, and control adult human MABs (hMABs) demonstrating down-regulation of pluripotency markers (SSEA4 and AP) and up-regulation of human MAB markers (red trace). (B) Affymetrix GeneChip microarray analysis showing unsupervised hierarchical clustering of HIDEMs, MABs, ESCs, fibroblasts (FIB), endothelial cells (END), mesenchymal stem cells (MSC), smooth muscle (SM) cells, neural progenitor cells (NPC), and iPSCs. Data were meta-analyzed as described in the Supplementary Materials. (C) Coculture assay of green fluorescent protein–positive (GFP+) HIDEMs and C2C12 myoblasts: green fluorescent myotubes are present in vitro after 3 days in differentiation medium (live imaging). Scale bar, 70 μm. (D) Immunofluorescence of the same coculture assay shown in (C) depicting a GFP+ myotube containing three HIDEM nuclei (arrows). Scale bar, 30 μm (see also fig. S1D). The bar graph quantifies the contribution of human nuclei to myotube formation in vitro. (E) Immunofluorescence showing early in vitro myogenic differentiation of HIDEMs 2 days after tamoxifen-induced overexpression of MyoD-ER. Scale bar, 50 μm. (F) Myogenic conversion of two representative HIDEM lines 5 days after tamoxifen administration. Scale bar, 100 μm. (G) RT-PCR analysis of SGCA and myogenic regulatory factor (MYOD and MYOGENIN) transcripts in terminally differentiated MyoD-ER–transduced HIDEMs {an endothelial cell line [human umbilical cord endothelial cell (HUVEC)] was used as a negative control}. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

To compare the molecular phenotype of HIDEMs with that of MABs and other cell types (including iPSCs), we first performed gene expression profiling of HIDEMs (n = 6) and human MABs (n = 3) using an Affymetrix GeneChip, which revealed a marked similarity between the two populations (Fig. 4B). In addition, we downloaded from the Gene Expression Omnibus (GEO) public repository 82 different data sets and performed meta-analysis using hierarchical clustering and principal components analysis (Fig. 4B, fig. S2, and Supplementary Materials). Both analyses revealed that gene expression profiles of HIDEMs are very similar to MABs and show some similarities with mesoderm cells (mesenchymal stem cells, fibroblasts, and smooth muscle and endothelial cells). There was far less correlation between gene expression profiles of HIDEMs and those of neural progenitors, ESCs, and iPSCs.

HIDEMs do not spontaneously differentiate into skeletal myocytes in vitro, but, like embryonic MABs (32), they can be induced to fuse with or differentiate into skeletal myocytes/myotubes by coculture with myoblasts or by expression of the myogenic regulator MyoD, respectively (Fig. 4, C to G). Indeed, upon transduction with a lentiviral vector containing tamoxifen-inducible MyoD (MyoD-ER; Supplementary Materials) (33), HIDEMs undergo marked (that is, >90% of the total cell population) myogenic differentiation (Fig. 4F). Additionally, differentiation toward a more mature vascular lineage could be induced by transforming growth factor–β administration; formation of a vascular-like network was observed spontaneously and upon coculture with human endothelial cells (fig. S1). Together, these results demonstrate generation of a human mesoderm progenitor cell type with MAB characteristics from healthy human iPSCs (see figs. S1 and S2).

Finally, we tested the possibility of deriving HIDEMs from certified vector integration–free (VIF) human iPSCs (see Materials and Methods). We obtained cells with features comparable to those of HIDEMs derived from iPSCs generated with viral vectors, demonstrating that the presence of exogenous factors does not sustain their proliferative capability (Fig. 3, D and E, and fig. S1).

Generating LGMD2D iPSCs and genetically correcting HIDEMs

After validation of the above protocol with healthy human donor iPSCs, fibroblasts or myoblasts obtained from four LGMD2D patients (patients 1 to 4; representative example in Fig. 5A) were reprogrammed using retroviral vectors carrying SOX2, KLF4, and OCT4 ± cMYC complementary DNAs (cDNAs) (see Supplementary Materials). In the absence of cMYC, fewer iPSC colonies were obtained, but these were indistinguishable from those obtained in the presence of cMYC. Colonies of iPSCs started to appear about 30 days after transduction with the viral vectors carrying reprogramming factor cDNAs; the reprogramming efficiency was 0.005% 45 days after transduction with the viral vectors (cells cultured in medium containing valproate and 3 to 5% O2) (34, 35). Clonal lines were established from four different LGMD2D patients, with morphology comparable to that for human ESCs (Fig. 5B). Pluripotency was assessed by AP staining, expression of specific transcription factors, and formation of embryoid bodies and teratomas (Fig. 5B and fig. S3). We detected relatively low levels of KLF4 expression (Fig. 5C), in line with recent reports (36), but this did not affect the pluripotency of our iPSC lines. Derivation and characterization of LGMD2D HIDEMs revealed that karyotype, proliferative capacity, surface marker expression, and myogenic differentiation were comparable with that of HIDEMs derived from healthy control individuals (Fig. 5C and fig. S4). No reactivation of the exogenous transgenes was observed, although endogenous SOX2 expression remained high in iPSCs derived from one patient (expression is shown in fig. S4), but this did not interfere with differentiation, as recently reported (37). No tumors developed in tumorigenic assays (0 of 36 immunodeficient mice transplanted with LGMD2D HIDEMs developed tumors) whether or not cMYC was present in the original reprogramming cocktail.

Fig. 5

Reprogramming LGMD2D cells to iPSCs and derivation of HIDEMs. (A) Representative morphology of a LGMD2D cellular population obtained after culture of a skeletal muscle biopsy. Scale bar, 50 μm. (B) Reprogramming of LGMD2D cells to iPSCs using the factors OCT4, SOX2, and KLF4 ± cMYC (two of four lines were not transduced with cMYC). Upper images show morphology (phase), AP staining (blue), and Nanog expression (green) in LGMD2D iPSCs. White scale bar, 0.9 mm; black scale bar, 0.8 mm. Lower panels show a teratoma formation assay performed with cell colonies depicted in the upper panels (see the Supplementary Materials). Two left panels show the teratoma mass before and after resection from a NOD/scid mouse; below these two images is an H&E-stained section from the resected teratoma, with fields inside the white boxes showing the different tissues (representative of the three germ layers) into which the teratoma can differentiate. Scale bar, 250 μm. (C) LGMD2D HIDEMs. The top two images depict the morphology and AP staining of mature HIDEMs (scale bar, 50 μm); below these are three images showing the correct karyotype in three representative HIDEM populations from patients 1, 2, and 4. The bar graph below the karyotypes shows expression of total and exogenous reprogramming factors (OCT4, SOX2, and KLF4) by LGMD2D iPSCs and the HIDEMs derived from them. The data shown are the average of cells derived from four different patients (data showing values of each patient are available in fig. S3). The curves illustrate proliferation of three different LGMD2D HIDEM lines versus primary human MAB control cells (black line). Histograms show surface markers detected by FACS analysis for HIDEMs derived from patient 1. Bottom panel shows MyoD-ER–mediated conversion to a myogenic fate of three different HIDEM lines (left column) and fusion of a representative population (not transduced with MyoD-ER and marked with GFP) with C2C12 myoblasts (right column) Scale bar, 250 μm. (D) Myogenic differentiation via tamoxifen-induced MyoD-ER nuclear translocation into genetically corrected LGMD2D HIDEMs. Shown is the muscle-specific SGCA lentiviral vector (details in fig. S3C). Immunofluorescence panel shows SGCA expression only in a differentiated myotube (white arrow and inset). Scale bar, 40 μm. Western blot confirms immunofluorescence, demonstrating restoration of SGCA expression in genetically corrected and differentiated HIDEMs.

To genetically correct LGMD2D HIDEMs, we developed a new lentiviral vector carrying the human α-sarcoglycan cDNA (SGCA) under transcriptional control of the muscle-specific myosin light chain 1F promoter and enhancer (Fig. 5D and fig. S4). As shown in Fig. 5D and fig. S4, the transgene is selectively expressed in myotubes generated from genetically corrected LGMD2D HIDEMs previously transduced with the MyoD-ER lentivector (as opposed to surrounding cells that are undifferentiated). These data show that it is possible to reprogram adult somatic cells from LGMD2D patients to pluripotency and to genetically correct MABs derived from LGMD2D iPSCs. They also show that the genetically corrected MABs derived from LGMD2D iPSCs undergo terminal myogenic differentiation with correct and specific expression of the therapeutic transgene (Fig. 5D and fig. S4).

Additionally, we have also generated HIDEMs from iPSCs derived from DMD patients and genetically corrected them with a human artificial chromosome containing the entire dystrophin locus (DYS-HAC; fig. S5) (38). We have recently shown efficacy of combined mouse MAB transplantation and DYS-HAC–mediated genetic correction in mdx mice (24).

Transplantation of iPSC-derived MAB-like cells in Sgca-null/scid/beige mice

There are no large-animal models of LGMD2D, and the only available preclinical model is the Sgca-null mouse (31). To transplant human cells in this model, we crossed the severe combined immunodeficient (scid)/beige mouse with the Sgca-null mouse, generating a new dystrophic and immune-deficient triple mutant: the Sgca-null/scid/beige mouse (fig. S6). Phenotypically, Sgca-null/scid/beige mice showed reduced motility and curving of the vertebral column (kyphosis). Histologically, these mice show an absence of Sgca and typical signs of progressive muscular dystrophy, such as regenerating and necrotic myofibers, inflammatory infiltrates in muscle, fibrosis, and elevated creatine kinase (fig. S6).

MyoD-ER–transduced healthy HIDEMs (106) (n = 2 lines tested) and genetically corrected LGMD2D HIDEMs (n = 2 lines tested) were marked with a lentiviral vector expressing green fluorescent protein (GFP) and were transplanted intramuscularly in the tibialis anterior muscle of juvenile Sgca-null/scid/beige mice (see Supplementary Materials). This resulted in colonization of the transplanted muscle (Fig. 6A), with donor cells observed inside recipient skeletal muscle fibers 7 days after transplantation (Fig. 6B). One month after transplantation, we calculated that the percentage of cells that had engrafted and survived in the host muscle was about 5 to 7% of the total number injected (by counting an average of 35 cells per 7-μm section). This corresponded to 53 ± 14 (SEM) SGCA+ fibers per muscle section (Fig. 6C). Moreover, reconstitution of the dystrophin-associated protein complex was demonstrated by coexpression of β- and γ-sarcoglycans (SGCB and SGCG; Fig. 6C).

Fig. 6

Transplantation of HIDEMs into Sgca-null/scid/beige mice. (A) GFP fluorescence 7 days after intramuscular injection of 106 genetically corrected LGMD2D HIDEMs into the tibialis anterior muscle of Sgca-null/scid/beige mice. Scale bar, 1 mm. (B) (Top) Immunofluorescence staining of a section from the muscle shown in (A) demonstrating engraftment of genetically corrected LGMD2D HIDEMs as revealed by lamin A/C+ nuclei (lamin A/C marks the human nuclear lamina). (Bottom) Magnification of the area inside the white box in top image showing a cluster of myofibers containing donor human cell nuclei. Scale bar, 500 μm. (C) Immunofluorescence showing a cluster of SGCA+ myofibers containing human nuclei 1 month after intramuscular transplantation of genetically corrected LGMD2D HIDEMs (quantified in the bar graph; error bars show SD and the number corresponds to an average of 2% of tibialis anterior myofibers). Scale bar, 60 μm. Bottom images show the same cluster in serial section stained for β- and γ-sarcoglycan (SGCB and SGCG). (D) Intra-arterial transplantation of genetically corrected LGMD2D HIDEMs. Left panels show blood vessel–associated GFP+ cells 6 hours after injection of LGMD2D HIDEMs into the femoral artery. Scale bar, 0.5 mm. Top right and middle panels show immunofluorescence of human cells in-between mouse myofibers (scale bar, 50 μm); lower panel depicts a human fluorescent cell outside CD31+ blood vessels 12 hours after transplantation (scale bar, 90 μm). (E) The bar graph illustrates quantitative real-time PCR analysis of human telomerase DNA to measure engraftment (fold increase) of either HIDEMs or the original cells (before reprogramming) from healthy donors or LGMD2D patients 24 hours after intra-arterial transplantation (injected in the right or in the left femoral artery, respectively; ***P < 0.0005, unpaired t test). (F) Representative example of SGCA+ myofibers containing human nuclei 1 month after intra-arterial transplantation of genetically corrected LGMD2D HIDEMs. Scale bar, 50 μm. (G) RT-PCR confirming SGCA expression 1 month after intramuscular (IM) and intra-arterial (IA) injection. TA, tibialis anterior muscle. GC, gastrocnemius muscle.

Intra-arterial transplantation of genetically corrected LGMD2D patient-derived HIDEMs resulted in colonization of skeletal muscle downstream of the injection site (Fig. 6D), with cells migrating out of the blood vessels within 12 hours after transplantation (Fig. 6D). These data were confirmed by quantitative PCR of DNA performed 24 hours after transplantation, comparing healthy control and LGMD2D HIDEMs (right leg) with the cells from which they were originally derived (fibroblasts or myoblasts that were then reprogrammed to iPSCs; left leg) (Fig. 6E) (Supplementary Materials). All HIDEMs showed greater engraftment compared to the fibroblasts or myoblasts from which they were derived, although we observed variability among different iPSC lines. One month after intra-arterial transplantation, SCGA expression was detected by immunofluorescence and reverse transcription–PCR (RT-PCR) (Fig. 6, F and G).

We then investigated the possibility of enhancing HIDEM engraftment by transplanting mouse cells instead of human. We generated and transplanted murine iPSC-derived MABs (MIDEMs; n = 18; fig. S7) and detected five to sixfold more SGCA+ myofibers in mouse muscle compared to transplantation with HIDEMs (SGCA+ myofibers: 286 ± 41 versus 53 ± 14, mean ± SEM; Fig. 7, A and B). We saw a concomitant reduction in fibrotic-adipose tissue in transplanted muscle (26.24% less than nontransplanted muscle, P < 0.05, n = 6; Fig. 7C). These data suggest that species-specific variables, other than the adaptive immune system, control donor cell engraftment. As a consequence of this enhanced engraftment, we detected functional amelioration of motor capacity using a treadmill test to exhaustion (24) in animals transplanted with MIDEMs. Intramuscularly and intra-arterially transplanted mice showed enhanced motor capacity after treatment, running from 48 to 62% more than their baseline performance and from 12 to 22% more than untreated animals 35 days after transplantation (P < 0.05 and P < 0.005, respectively; Fig. 7D). To validate these findings, we measured the tetanic force of the tibialis anterior muscle and force of contraction on isolated muscle fibers 4 months after transplantation. Figure 7E shows that in tibialis anterior muscles from mice transplanted intramuscularly and intra-arterially, the tetanic force was significantly higher than in untreated mice (67% and 83%, respectively; P < 0.05). Individual muscle fibers (n = 119) were then dissected from the gastrocnemius muscle of the same mice, and the analysis demonstrated that GFP+ myofibers developed greater force than did muscle fibers from untreated mice (Fig. 7E).

Fig. 7

Transplantation of murine iPSC-derived MABs into Sgca-null/scid/beige mice. (A) Stereoscopic pictures of GFP+ myofibers 1 month after transplantation of murine iPSC-derived MAB-like cells (MIDEMs). Scale bar, 1 mm. (B) Transverse sections from the muscle shown in (A), showing large areas of GFP- and Sgca-positive myofibers. (C) Quantification of fibrosis in transplanted versus control mice showing a reduction in fibrosis in transplanted mice (n = 3). *P < 0.05, Student’s t test. The two images show representative Masson trichrome staining of tibialis anterior muscles from transplanted and control Sgca-null/scid/beige mice (blue, fibrotic infiltrate). Scale bar, 250 μm. (D) Time to exhaustion on a treadmill test for transplanted Sgca-null/scid/beige mice (n = 13; 106 cells injected bilaterally in tibialis anterior, gastrocnemius, and quadriceps muscles) versus nontransplanted dystrophic (n = 8) and nondystrophic (n = 5) control mice. The data show functional amelioration of dystrophic muscle in mice transplanted with MIDEMs (12 to 22% more than nontransplanted animals 35 days after transplantation). Note that data are presented as average motor capacity relative to baseline performances measured until the day before transplantation. *P < 0.05; **P < 0.005, one-way ANOVA. (E) Force measurements 4 months after MIDEM transplantation. (Left graph) Normalized tetanic force of isolated tibialis anterior muscles from intramuscular and intra-arterially transplanted mice together with control nontransplanted dystrophic and nondystrophic mice (n ≥ 3 per group). (Right graph) Mean values of specific force for a population of single myofibers dissected from transplanted and nontransplanted gastrocnemius muscles (together with the controls; n values above columns). The arrow indicates a representative picture of a GFP+ myofiber analyzed in the assay. Scale bar, 60 μm. Error bars represent means ± SD. *P < 0.05; ***P < 0.0005, one-way ANOVA and Student-Newman-Keuls test. ns, not significant. (F) Cryosection of MIDEM-transplanted tibialis anterior muscle stained for CD31 (Pecam; brown, immunohistochemistry; to mark blood vessels) and AP (blue, enzymatic reaction). A serial section shows the presence of GFP+ myofibers and interstitial cells, some of which colocalize with the vessels marked as described above. Scale bar, 80 μm. The bar graph quantifies the total number of AP+ cells per section of tibialis anterior muscle of 8-month-old Sgca-null/scid/beige mice after IM transplantation with MIDEMs. Error bars represent means ± SEM. *P < 0.05; **P < 0.005, one-way ANOVA and Tukey’s test.

Finally, to determine whether the transplanted cells were able to contribute to the pool of AP+ pericytes in vivo, we searched for AP+ and GFP+ MIDEMs in the skeletal muscle interstitium of Sgca-null/scid/beige mice. As shown in Fig. 7F, double-positive donor cells were clearly identifiable near GFP+ myofibers, indicating donor cell contribution to muscle regeneration together with replenishment of the pericyte niche in vivo. Notably, the number of AP+ cells per tibialis anterior section was higher than that observed in untreated Sgca-null/scid/beige mice (535.5 ± 39.56 versus 344 ± 36.8, mean ± SEM; n = 6; P < 0.05) and was closer to the number of AP+ cells in wild-type animals (666.5 ± 47.6; n = 3; Fig. 7F).


Previous studies from our laboratory demonstrated rescue of dystrophic Sgca-null mice by intra-arterial transplantation of murine MABs (27). We then decided to apply this strategy using human genetically corrected MABs from LGMD2D patients. However, we found that LGMD2D patients have a reduced number of AP+ pericytes in vivo, and thus, obtaining pericyte-derived MABs directly from patients for genetic correction in vitro and autologous transplantation was not possible. To overcome this problem, we developed a strategy that allowed the derivation and propagation in culture of a population of MAB-like mesodermal progenitor cells derived from human iPSCs (HIDEMs) generated from adult somatic cells. The reproducibility of this protocol was validated using 10 different human iPSC lines generated in four different laboratories using different approaches. Notably, potential sources of variation among different HIDEM lines (for example, age and sex of donors and residual expression of reprogramming factors) did not correlate with reprogramming or differentiation efficiency (37). HIDEMs were also derived from iPSCs generated with three reprogramming factors (without cMYC) and from VIF iPSCs. We succeeded in deriving iPSC lines from four LGMD2D patients. We derived iPSCs from myoblasts from patient 1 that were similar to those derived from fibroblasts from healthy and dystrophic individuals. HIDEMs derived from LGMD2D patients were easily transduced with lentiviral vectors, resulting in a cell population that could be genetically corrected and expanded and that was clonogenic, nontumorigenic, and readily transplantable.

To test the therapeutic potential of genetically corrected HIDEMs for future transplantation into LGMD2D patients, we generated a new dystrophic and immune-deficient mouse: the Sgca-null/scid/beige mouse. Intramuscular or intra-arterial injection of genetically corrected HIDEMs resulted in their engraftment in dystrophic skeletal muscle and production of clusters of SGCA+ myofibers. Variable levels of engraftment of human cells in mouse dystrophic muscle were observed, possibly due to different levels of inflammation and sclerosis in the mouse recipients and to different expression levels of adhesion proteins (for example, integrins and selectins) by HIDEMs from different human subjects. The HIDEMs we isolated did not give rise to tumors upon subcutaneous, intramuscular, and intra-arterial transplantation into immune-deficient mice.

Recently, other laboratories have reported the derivation of myogenic progenitors from human iPSCs [for example, (14)]. These progenitors differentiate robustly in vivo and may be the choice for treating localized forms of muscle disorders where intramuscular transplantation into several sites of the few affected muscles is sufficient. The advantage of HIDEMs is that they can be delivered through the arterial circulation and thus are able to reach muscles throughout the body. However, more work is needed to assess the safety and to improve engraftment upon systemic delivery of genetically engineered HIDEMs before they enter clinical testing.

Recent adeno-associated virus–based gene therapy trials have shown promise for treating LGMD2D (39). Nevertheless, immunity and the loss of transgene expression are still hurdles that need to be overcome (40, 41). Similarly, an immune response also might be elicited by transplanted allogeneic HIDEMs or MABs, although this is still being investigated. The limited availability of adult tissue-specific muscle progenitor cells is a major obstacle for cell therapies. Reprogramming of adult somatic cells to form iPSCs followed by lineage-specific commitment and differentiation may solve the problem of the limited supply of muscle progenitor cells.

Deriving patient-specific iPSCs and expanding their differentiated progeny may provide a useful strategy for gene and cell therapies. Although very preliminary, our study suggests that genetically corrected MABs generated from iPSCs derived from the fibroblasts or myoblasts of LGMD2D patients could be useful for autologous transplantation and that this approach might also be applicable for treating other recessive muscular dystrophies.

Materials and Methods

Cell cultures

Human MABs and HIDEMs were cultured in MegaCell DMEM (Dulbecco’s modified Eagle’s medium) (Sigma) as described (42). Alternatively, the same cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Sigma) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, human basic fibroblast growth factor (5 ng/ml), penicillin (100 IU/ml), streptomycin (100 mg/ml), 0.5 μM oleic and linoleic acids (Sigma), 1.5 μM Fe++ [Iron(II) chloride tetrahydrate, Sigma; or Fer-In-Sol, Mead Johnson], 0.12 μM Fe+++ [Iron(III) nitrate nonahydrate, Sigma; or Ferlixit, Aventis], and 1% insulin/transferrin/selenium (Gibco).

iPSCs were cultured as described (1, 2, 43). VIF human iPSCs (Gibco) were a certified zero-footprint line generated from cord blood–derived CD34+ progenitors with a three-plasmid and seven-factor Epstein-Barr virus nuclear antigen (EBNA)–based episomal system. The other healthy donor iPSC lines used in this study have been described in (43). The murine iPSCs used here were characterized and cultured as previously described (38). Additional details are available in the Supplementary Materials.

LGMD2D samples

LGMD2D skeletal muscle cells and biopsies were provided by the biobanks of M. Moggio (Telethon Genetic BioBank Network; Ospedale Maggiore Policlinico, Milan, Italy), M. Mora (Telethon Genetic BioBank Network; Istituto Neurologico Carlo Besta, Milan, Italy), and B. Schoser and P. Schneiderat [Munich Tissue Culture Collection (MTCC), Friedrich-Baur Institute, Munich, Germany]. We are also grateful to J. Diaz-Manera (Hospital Santa Creu i Sant Pau, Barcelona, Spain) and S. Previtali (San Raffaele Scientific Institute, Milan, Italy) for providing LGMD2D slides. See table S1 for additional details.

Viral vectors and reprogramming to iPSCs

Generation of iPSCs from human cells was done with a standard retrovirus-based system previously published (2). MyoD-ER construct was provided by J. S. Chamberlain (University of Washington, Seattle, WA) and used as previously described (33). Human muscle-specific SGCA lentivector (pLentiMLC1F/SGCA) construction and more details are available in the Supplementary Materials.

Generation of iPSC-derived MAB-like cells

Substantial modification of the available protocols to generate vascular cells from ESCs [for example, in (44)] facilitated the initial setup of this method. The main steps of the protocol for HIDEM derivation are summarized here:

1. Dissociation of iPSCs colonies to single-cell suspension (week 1):

a. 10 μM ROCK inhibitor for 1 hour in iPSC medium (see above).

b. 30 to 120 min at 37°C and 5% CO2 in dissociation medium [0.5 mM EDTA, 0.1 mM β-mercaptoethanol, 3% FBS in phosphate-buffered saline (PBS) without Ca2+ and Mg2+].

c. Gently shake dishes every 15 min to dissociate colonies.

d. Collect and gently resuspend cells with a P1000 tip to favor dissociation.

2. Seed 6 × 104/cm2 cells obtained in step 1 on a Matrigel (growth factor–reduced)-coated dish (about 6 × 105 cells/3.5-cm dish; week 1) in α-MEM (Gibco) containing antibiotics (penicillin/streptomycin), 10% FBS, nucleotides, and 0.2% β-mercaptoethanol for 1 week at 37°C, 5% CO2, and 3 to 5% O2.

3. Dissociate culture (as described in step 1), gently scrape dish surface with a cell scraper, filter solution using a 40-μm strainer, and seed 2.5 × 104 cells/cm2 with medium and conditions as in step 2 (week 2).

4. If human MAB-like cells are present [see Fig. 3B and (42)], wait up to 10 days from step 3, trypsinize cells (5 min at 37°C, 5% CO2, and 3 to 5% O2), and seed them on a Matrigel-coated dish at about 80% confluency in human MAB complete medium (either MegaCell DMEM or IMDM base, see above; week 3).

5. Split cells (with trypsin from now on) when they reach 100% confluency to have again a culture at 80% confluency, from now on plastic and in human MAB medium (weeks 3 to 4).

From now on, culture HIDEMs exactly like human MABs, as described above and detailed in (42). Transduce the cells with lentiviral MyoD-ER (with a maximum multiplicity of infection of 5) and administer 4OH- or standard tamoxifen to obtain robust myogenic differentiation.

Differentiation of murine iPSCs to MIDEMs was done following the above protocol. The main difference with HIDEM generation protocol was the introduction of a purification step after point no. 5 (see above): Cells were indeed negatively FACS-sorted for SSEA1 (see below) to remove residual pluripotent cells.

Proliferation and differentiation assays

Growth curves and telomeric repeat amplification protocol (TRAP) have been performed as recently described (24), as skeletal and smooth muscle differentiation (32, 42). Details for embryoid body formation and differentiation are available in the Supplementary Materials.

Surface marker analysis and gene expression profiling

A detailed report of the procedures, antibodies, and meta-analysis is available in the Supplementary Materials. Raw data of HIDEM and control human MAB gene expression profiling are available in the GEO repository.


Scid, scid/beige, nonobese diabetic (NOD)/scid, NOD/scid/γ chain knockout (NSG), and nude mice were purchased from Charles River Laboratories and were housed in San Raffaele Scientific Institute animal house together with Sgca-null/scid/beige. All mice were kept in specific pathogen–free conditions, and all procedures involving living animals conformed to Italian law (D.L.vo 116/92 and subsequent additions) and were approved by the San Raffaele Institutional Review Board.

Generation of Sgca-null/scid/beige mouse is detailed in the Supplementary Materials. Briefly, females homozygous for Sgca mutation (Sgca−/−) were bred with homozygous scid/beige−/− males. The resulting F1 heterozygous females were crossed with scid/beige−/− males. In F2 mice (and in subsequent generations), we verified Sgca and scid mutation (beige mutation was genotyped by Charles River Laboratories), leucopenia, and the absence of B and T lymphocytes. Then, we isolated Sgca+/−/scid/beige−/− females and crossed them with scid/beige−/− males for three generations. In F5, Sgca−/−scid/beige−/− males and females were bred together to generate mice homozygous for both scid/beige and Sgca mutations. Sgca+/+ and Sgca−/− immunocompetent mice, as well as Sgca+/+ immune-deficient matched controls, were also maintained in the colony. Animals of all genotypes presented an average of 68.7 ± 2.2% (SD; n = 13) of CB17 background according to single-nucleotide polymorphism analysis (Mouse 348 SNP panel, Charles River Laboratories).

Transplantation, tumorigenic, and teratoma formation assays

Intramuscular (n = 25 Sgca-null/scid/beige mice) and intra-arterial (n = 15 Sgca-null/scid/beige mice) injections were done as previously described (24). When MyoD-ER–expressing cells were transplanted, tamoxifen (33 μg/g) was given once a day (intraperitoneally or subcutaneously) for a total of 7 days starting from 1 day before transplantation. Further information regarding cell transplantation, together with a detailed description of tumorigenic and teratoma formation assay, is available in the Supplementary Materials.

PCR and immunoblotting

Genotyping PCR for Sgca and scid mutations was done as already described (24, 31). Genotyping PCR for the beige (Lystbg) mutation was performed by Charles River Laboratories. Primers, quantitative real-time PCRs, and Western blot are detailed in the Supplementary Materials.

Histology, histochemistry, immunofluorescence, and karyotype analysis

Tissue sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich) and Masson trichrome (Bio-Optica) following protocol provided by the manufacturers. AP was detected as already described (19) or with the protocol available with the PermaBlue/AP kit (Histo-line laboratories). Immunofluorescence is detailed in the Supplementary Materials. Karyotype analyses were performed and certified by Synlab Diagnostic Services Srl (Italy) with QFQ staining (n = 50 metaphases per sample).

Functional measurements: Motor capacity and force of contraction

Control untransplanted (vehicle: PBS) Sgca+/+/scid/beige (n = 5 for intramuscular; n = 8 for intra-arterial), untransplanted (vehicle) Sgca-null/scid/beige (n = 8 for intramuscular; n = 8 for intra-arterial), and transplanted Sgca-null/scid/beige (n = 8 for intramuscular; n = 5 for intra-arterial) were tested for functional recovery on a treadmill (Columbus Instruments), as recently reported (24). Mechanics of isolated muscles and single-fiber analysis were performed as previously described (24), and details are available in the Supplementary Materials.

Statistical analysis

We expressed values as means ± SEM or SD. We assessed significance of the differences between means by Student’s t test, and when more than two groups had to be compared, we used one-way analysis of variance (ANOVA) followed by Tukey’s or Student-Newman-Keuls post test to determine which groups were statistically significantly different from the others. A probability of less than 5% (P < 0.05) was considered to be statistically significant. Data were analyzed with Microsoft Excel 14.1.3 and GraphPad Prism 5.

Supplementary Materials

Materials and Methods

Fig. S1. Additional characterization of HIDEMs derived from healthy donors.

Fig. S2. Gene expression profiling of HIDEMs.

Fig. S3. Additional characterization of iPSCs derived from LGMD2D patients.

Fig. S4. Additional characterization of HIDEMs derived from LGMD2D patients.

Fig. S5. Generation and characterization of HIDEMs from DMD and DMD(DYS-HAC) iPSCs.

Fig. S6. Generation and characterization of Sgca-null/scid/beige mouse.

Fig. S7. Derivation of mesoangioblast-like cells from murine iPSCs (MIDEMs).

Table S1. Characteristics of LGMD2D patients.

Table S2. Characteristics of healthy controls.


References and Notes

  1. Acknowledgments: We thank S. Previtali, J. Diaz-Manera, the Telethon Network of Genetic Biobanks, and the MTCC for providing samples; M. Noviello, D. Moi, A. Lombardo, and D. Becker for help and reagents; D. Sassoon, L. Wrabetz, and S. Maffioletti for helpful discussions; K. English for critical reading of the manuscript; and J. Chamberlain for providing the MyoD-ER lentiviral vector. Funding: This work was supported by European Research Council, European Community 7th Framework project OPTISTEM (contract number Health-F5-2009-223098), Duchenne Parent Project Italy, Telethon Network of Genetic Biobanks (GTB07001F; to M.M.), and Cariplo Foundation (to R.B.). Author contributions: F.S.T. wrote the manuscript and conceived and carried out most of the experimental work and analysis with the help of M.F.M.G.; L.P., S.B., F.U., M.C., S.A., R.T., M.R., G. Calderazzi, H.H., and O.C. performed in vitro and in vivo experiments and interpreted data; E.T. and V.A. performed microarray experiments; E.L. and R.B. performed muscle physiology assays; M.M., P.S., B.S., M.O., M.S., Y.T., and V.B. provided samples and discussed results; G. Cossu coordinated the project and wrote the manuscript with F.S.T. Competing interests: F.S.T. and G.C. have filed a U.S. provisional patent application 61/588,269 detailing the strategy described in this article, “Re-establishment and genetic correction of progenitors from limb-girdle muscular dystrophy via reprogramming of autologous cells.” Data and materials availability: The microarray data have been deposited in the National Center for Biotechnology Information GEO (GSE36098).
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