transplantation of genetically corrected human ipsc ... fs, sci transl med. 2012 .pdf · francesco...

DOI: 10.1126/scitranslmed.3003541, 140ra89 (2012);4 Sci Transl Med

et al.Francesco Saverio TedescoProgenitors in Mice with Limb-Girdle Muscular DystrophyTransplantation of Genetically Corrected Human iPSC-Derived

Editor's Summary

dystrophy.cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscularThis strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells.mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength

-sarcoglycan. Using mouse iPSC-derivedαskeletal muscle, engrafted, and formed muscle fibers expressing mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse -sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived

α, which encodes SGCAwere then genetically corrected in vitro using a viral vector expressing the defective gene human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells thatTo overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain

cells.mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number ofmesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous

explore whether genetically correctedet al.therapy in animal models of muscular dystrophy. In a new study, Tedesco corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a celltransplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers bymobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several

Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired

Muscle Progenitors Find Their Way Home

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R E S EARCH ART I C L E

MUSCULAR DYSTROPHY

Transplantation of Genetically Corrected HumaniPSC-Derived Progenitors in Mice withLimb-Girdle Muscular DystrophyFrancesco Saverio Tedesco,1,2,3* Mattia F. M. Gerli,1,2 Laura Perani,2† Sara Benedetti,1,2†

Federica Ungaro,4† Marco Cassano,5†‡ Stefania Antonini,1,2,6† Enrico Tagliafico,7,8

Valentina Artusi,7,8 Emanuela Longa,9,10 Rossana Tonlorenzi,2 Martina Ragazzi,1,2

Giorgia Calderazzi,2,6 Hidetoshi Hoshiya,1,2 Ornella Cappellari,1,2 Marina Mora,11

Benedikt Schoser,12 Peter Schneiderat,12 Mitsuo Oshimura,13 Roberto Bottinelli,9,10

Maurilio Sampaolesi,5,14 Yvan Torrente,15 Vania Broccoli,4 Giulio Cossu1,2,6*

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

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INTRODUCTION
Induced pluripotent stem cells (iPSCs) are the product of reprogram-ming adult somatic cells to an embryonic stem cell (ESC)–like stateusing specific transcription factors (1, 2). They show extensive self-renewal and generate differentiated progeny representing all three germlayers. Deriving patient-specific iPSCs to study diseases in vitro is al-

1Department of Cell and Developmental Biology and Centre for Stem Cells andRegenerative Medicine, University College London, WC1E 6DE London, UK. 2Division ofRegenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute,20132 Milan, Italy. 3University College London Hospitals NHS Foundation Trust, UCH, NW12BU London, UK. 4Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan,Italy. 5Stem Cell Interdepartmental Institute, KU Leuven, 3000 Leuven, Belgium. 6Depart-ment of Biology, University of Milan, 20133 Milan, Italy. 7Department of BiomedicalSciences, University of Modena and Reggio Emilia, 41125 Modena, Italy. 8Center forGenome Research, University of Modena and Reggio Emilia, 41125 Modena, Italy.9Department of Molecular Medicine and Interuniversity Institute of Myology, University ofPavia, 27100 Pavia, Italy. 10Fondazione Salvatore Maugeri (IRCCS), Scientific Institute ofPavia, 27100 Pavia, Italy. 11National Neurological Institute “C. Besta,” 20126 Milan, Italy.12Department of Neurology, Ludwig-Maximilians-University, 81377 Munich, Germany.13Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction,Tottori University, Yonago 683-8503, Japan. 14Human Anatomy, University of Pavia, 27100Pavia, Italy. 15Department of Neurological Science, University of Milan, Fondazione IRCCSPoliclinico Mangiagalli-Regina Elena, 20122 Milan, Italy.*To whom correspondence should be addressed. E-mail: [email protected] (F.S.T.);[email protected] (G.C.)†These authors contributed equally to this work.‡Present address: School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne,CH-1015 Lausanne, Switzerland.

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ready under way (3). Genetic correction of patient-specific iPSCs forautologous cell transplantation may be a promising strategy for treatinga variety of diseases including the muscular dystrophies (4). A criticalstep in designing iPSC-based protocols for treating skeletal muscle dis-orders is the development of techniques for inducing iPSCs to becommitted to a muscle-specific progenitor cell fate. Recent studies havedescribed the generation of satellite cells (the main progenitor cellsresident in skeletal muscle that are responsible for muscle regenera-tion) and their in vitro–activated progeny (myoblasts) from murineiPSCs and from murine and human ESCs (5–7). However, these pro-genitor cells have the same limitations as satellite cells in adult musclefor the purposes of cell therapy; that is, they cannot be delivered to mus-cle systemically and, in addition, they have poor survival and limitedmigration capabilities (8). Other mesoderm cell types have been shownto 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 (8–14). Moreover,mesenchymal stem cells and vasculogenic pericytes derived from hu-man ESCs and iPSCs have been shown to ameliorate limb ischemiaafter transplantation into mice with a ligated femoral artery (15, 16).

Human pericytes have been shown to contribute to regeneration ofmesodermal tissues (17, 18). Mesoangioblasts (MABs), which arederived from alkaline phosphatase–positive (AP+) human skeletal musclepericytes, are a valuable cell population because, when they are deliveredsystemically in the arterial circulation they colonize and contribute to

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muscle regeneration of the dystrophic muscle (19). Moreover, lineage-tracing experiments in the mouse demonstrated that they naturallycontribute to skeletal muscle growth and regeneration (20). However,human MABs have a limited life span, and the need to obtain billionsof cells to treat all of the skeletal muscles of patients with muscular dys-trophy challenges the proliferative capabilities of these cells. The possi-bility of deriving MABs from iPSCs offers the advantage of producingunlimited numbers of myogenic progenitor cells that can be deliveredsystemically.

On the basis of evidence of safety and efficacy in mouse models oflimb-girdle muscular dystrophy 2D (LGMD2D) and Duchenne’s mus-cular dystrophy (DMD) (a-sarcoglycan–null mice and mdx mice, re-spectively) and a dog model of DMD (19, 21–27), allogeneic humanleukocyte antigen (HLA)–matched MABs have been expanded underclinical-grade conditions and are currently being transplanted into DMDpatients 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), weset out to isolate human MABs from several patients but invariablyfailed to derive and/or expand cell populations with a MAB phenotype.Further analysis showed that these patients have a reduced number ofAP+ pericytes. To overcome this problem, we developed a new protocolto derive MAB-like cells initially from iPSCs derived from healthy pa-tients and subsequently from iPSCs derived from myoblasts and fibro-blasts from LGMD2D patients. We derived iPSCs from the skeletalmuscle cells of LGMD2D patients, generated MABs from these iPSCs,and expanded theMABs in vitro (Fig. 1).We then transduced theMABswith a lentiviral vector carrying the wild-type human a-sarcoglycan gene(SGCA) and transplanted the corrected cells into a-sarcoglycan–nullimmunodeficient mice. We then measured expression of humana-sarcoglycan and showed functional amelioration of some of themotor and force deficits of the a-sarcoglycan–null immunodeficientmice after transplantation with MABs derived from mouse iPSCs.

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RESULTS

LGMD2D patients have reduced numbers of pericytesTo test the therapeutic potential of using human MABs for LGMD2Dcell therapy, we first attempted to isolate them from muscle biopsiesfrom LGMD2D patients (table S1). Unfortunately, the isolation of AP+

pericyte-derivedMABswas not successful, and the vast majority of cellsgrowing in culture from the biopsy were CD56+ (NCAM1) myoblasts(Fig. 2A), which proliferated very slowly (30). Next, we tried to purifyMABs from LGMD2D skeletal muscle cell preparations from biobanks(Fig. 2A and table S1) based on expression of themarkers AP andCD56(AP and CD56 are expressed by 20 to 40% and 3 to 8% of human adultMABs, respectively, in donor samples from healthy individuals after ini-tial expansion; n =∼30 samples analyzed for the preclinical studies of theclinical trial mentioned above). In four LGMD2D patients, the AP+ cellseitherwere greatly reduced innumber or couldnot differentiate intomyo-tubes in vitro (Fig. 2B). Furthermore, the muscle biopsy from patient 5contained mainly AP− and CD56− cells (presumably fibroblasts).

To explain this finding, we quantified the number of AP+ pericytesin sections from seven different LGMD2D skeletal muscle biopsies (fiveof which were obtained from the muscles used to generate the cells de-scribed above; see tables S1 and S2). The results showed a strong reduc-tion in the number of AP+ cells in comparisonwith age-matched healthycontrols (54.7%; Fig. 2, C and D), suggesting a possible disease-specificcellular depletionor functional alteration inAP+pericytes fromLGMD2Dpatients. Notably, a similar reduction in AP+ pericytes was observed ina-sarcoglycan–null (Sgca-null) mice (31) (Fig. 2E).

Generation of MAB-like progenitor cells from human iPSCsOne strategy to overcome the limited availability of MABs fromLGMD2D patients is to derive iPSCs from the patients and then gen-erate MABs from the iPSCs in vitro. To prove the feasibility of thisstrategy, we developed a method that allows easy, robust, and relatively

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fast derivation (<3 weeks) of mesodermalprogenitor cells similar to MABs fromhealthydonorhuman iPSCs,whichwe referto as HIDEMs (human iPSC-derivedMAB-like stem/progenitor cells; detailed inFig. 3A). This protocol results in a homo-geneous population of clonogenic (15.03 ±7.38% SEM of expandable clones derivedfrom single cells using limiting dilution;n = 6 HIDEM lines) nontumorigenic cells(0 of 27 immunodeficientmice transplantedwiththesecellsdevelopedtumors).Thismeth-odavoidedhaving topurify [by fluorescence-activated cell sorting (FACS)] progeny fromiPSC-derived embryoid bodies.

HIDEMs resembled human MABs inmorphology, AP expression, and prolifer-ative capacity (Fig. 3, B to E). Karyotypeanalysis demonstrated correct maintenanceof ploidy in the HIDEMs after extensivepassaging in culture (>20 population dou-blings; Fig. 3F). Immunofluorescence andquantitative real-time polymerase chain re-action (PCR) analyses revealed the absenceof expression of the reprogramming factors,

Fig. 1. iPSC-based cell therapy. (A) Fibroblasts and myoblasts were first isolated from muscle biopsiesof 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 toinduce 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 correctthe 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-deficientmouse model of LGMD2D (Sgca-null/scid/beige). The figure was produced using Servier Medical Art(http://www.servier.com).
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Fig. 2. Reduction of AP+ pericytes in LGMD2D. (A) The histograms show FACSanalysis for AP and CD56 staining of six skeletal muscle cell preparations fromone healthy donor and five LGMD2D patients (Pt. 1 to 5). The first two histo-grams are of cells isolated and cultured from muscle biopsies, whereas theremaining four histograms are of cells obtained from tissue banks (seeMaterials and Methods). (B) The images depict in vitro skeletal muscle differ-entiation of the samples in (A). MyHC, myosin heavy chain. Scale bar, 80 mm.(C) Hematoxylin and eosin (H&E) staining (pink-purple) combined with APstaining (blue) of skeletal muscle sections from the patients shown in (A)and (B), demonstrating reduced numbers of AP+ pericytes in sections fromLGMD2D patients. Black arrows indicate AP staining for the control andLGMD2D patient 1. Scale bar, 100 mm. Images in the lower row contain mag-nifications of the fields within the white rectangles. (D) Bar graph quantifyingthe reduction of AP in LGMD2D patients (Pt., black bars) shown in (C) (plus threeadditional 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) controlmice (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 mm.***P < 0.0005, unpaired t test.
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showing that they had been silenced and therefore could not interferewith differentiation and/or tumorigenesis (Fig. 3, G andH; Supplemen-tary Materials).

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Surface marker analysis (Fig. 4A) revealed up-regulation of MABmarkers during the derivation process, in particular CD13, CD44,CD49b, and CD146 (a perivascular marker), and down-regulation

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Fig. 3. Generation and characterization of HIDEMs derived from healthydonors. (A) Scheme of the differentiation protocol from the original fibro-

cause they were purchased as iPSCs. Control primary mouse embryonicfibroblasts (MEFs) and a negative control (CT) are shown. (F) Karyotype

blast (or myoblast) donor cells to the generation of HIDEMs. (B and C)Phase-contrast morphology (B) and AP staining (C) of HIDEMs derived fromhealthy donors 1, 3, and 4 and human adult MABs from a healthy individualat the same passage number (p7 or 8) in culture showing comparable fea-tures. Scale bars, 50 mm. (D) Growth curves of two HIDEM lines (one of whichwas derived from VIF iPSCs) and control human MABs showing comparableproliferation rates. (E) Representative (n = 3) gel containing a ladder ofPCR products showing telomerase activity of donor fibroblasts before re-programming (f), iPSCs (i), and HIDEMs (h) assayed by a telomeric repeatamplification protocol (TRAP). VIF HIDEMs do not have a fibroblast lane be-

analysis showing correct ploidy in HIDEMs, which were generated fromiPSCs derived from two representative healthy donors (donor 1: 46,XX;donor 3: 46,XY) after >20 population doublings. (G) Immunofluorescenceanalysis for the reprogramming factors (SOX2, cMYC, and OCT4) and forNanog showing their absence in HIDEMs. Scale bar, 30 mm. Insets show pos-itive control cells: iPSC colonies for SOX2, OCT4, and Nanog, and HeLa cellsfor cMYC. (H) Bar graph depicting a representative example of a quantita-tive real-time PCR analysis of total and exogenous SOX2, OCT4, and KLF4transcripts from iPSCs (black bar), immature HIDEMs (red bar), and matureHIDEMs (green bar).




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Fig. 4. Molecular signa-ture and skeletal muscledifferentiation of HIDEMs.(A) FACS analysis of undif-ferentiated iPSCs, partiallydifferentiated (immature)HIDEMs, differentiated(mature) HIDEMs, and con-trol adult human MABs(hMABs) demonstratingdown-regulation of pluri-potency markers (SSEA4and AP) and up-regulationof human MAB markers(red trace). (B) AffymetrixGeneChip microarray anal-ysis showing unsupervisedhierarchical clustering ofHIDEMs, MABs, ESCs, fibro-blasts (FIB), endothelialcells (END), mesenchymalstem cells (MSC), smoothmuscle (SM) cells, neuralprogenitor cells (NPC), andiPSCs. Data were meta-analyzed as described inthe Supplementary Mate-rials. (C) Coculture assay ofgreen fluorescent protein–positive (GFP+) HIDEMs andC2C12 myoblasts: greenfluorescent myotubes arepresent in vitro after 3 daysin differentiation medium(live imaging). Scale bar,70 mm. (D) Immunofluo-rescence of the same co-culture assay shown in (C)depicting a GFP+ myotubecontaining three HIDEMnuclei (arrows). Scale bar,30 mm (see also fig. S1D).The bar graph quantifiesthe contribution of humannuclei to myotube forma-tion in vitro. (E) Immuno-fluorescence showingearly in vitro myogenicdifferentiation of HIDEMs2 days after tamoxifen-induced overexpression ofMyoD-ER. Scale bar, 50mm. (F) Myogenic conver-sion of two representativeHIDEM lines 5 days after ta-moxifen administration.Scale bar, 100 mm. (G)RT-PCR analysis of SGCA andmyogenic regulatory factor(MYODandMYOGENIN) tran-scripts in terminally differ-
entiated 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.
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of pluripotency markers such as SSEA4. HIDEMs, like MABs takendirectly from healthy human muscle, are CD56-negative, negative, orweakly positive for endothelial markers such as Flk1, and also showvariable positivity for AP (19) after a transient down-regulation duringearly differentiation (an enzyme assay confirmed AP detection in sam-ples showing a reduced AP signal by FACS analysis; fig. S1).

To compare themolecular phenotype ofHIDEMswith that ofMABsand other cell types (including iPSCs), we first performed gene expres-sion profiling of HIDEMs (n = 6) and human MABs (n = 3) using anAffymetrix GeneChip, which revealed a marked similarity between thetwo populations (Fig. 4B). In addition, we downloaded from the GeneExpression Omnibus (GEO) public repository 82 different data sets andperformed meta-analysis using hierarchical clustering and principalcomponents analysis (Fig. 4B, fig. S2, and Supplementary Materials).Both analyses revealed that gene expression profiles of HIDEMs arevery similar to MABs and show some similarities with mesoderm cells(mesenchymal stem cells, fibroblasts, and smoothmuscle and endothe-lial cells). Therewas far less correlation between gene expression profilesof HIDEMs and those of neural progenitors, ESCs, and iPSCs.

HIDEMs do not spontaneously differentiate into skeletal myo-cytes in vitro, but, like embryonic MABs (32), they can be inducedto fuse with or differentiate into skeletal myocytes/myotubes by co-culture with myoblasts or by expression of the myogenic regulatorMyoD, respectively (Fig. 4, C to G). Indeed, upon transduction with alentiviral 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 lineagecould be induced by transforming growth factor–b administration;formation of a vascular-like network was observed spontaneouslyand upon coculture with human endothelial cells (fig. S1). Together,these results demonstrate generation of a human mesoderm progen-itor cell type with MAB characteristics from healthy human iPSCs (seefigs. S1 and S2).

Finally, we tested the possibility of deriving HIDEMs from certifiedvector integration–free (VIF) human iPSCs (see Materials and Meth-ods). We obtained cells with features comparable to those of HIDEMsderived from iPSCs generated with viral vectors, demonstrating thatthe presence of exogenous factors does not sustain their proliferativecapability (Fig. 3, D and E, and fig. S1).

Generating LGMD2D iPSCs and genetically correcting HIDEMsAfter validation of the above protocol with healthy human donor iPSCs,fibroblasts or myoblasts obtained from four LGMD2D patients (pa-tients 1 to 4; representative example in Fig. 5A) were reprogrammedusing retroviral vectors carrying SOX2, KLF4, and OCT4 ± cMYCcomplementary DNAs (cDNAs) (see Supplementary Materials). Inthe absence of cMYC, fewer iPSC colonies were obtained, but thesewere indistinguishable from those obtained in the presence of cMYC.Colonies of iPSCs started to appear about 30 days after transductionwith the viral vectors carrying reprogramming factor cDNAs; the re-programming efficiency was 0.005% 45 days after transduction withthe viral vectors (cells cultured in medium containing valproate and 3to 5% O2) (34, 35). Clonal lines were established from four differentLGMD2D patients, with morphology comparable to that for humanESCs (Fig. 5B). Pluripotency was assessed by AP staining, expressionof specific transcription factors, and formation of embryoid bodiesand teratomas (Fig. 5B and fig. S3). We detected relatively low levels

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of KLF4 expression (Fig. 5C), in line with recent reports (36), but thisdid not affect the pluripotency of our iPSC lines. Derivation and char-acterization of LGMD2D HIDEMs revealed that karyotype, prolifera-tive capacity, surface marker expression, and myogenic differentiationwere comparable with that of HIDEMs derived from healthy controlindividuals (Fig. 5C and fig. S4). No reactivation of the exogenoustransgenes was observed, although endogenous SOX2 expression re-mained high in iPSCs derived from one patient (expression is shownin fig. S4), but this did not interfere with differentiation, as recentlyreported (37). No tumors developed in tumorigenic assays (0 of 36immunodeficient mice transplanted with LGMD2D HIDEMs devel-oped tumors) whether or not cMYC was present in the original re-programming cocktail.

To genetically correct LGMD2D HIDEMs, we developed a newlentiviral vector carrying the human a-sarcoglycan cDNA (SGCA)under transcriptional control of the muscle-specific myosin light chain1F promoter and enhancer (Fig. 5D and fig. S4). As shown in Fig. 5Dand fig. S4, the transgene is selectively expressed in myotubes gener-ated from genetically corrected LGMD2D HIDEMs previously trans-duced with the MyoD-ER lentivector (as opposed to surrounding cellsthat are undifferentiated). These data show that it is possible to re-program adult somatic cells from LGMD2D patients to pluripotencyand to genetically correct MABs derived from LGMD2D iPSCs. Theyalso show that the genetically corrected MABs derived from LGMD2DiPSCs undergo terminal myogenic differentiation with correct and spe-cific expression of the therapeutic transgene (Fig. 5D and fig. S4).

Additionally, we have also generated HIDEMs from iPSCs derivedfromDMDpatients and genetically corrected themwith a human artificialchromosome containing the entire dystrophin locus (DYS-HAC; fig. S5)(38). We have recently shown efficacy of combined mouse MAB trans-plantation andDYS-HAC–mediated genetic correction inmdxmice (24).

Transplantation of iPSC-derived MAB-like cellsin Sgca-null/scid/beige miceThere are no large-animal models of LGMD2D, and the only availablepreclinical model is the Sgca-null mouse (31). To transplant humancells in this model, we crossed the severe combined immunodeficient(scid)/beige mouse with the Sgca-null mouse, generating a new dys-trophic and immune-deficient triple mutant: the Sgca-null/scid/beigemouse (fig. S6). Phenotypically, Sgca-null/scid/beige mice showed re-duced motility and curving of the vertebral column (kyphosis). His-tologically, these mice show an absence of Sgca and typical signs ofprogressive muscular dystrophy, such as regenerating and necroticmyofibers, inflammatory infiltrates in muscle, fibrosis, and elevatedcreatine kinase (fig. S6).

MyoD-ER–transduced healthy HIDEMs (106) (n = 2 lines tested)and genetically corrected LGMD2D HIDEMs (n = 2 lines tested) weremarked with a lentiviral vector expressing green fluorescent protein(GFP) and were transplanted intramuscularly in the tibialis anteriormuscle of juvenile Sgca-null/scid/beige mice (see Supplementary Ma-terials). This resulted in colonization of the transplanted muscle (Fig.6A), with donor cells observed inside recipient skeletal muscle fibers7 days after transplantation (Fig. 6B). One month after transplanta-tion, we calculated that the percentage of cells that had engraftedand survived in the host muscle was about 5 to 7% of the total num-ber injected (by counting an average of 35 cells per 7-mm section). Thiscorresponded to 53 ± 14 (SEM) SGCA+ fibers per muscle section (Fig.6C). Moreover, reconstitution of the dystrophin-associated protein




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Fig. 5. ReprogrammingLGMD2D cells to iPSCs andderivation of HIDEMs. (A) Rep-resentative morphology ofa LGMD2D cellular popula-tion obtained after culture ofa skeletal muscle biopsy.Scale bar, 50 mm. (B) Repro-gramming of LGMD2D cellsto iPSCs using the factorsOCT4, SOX2, and KLF4 ±cMYC (two of four lines werenot transduced with cMYC).Upper images show mor-phology (phase), AP staining(blue), and Nanog expres-sion (green) in LGMD2D iPSCs.White scale bar, 0.9 mm; blackscale bar, 0.8 mm. Lowerpanels show a teratoma for-mation assay performed withcell colonies depicted in theupper panels (see the Supple-mentary Materials). Two leftpanels show the teratomamass before and after resec-tion from a NOD/scid mouse;below these two images isan H&E-stained section fromthe resected teratoma, withfields inside the white boxesshowing the different tissues(representative of the threegerm layers) into which theteratoma can differentiate.Scalebar, 250mm. (C) LGMD2DHIDEMs. The top two imagesdepict the morphology andAP staining of mature HIDEMs(scale bar, 50 mm); below theseare three images showing thecorrect karyotype in three rep-resentative HIDEM popula-tions from patients 1, 2, and4. Thebargraphbelowthekar-yotypes shows expression oftotal and exogenous repro-gramming factors (OCT4,SOX2, and KLF4) by LGMD2DiPSCs and the HIDEMs derivedfrom them. The data shownare the averageof cells derivedfrom four different patients(data showing values of eachpatient are available in fig.S3). The curves illustrate pro-liferation of three differentLGMD2D HIDEM lines versus
primary humanMAB control cells (black line). Histograms show surfacemark-ers detected by FACS analysis for HIDEMs derived from patient 1. Bottompanel shows MyoD-ER–mediated conversion to a myogenic fate of threedifferent HIDEM lines (left column) and fusion of a representative population(not transducedwithMyoD-ER andmarkedwithGFP)with C2C12myoblasts(right column) Scale bar, 250 mm. (D)Myogenic differentiation via tamoxifen-
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induced MyoD-ER nuclear translocation into genetically corrected LGMD2DHIDEMs. Shown is the muscle-specific SGCA lentiviral vector (details in fig.S3C). Immunofluorescence panel shows SGCA expression only in a differen-tiated myotube (white arrow and inset). Scale bar, 40 mm. Western blotconfirms immunofluorescence, demonstrating restoration of SGCA expres-sion in genetically corrected and differentiated HIDEMs.




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Fig. 6. Transplantation of HIDEMs into Sgca-null/scid/beige mice. (A) GFPfluorescence 7 days after intramuscular injection of 106 genetically correctedLGMD2D HIDEMs into the tibialis anterior muscle of Sgca-null/scid/beigemice. Scale bar, 1 mm. (B) (Top) Immunofluorescence staining of a sectionfrom the muscle shown in (A) demonstrating engraftment of geneticallycorrected LGMD2D HIDEMs as revealed by lamin A/C+ nuclei (lamin A/Cmarks the human nuclear lamina). (Bottom) Magnification of the area insidethe white box in top image showing a cluster of myofibers containing donorhuman cell nuclei. Scale bar, 500 mm. (C) Immunofluorescence showing acluster of SGCA+ myofibers containing human nuclei 1 month after intra-muscular transplantation of genetically corrected LGMD2D HIDEMs (quanti-fied in the bar graph; error bars show SD and the number corresponds to anaverage of 2% of tibialis anterior myofibers). Scale bar, 60 mm. Bottom im-ages show the same cluster in serial section stained for b- and g-sarcoglycan(SGCB and SGCG). (D) Intra-arterial transplantation of genetically corrected
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LGMD2D HIDEMs. Left panels show blood vessel–associated GFP+ cells6 hours after injection of LGMD2D HIDEMs into the femoral artery. Scalebar, 0.5 mm. Top right and middle panels show immunofluorescence of hu-man cells in-between mouse myofibers (scale bar, 50 mm); lower panel de-picts a human fluorescent cell outside CD31+ blood vessels 12 hours aftertransplantation (scale bar, 90 mm). (E) The bar graph illustrates quantitativereal-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 trans-plantation (injected in the right or in the left femoral artery, respectively;***P < 0.0005, unpaired t test). (F) Representative example of SGCA+ myo-fibers containing human nuclei 1 month after intra-arterial transplantationof genetically corrected LGMD2D HIDEMs. Scale bar, 50 mm. (G) RT-PCRconfirming SGCA expression 1 month after intramuscular (IM) and intra-arterial (IA) injection. TA, tibialis anterior muscle. GC, gastrocnemius muscle.




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complex was demonstrated by coexpression of b- and g-sarcoglycans(SGCB and SGCG; Fig. 6C).

Intra-arterial transplantation of genetically corrected LGMD2Dpatient-derived HIDEMs resulted in colonization of skeletal muscledownstream of the injection site (Fig. 6D), with cells migrating out ofthe blood vessels within 12 hours after transplantation (Fig. 6D). Thesedata were confirmed by quantitative PCR of DNA performed 24 hoursafter transplantation, comparing healthy control and LGMD2DHIDEMs(right leg) with the cells from which they were originally derived (fibro-blasts or myoblasts that were then reprogrammed to iPSCs; left leg)(Fig. 6E) (Supplementary Materials). All HIDEMs showed greater en-graftment compared to the fibroblasts or myoblasts from which theywere derived, although we observed variability among different iPSClines. One month after intra-arterial transplantation, SCGA expressionwas detected by immunofluorescence and reverse transcription–PCR(RT-PCR) (Fig. 6, F and G).

We then investigated the possibility of enhancing HIDEM engraft-ment by transplantingmouse cells instead of human.We generated andtransplanted murine iPSC-derived MABs (MIDEMs; n = 18; fig. S7)and detected five to sixfold more SGCA+ myofibers in mouse musclecompared to transplantation with HIDEMs (SGCA+ myofibers: 286 ±41 versus 53 ± 14, mean ± SEM; Fig. 7, A and B).We saw a concomitantreduction in fibrotic-adipose tissue in transplanted muscle (26.24% lessthan nontransplantedmuscle, P < 0.05, n = 6; Fig. 7C). These data sug-gest that species-specific variables, other than the adaptive immune sys-tem, control donor cell engraftment. As a consequence of this enhancedengraftment, we detected functional amelioration of motor capacityusing a treadmill test to exhaustion (24) in animals transplanted withMIDEMs. Intramuscularly and intra-arterially transplanted mice showedenhanced motor capacity after treatment, running from 48 to 62%morethan their baseline performance and from 12 to 22% more than un-treated animals 35 days after transplantation (P < 0.05 and P < 0.005,respectively; Fig. 7D). To validate these findings, wemeasured the tetanicforce of the tibialis anterior muscle and force of contraction on isolatedmuscle fibers 4 months after transplantation. Figure 7E shows that intibialis anterior muscles from mice transplanted intramuscularly andintra-arterially, the tetanic force was significantly higher than in un-treated mice (67% and 83%, respectively; P < 0.05). Individual musclefibers (n = 119) were then dissected from the gastrocnemius muscle ofthe samemice, and the analysis demonstrated thatGFP+myofibers devel-oped greater force than didmuscle fibers from untreatedmice (Fig. 7E).

Finally, to determine whether the transplanted cells were ableto contribute to the pool of AP+ pericytes in vivo, we searched forAP+ and GFP+ MIDEMs in the skeletal muscle interstitium of Sgca-null/scid/beige mice. As shown in Fig. 7F, double-positive donor cellswere clearly identifiable near GFP+ myofibers, indicating donor cellcontribution to muscle regeneration together with replenishment ofthe pericyte niche in vivo. Notably, the number of AP+ cells per tibialisanterior 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-typeanimals (666.5 ± 47.6; n = 3; Fig. 7F).

DISCUSSION

Previous studies from our laboratory demonstrated rescue of dystrophicSgca-null mice by intra-arterial transplantation of murine MABs (27).

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We then decided to apply this strategy using human genetically correctedMABs from LGMD2D patients. However, we found that LGMD2Dpatients have a reduced number of AP+ pericytes in vivo, and thus, ob-taining pericyte-derived MABs directly from patients for genetic cor-rection in vitro and autologous transplantation was not possible. Toovercome this problem, we developed a strategy that allowed the deriva-tion andpropagation in culture of a population ofMAB-likemesodermalprogenitor cells derived from human iPSCs (HIDEMs) generated fromadult somatic cells. The reproducibility of this protocol was validatedusing 10 different human iPSC lines generated in four different labora-tories using different approaches. Notably, potential sources of variationamong different HIDEM lines (for example, age and sex of donors andresidual expression of reprogramming factors) did not correlate withreprogramming or differentiation efficiency (37). HIDEMs were alsoderived from iPSCs generated with three reprogramming factors (with-out cMYC) and from VIF iPSCs. We succeeded in deriving iPSC linesfrom four LGMD2D patients. We derived iPSCs from myoblasts frompatient 1 that were similar to those derived from fibroblasts fromhealthy and dystrophic individuals. HIDEMs derived from LGMD2Dpatients were easily transduced with lentiviral vectors, resulting in a cellpopulation that could be genetically corrected and expanded and thatwas clonogenic, nontumorigenic, and readily transplantable.

To test the therapeutic potential of genetically corrected HIDEMsfor future transplantation into LGMD2D patients, we generated a newdystrophic and immune-deficient mouse: the Sgca-null/scid/beigemouse. Intramuscular or intra-arterial injection of geneticallycorrected HIDEMs resulted in their engraftment in dystrophic skeletalmuscle and production of clusters of SGCA+ myofibers. Variablelevels of engraftment of human cells in mouse dystrophic muscle wereobserved, possibly due to different levels of inflammation and sclerosisin the mouse recipients and to different expression levels of adhesionproteins (for example, integrins and selectins) by HIDEMs from dif-ferent human subjects. The HIDEMs we isolated did not give rise totumors upon subcutaneous, intramuscular, and intra-arterial trans-plantation into immune-deficient mice.

Recently, other laboratories have reported the derivation of my-ogenic progenitors from human iPSCs [for example, (14)]. Theseprogenitors differentiate robustly in vivo and may be the choice fortreating localized forms of muscle disorders where intramusculartransplantation into several sites of the few affected muscles is suffi-cient. The advantage of HIDEMs is that they can be delivered throughthe arterial circulation and thus are able to reach muscles throughoutthe body. However, more work is needed to assess the safety and toimprove engraftment upon systemic delivery of genetically engineeredHIDEMs before they enter clinical testing.

Recent adeno-associated virus–based gene therapy trials have shownpromise for treating LGMD2D (39). Nevertheless, immunity and theloss of transgene expression are still hurdles that need to be overcome(40, 41). Similarly, an immune response also might be elicited bytransplanted allogeneic HIDEMs or MABs, although this is still beinginvestigated. The limited availability of adult tissue-specific muscleprogenitor cells is a major obstacle for cell therapies. Reprogrammingof adult somatic cells to form iPSCs followed by lineage-specific com-mitment and differentiation may solve the problem of the limited sup-ply of muscle progenitor cells.

Deriving patient-specific iPSCs and expanding their differentiatedprogeny may provide a useful strategy for gene and cell therapies. Al-though very preliminary, our study suggests that genetically corrected




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Fig. 7. Transplantation of murine iPSC-derived MABs into Sgca-null/scid/+

transplantation. (Left graph) Normalized tetanic force of isolated tibialis an-
beigemice. (A) Stereoscopic pictures of GFP myofibers 1month after trans-plantation of murine iPSC-derived MAB-like cells (MIDEMs). Scale bar, 1 mm.(B) Transverse sections from themuscle shown in (A), showing large areas ofGFP- and Sgca-positive myofibers. (C) Quantification of fibrosis in trans-planted versus control mice showing a reduction in fibrosis in transplantedmice (n = 3). *P < 0.05, Student’s t test. The two images show representativeMasson trichrome staining of tibialis anteriormuscles from transplanted andcontrol Sgca-null/scid/beige mice (blue, fibrotic infiltrate). Scale bar, 250 mm.(D) Time to exhaustion on a treadmill test for transplanted Sgca-null/scid/beigemice (n = 13; 106 cells injected bilaterally in tibialis anterior, gastrocne-mius, and quadriceps muscles) versus nontransplanted dystrophic (n = 8)and nondystrophic (n = 5) control mice. The data show functional ameliora-tion of dystrophic muscle in mice transplanted with MIDEMs (12 to 22%more than nontransplanted animals 35 days after transplantation). Note thatdata are presented as average motor capacity relative to baseline perform-ances measured until the day before transplantation. *P < 0.05; **P <0.005, one-way ANOVA. (E) Force measurements 4 months after MIDEM
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terior muscles from intramuscular and intra-arterially transplanted micetogether with control nontransplanted dystrophic and nondystrophic mice(n≥ 3 per group). (Right graph)Mean values of specific force for a populationof single myofibers dissected from transplanted and nontransplanted gas-trocnemius muscles (together with the controls; n values above columns).The arrow indicates a representative picture of a GFP+ myofiber analyzedin the assay. Scale bar, 60 mm. Error bars represent means ± SD. *P < 0.05;***P < 0.0005, one-way ANOVA and Student-Newman-Keuls test. ns, not sig-nificant. (F) Cryosection of MIDEM-transplanted tibialis anterior musclestained for CD31 (Pecam; brown, immunohistochemistry; to mark bloodvessels) and AP (blue, enzymatic reaction). A serial section shows the pres-ence of GFP+myofibers and interstitial cells, some of which colocalize withthe vessels marked as described above. Scale bar, 80 mm. The bar graphquantifies the total number of AP+ cells per section of tibialis anteriormuscle of 8-month-old Sgca-null/scid/beigemice after IM transplantationwith MIDEMs. Error bars representmeans ± SEM. *P < 0.05; **P < 0.005, one-way ANOVA and Tukey’s test.

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MABs generated from iPSCs derived from the fibroblasts or myoblastsof LGMD2D patients could be useful for autologous transplantationand that this approach might also be applicable for treating other re-cessive muscular dystrophies.

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MATERIALS AND METHODS

Cell culturesHuman MABs and HIDEMs were cultured in MegaCell DMEM(Dulbecco’s modified Eagle’s medium) (Sigma) as described (42). Al-ternatively, the same cells were cultured in Iscove’s modified Dulbecco’smedium (IMDM; Sigma) containing 10% fetal bovine serum (FBS),2 mM glutamine, 0.1 mM b-mercaptoethanol, 1% nonessential aminoacids, human basic fibroblast growth factor (5 ng/ml), penicillin(100 IU/ml), streptomycin (100 mg/ml), 0.5 mM oleic and linoleicacids (Sigma), 1.5 mM Fe++ [Iron(II) chloride tetrahydrate, Sigma;or Fer-In-Sol, Mead Johnson], 0.12 mM Fe+++ [Iron(III) nitrate nona-hydrate, 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–derivedCD34+ progenitors with a three-plasmid and seven-factor Epstein-Barr virus nuclear antigen (EBNA)–based episomal system. The otherhealthy donor iPSC lines used in this study have been described in(43). The murine iPSCs used here were characterized and cultured aspreviously described (38). Additional details are available in the Sup-plementary Materials.

LGMD2D samplesLGMD2D skeletal muscle cells and biopsies were provided by the bio-banks of M. Moggio (Telethon Genetic BioBank Network; OspedaleMaggiore Policlinico, Milan, Italy), M. Mora (Telethon Genetic BioBankNetwork; Istituto Neurologico Carlo Besta, Milan, Italy), and B. Schoserand 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 (SanRaffaele Scientific Institute, Milan, Italy) for providing LGMD2D slides.See table S1 for additional details.

Viral vectors and reprogramming to iPSCsGeneration of iPSCs from human cells was done with a standardretrovirus-based system previously published (2). MyoD-ER constructwas provided by J. S. Chamberlain (University of Washington, Seattle,WA) and used as previously described (33). Human muscle-specificSGCA lentivector (pLentiMLC1F/SGCA) construction and more de-tails are available in the Supplementary Materials.

Generation of iPSC-derived MAB-like cellsSubstantial modification of the available protocols to generate vascularcells from ESCs [for example, in (44)] facilitated the initial setup ofthis method. The main steps of the protocol for HIDEM derivationare summarized here:

1. Dissociation of iPSCs colonies to single-cell suspension (week 1):a. 10 mMROCK inhibitor for 1 hour in iPSCmedium (see above).b. 30 to 120 min at 37°C and 5% CO2 in dissociation medium

[0.5 mM EDTA, 0.1 mM b-mercaptoethanol, 3% FBS in phosphate-buffered saline (PBS) without Ca2+ and Mg2+].

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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 a-MEM (Gibco) containing antibiotics (penicillin/streptomycin),10% FBS, nucleotides, and 0.2% b-mercaptoethanol for 1 week at37°C, 5% CO2, and 3 to 5% O2.

3. Dissociate culture (as described in step 1), gently scrape dish sur-face with a cell scraper, filter solution using a 40-mm strainer, and seed2.5 × 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)], waitup to 10 days from step 3, trypsinize cells (5 min at 37°C, 5% CO2, and3 to 5% O2), and seed them on a Matrigel-coated dish at about 80%confluency in human MAB complete medium (either MegaCell DMEMor 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 onplastic and in human MAB medium (weeks 3 to 4).

From now on, culture HIDEMs exactly like human MABs, asdescribed above and detailed in (42). Transduce the cells with lentiviralMyoD-ER (with a maximummultiplicity of infection of 5) and administer4OH- or standard tamoxifen to obtain robust myogenic differentiation.

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

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

Surface marker analysis and gene expression profilingA detailed report of the procedures, antibodies, and meta-analysis isavailable in the Supplementary Materials. Raw data of HIDEM andcontrol human MAB gene expression profiling are available in theGEO repository.

MiceScid, scid/beige, nonobese diabetic (NOD)/scid, NOD/scid/g chainknockout (NSG), and nude mice were purchased from Charles RiverLaboratories and were housed in San Raffaele Scientific Instituteanimal house together with Sgca-null/scid/beige. All mice were keptin specific pathogen–free conditions, and all procedures involving liv-ing animals conformed to Italian law (D.L.vo 116/92 and subsequentadditions) and were approved by the San Raffaele Institutional ReviewBoard.

Generation of Sgca-null/scid/beige mouse is detailed in the Sup-plementary Materials. Briefly, females homozygous for Sgca mutation(Sgca−/−) were bred with homozygous scid/beige−/− males. The result-ing F1 heterozygous females were crossed with scid/beige−/− males.In F2 mice (and in subsequent generations), we verified Sgca and scidmutation (beige mutation was genotyped by Charles River Laboratories),leucopenia, and the absence of B and T lymphocytes. Then, we isolatedSgca+/−/scid/beige−/− females and crossed them with scid/beige−/− males




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for three generations. In F5, Sgca−/−scid/beige−/− males and femaleswere bred together to generate mice homozygous for both scid/beigeand Sgca mutations. Sgca+/+ and Sgca−/− immunocompetent mice, aswell as Sgca+/+ immune-deficient matched controls, were also main-tained in the colony. Animals of all genotypes presented an average of68.7 ± 2.2% (SD; n = 13) of CB17 background according to single-nucleotide polymorphism analysis (Mouse 348 SNP panel, CharlesRiver Laboratories).

Transplantation, tumorigenic, and teratomaformation assaysIntramuscular (n = 25 Sgca-null/scid/beige mice) and intra-arterial(n = 15 Sgca-null/scid/beige mice) injections were done as previouslydescribed (24). When MyoD-ER–expressing cells were transplanted,tamoxifen (33 mg/g) was given once a day (intraperitoneally or sub-cutaneously) for a total of 7 days starting from 1 day before transplan-tation. Further information regarding cell transplantation, togetherwith a detailed description of tumorigenic and teratoma formation as-say, is available in the Supplementary Materials.

PCR and immunoblottingGenotyping PCR for Sgca and scid mutations was done as alreadydescribed (24, 31). Genotyping PCR for the beige (Lystbg) mutationwas performed by Charles River Laboratories. Primers, quantitativereal-time PCRs, and Western blot are detailed in the SupplementaryMaterials.

Histology, histochemistry, immunofluorescence, andkaryotype analysisTissue sections were stained with hematoxylin and eosin (H&E)(Sigma-Aldrich) and Masson trichrome (Bio-Optica) following pro-tocol provided by the manufacturers. AP was detected as already de-scribed (19) or with the protocol available with the PermaBlue/AP kit(Histo-line laboratories). Immunofluorescence is detailed in the Sup-plementary Materials. Karyotype analyses were performed and certi-fied by Synlab Diagnostic Services Srl (Italy) with QFQ staining (n =50 metaphases per sample).

Functional measurements: Motor capacityand force of contractionControl untransplanted (vehicle: PBS) Sgca+/+/scid/beige (n = 5 forintramuscular; n = 8 for intra-arterial), untransplanted (vehicle) Sgca-null/scid/beige (n = 8 for intramuscular; n = 8 for intra-arterial), andtransplanted Sgca-null/scid/beige (n = 8 for intramuscular; n = 5 forintra-arterial) were tested for functional recovery on a treadmill(Columbus Instruments), as recently reported (24). Mechanics of iso-lated muscles and single-fiber analysis were performed as previously de-scribed (24), and details are available in the Supplementary Materials.

Statistical analysisWe expressed values as means ± SEM or SD. We assessed significanceof the differences between means by Student’s t test, and when morethan two groups had to be compared, we used one-way analysis ofvariance (ANOVA) followed by Tukey’s or Student-Newman-Keulspost test to determine which groups were statistically significantly dif-ferent from the others. A probability of less than 5% (P < 0.05) wasconsidered to be statistically significant. Data were analyzed withMicrosoft Excel 14.1.3 and GraphPad Prism 5.

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/4/140/140ra89/DC1Materials and MethodsFig. 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.Reference

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Acknowledgments: We thank S. Previtali, J. Diaz-Manera, the Telethon Network of GeneticBiobanks, and the MTCC for providing samples; M. Noviello, D. Moi, A. Lombardo, andD. Becker for help and reagents; D. Sassoon, L. Wrabetz, and S. Maffioletti for helpfuldiscussions; K. English for critical reading of the manuscript; and J. Chamberlain for pro-viding the MyoD-ER lentiviral vector. Funding: This work was supported by European Re-search Council, European Community 7th Framework project OPTISTEM (contract numberHealth-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 themanuscript and conceived and carried out most of the experimental work and analysiswith 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. performedmicroarray 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 projectand 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 viareprogramming of autologous cells.” Data and materials availability: The microarray data havebeen deposited in the National Center for Biotechnology Information GEO (GSE36098).

Submitted 1 December 2011Accepted 8 June 2012Published 27 June 201210.1126/scitranslmed.3003541

Citation: F. S. Tedesco, M. F. M. Gerli, L. Perani, S. Benedetti, F. Ungaro, M. Cassano, S. Antonini,E. Tagliafico, V. Artusi, E. Longa, R. Tonlorenzi, M. Ragazzi, G. Calderazzi, H. Hoshiya,O. Cappellari, M. Mora, B. Schoser, P. Schneiderat, M. Oshimura, R. Bottinelli, M. Sampaolesi,Y. Torrente, V. Broccoli, G. Cossu, Transplantation of genetically corrected human iPSC-derivedprogenitors in mice with limb-girdle muscular dystrophy. Sci. Transl. Med. 4, 140ra89 (2012).



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